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2002/657/EC: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (Text with EEA relevance) (notified under document number C(2002) 3044) 2002/657/EC: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (Text with EEA relevance) (notified under document number C(2002) 3044)
OJ L 221, , pp. 8–36
(ES, DA, DE, EL, EN, FR, IT, NL, PT, FI, SV) This document has been published in a special edition(s)
(CS, ET, LV, LT, HU, MT, PL, SK, SL, BG, RO, HR)
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Odločba Komisije z dne 14. avgusta 2002 o izvajanju Direktive Sveta 96/23/ES glede opravljanja analitskih metod in razlage rezultatov (notificirano pod dokumentarno številko K(2002)3044)Besedilo velja za EGP Odločba Komisije z dne 14. avgusta 2002 o izvajanju Direktive Sveta 96/23/ES glede opravljanja analitskih metod in razlage rezultatov (notificirano pod dokumentarno številko K(2002)3044)Besedilo velja za EGP
OJ L 221, , str. 8–36
(ES, DA, DE, EL, EN, FR, IT, NL, PT, FI, SV) Other special edition(s)
(CS, ET, LV, LT, HU, MT, PL, SK, BG, RO, HR)
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| Avis juridique important | Pomembno pravno obvestilo |
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| 32002D0657 | 32002D0657 |
| 2002/657/EC: Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (Text with EEA relevance) (notified under document number C(2002) 3044) | Odločba Komisije z dne 14. avgusta 2002 o izvajanju Direktive Sveta 96/23/ES glede opravljanja analitskih metod in razlage rezultatov (notificirano pod dokumentarno številko K(2002)3044)Besedilo velja za EGP |
| Official Journal L 221 , 17/08/2002 P. 0008 - 0036 | Uradni list L 221 , 17/08/2002 str. 0008 - 0036 |
| CS.ES poglavje 3 zvezek 36 str. 493 - 521 | |
| ET.ES poglavje 3 zvezek 36 str. 493 - 521 | |
| Commission Decision | |
| of 12 August 2002 | |
| implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results | |
| (notified under document number C(2002) 3044) | |
| (Text with EEA relevance) | |
| (2002/657/EC) | |
| THE COMMISSION OF THE EUROPEAN COMMUNITIES, | |
| Having regard to the Treaty establishing the European Community, | |
| Having regard to Council Directive 96/23/EC of 29 April 1996 on measures to monitor certain substances and residues thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and Decisions 89/187/EEC and 91/664/EEC(1), and in particular the second subparagraph of Article 15(1) thereof, | |
| Whereas: | |
| (1) The presence of residues in products of animal origin is a matter of concern for public health. | |
| (2) Commission Decision 98/179/EC of 23 February 1998 laying down detailed rules on official sampling for the monitoring of certain substances and residues thereof in live animals and animal products(2) provides that the analysis of samples is to be carried out exclusively by laboratories approved for official residue control by the competent national authority. | |
| (3) It is necessary to ensure the quality and comparability of the analytical results generated by laboratories approved for official residue control. This should be achieved by using quality assurance systems and specifically by applying of methods validated according to common procedures and performance criteria and by ensuring traceability to common standards or standards commonly agreed upon. | |
| (4) Council Directive 93/99/EEC of 29 October 1993 on the subject of additional measures concerning the official control of foodstuffs and Decision 98/179/EC(3) require official control laboratories to be accredited according to ISO 17025 (1) from January 2002 onwards. Pursuant to Decision 98/179/EC, participation in an internationally recognised external quality control assessment and accreditation scheme is required for approved laboratories. Moreover, approved laboratories must prove their competence by regular and successful participation in adequate proficiency testing schemes recognised or organised by the national or Community reference laboratories. | |
| (5) A network of Community reference laboratories, national reference laboratories and national control laboratories operates under Directive 96/23/EC to enhance coordination. | |
| (6) As a result of advances in analytical chemistry since the adoption of Directive 96/23/EC the concept of routine methods and reference methods has been superseded by criteria approach, in which performance criteria and procedures for the validation of screening and confirmatory methods are established. | |
| (7) It is necessary to determine common criteria for the interpretation of test results of official control laboratories in order to ensure a harmonised implementation of Directive 96/23/EC. | |
| (8) It is necessary to provide for the progressive establishment of minimum required performance limits (MRPL) of analytical method for substances for which no permitted limit has been established and in particular for those substances whose use is not authorised, or is specifically prohibited in the Community, in order to ensure harmonised implementation of Directive 96/23/EC. | |
| (9) Commission Decision 90/515/EEC of 26 September 1990 laying down the reference methods for detecting residues of heavy metals and arsenic(4), Commission Decision 93/256/EEC of 14 May 1993 laying down the methods to be used for detecting residues of substances having a hormonal or a thyrostatic action(5), and of Commission Decision 93/257/EEC of 15 April 1993 laying down the reference methods and the list of the national reference laboratories for detecting residues(6), as last amended by Decision 98/536/EC(7) have been re-examined before in order to take account of developments in scientific and technical knowledge, have been found outdated in their scope and provisions and should accordingly be repealed with this Decision. | |
| (10) In order to allow methods for the analysis of official samples to be adapted to the provisions of this Decision, a transitional period should be laid down. | |
| (11) The measures provided for in this Decision are in accordance with the opinion of the Standing Committee on the Food Chain and Animal Health, | |
| HAS ADOPTED THIS DECISION: | |
| Article 1 | |
| Subject matter and scope | |
| This Decision provides rules for the analytical methods to be used in the testing of official samples taken pursuant to Article 15(1), second sentence, of Directive 96/23/EC and specifies common criteria for the interpretation of analytical results of official control laboratories for such samples. | |
| This Decision shall not apply to substances for which more specific rules have been laid down in other Community legislation. | |
| Article 2 | |
| Definitions | |
| For the purpose of this Decision the definitions in Directive 96/23/EC and in the Annex to this decision shall apply. | |
| Article 3 | |
| Analytical methods | |
| The Member States shall ensure that official samples taken pursuant to Directive 96/23/EC are analysed using methods that: | |
| (a) are documented in test instructions, preferably according to ISO 78-2 (6); | |
| (b) comply with Part 2 of the Annex to this Decision; | |
| (c) have been validated according to the procedures described in Part 3 of the Annex; | |
| (d) comply with the relevant minimum required performance limits (MRPL) to be established in accordance with Article 4. | |
| Article 4 | |
| Minimum required performance limits | |
| The present Decision shall be reviewed to progressively establish the minimum required performance limits (MRPL) of analytical methods to be used for substances for which no permitted limit has been established. | |
| Article 5 | |
| Quality control | |
| The Member States shall ensure the quality of the results of the analysis of samples taken pursuant to Directive 96/23/EC, in particular by monitoring tests and/or calibration results according to Chapter 5.9 of ISO 17025 (1). | |
| Article 6 | |
| Interpretation of results | |
| 1. The result of an analysis shall be considered non-compliant if the decision limit of the confirmatory method for the analyte is exceeded. | |
| 2. If a permitted limit has been established for a substance, the decision limit is the concentration above which it can be decided with a statistical certainty of 1 - α that the permitted limit has been truly exceeded. | |
| 3. If no permitted limit has been established for a substance, the decision limit is the lowest concentration level at which a method can discriminate with a statistical certainty of 1 - α that the particular analyte is present. | |
| 4. For substances listed in Group A of Annex I to Directive 96/23/EC, the α error shall be 1 % or lower. For all other substances, the α error shall be 5 % or lower. | |
| Article 7 | |
| Repeal | |
| Decisions 90/515/EEC, 93/256/EEC and 93/257/EEC are repealed. | |
| Article 8 | |
| Transitional provisions | |
| The methods for the analysis of official samples of substances listed in Group A of Annex I to Directive 96/23/EC, which satisfy the criteria set out in Decisions 90/515/EEC, 93/256/EEC and 93/257/EEC may be used for up to two years after this Decision enters into force. Methods currently applied for substances listed in Group B of Annex I to Directive 96/23/EC shall comply with this Decision at the latest five years after the date of application of this Decision. | |
| Article 9 | |
| Date of application | |
| This Decision shall apply from 1 September 2002. | |
| Article 10 | |
| Addressees | |
| This Decision is addressed to the Member States. | |
| Done at Brussels, 12 August 2002. | |
| For the Commission | |
| David Byrne | |
| Member of the Commission | |
| (1) OJ L 125, 23.5.1996, p. 10. | |
| (2) OJ L 65, 5.3.1998, p. 31. | |
| (3) OJ L 290, 24.11.1993, p. 14. | |
| (4) OJ L 286, 18.10.1990, p. 33. | |
| (5) OJ L 118, 14.5.1993, p. 64. | |
| (6) OJ L 118, 14.5.1993, p. 75. | |
| (7) OJ L 251, 11.9.1998, p. 39. | |
| ANNEX | |
| PERFORMANCE CRITERIA, OTHER REQUIREMENTS AND PROCEDURES FOR ANALYTICAL METHODS | |
| 1. DEFINITIONS | |
| 1.1. Accuracy means the closeness of agreement between a test result and the accepted reference value (2). It is determined by determining trueness and precision. | |
| 1.2. Alpha (α) error means the probability that the tested sample is compliant, even though a non-compliant measurement has been obtained (false non-compliant decision). | |
| 1.3. Analyte means the substance that has to be detected, identified and/or quantified and derivatives emerging during its analysis. | |
| 1.4. Beta (β) error means the probability that the tested sample is truly non-compliant, even though a compliant measurement has been obtained (false compliant decision). | |
| 1.5. Bias means the difference between the expectation of the test result and an accepted reference value (2). | |
| 1.6. Calibration standard means a device for measurements that represents the quantity of substance of interest in a way that ties its value to a reference base. | |
| 1.7. Certified reference material (CRM) means a material that has had a specified analyte content assigned to it. | |
| 1.8. Co-chromatography means a procedure in which the extract prior to the chromatographic step(s) is divided into two parts. Part one is chromatographed as such. Part two is mixed with the standard analyte that is to be measured. Then this mixture is also chromatographed. The amount of added standard analyte has to be similar to the estimated amount of the analyte in the extract. This method is designed to improve the identification of an analyte when chromatographic methods are used, especially when no suitable internal standard can be used. | |
| 1.9. Collaborative study means analysing the same sample by the same method to determine the performance characteristics of the method. The study covers random measurement error and laboratory bias. | |
| 1.10. Confirmatory method means methods that provide full or complementary information enabling the substance to be unequivocally identified and if necessary quantified at the level of interest. | |
| 1.11. Decision limit (CCα) means the limit at and above which it can be concluded with an error probability of α that a sample is non-compliant. | |
| 1.12. Detection capability (CCβ) means the smallest content of the substance that may be detected, identified and/or quantified in a sample with an error probability of β. In the case of substances for which no permitted limit has been established, the detection capability is the lowest concentration at which a method is able to detect truly contaminated samples with a statistical certainty of 1 - β. In the case of substances with an established permitted limit, this means that the detection capability is the concentration at which the method is able to detect permitted limit concentrations with a statistical certainty of 1 - β. | |
| 1.13. Fortified sample material means a sample enriched with a known amount of the analyte to be detected. | |
| 1.14. Interlaboratory study (comparison) means organisation, performance and evaluation of tests on the same sample by two or more laboratories in accordance with predetermined conditions to determine testing performance. According to the purpose the study can be classified as collaborative study or proficiency study. | |
| 1.15. Internal Standard (IS) means a substance not contained in the sample with physical-chemical properties as similar as possible to those of the analyte that has to be identified and which is added to each sample as well as to each calibration standard. | |
| 1.16. Laboratory sample means a sample prepared for sending to a laboratory and intended for inspection or testing. | |
| 1.17. Level of interest means the concentration of substance or analyte in a sample that is significant to determine its compliance with legislation. | |
| 1.18. Minimum required performance limit (MRPL) means minimum content of an analyte in a sample, which at least has to be detected and confirmed. It is intended to harmonise the analytical performance of methods for substances for which no permitted limited has been established. | |
| 1.19. Performance characteristic means functional quality that can be attributed to an analytical method. This may be for instance specificity, accuracy, trueness, precision, repeatability, reproducibility, recovery, detection capability and ruggedness. | |
| 1.20. Performance criteria means requirements for a performance characteristic according to which it can be judged that the analytical method is fit for the purpose and generates reliable results. | |
| 1.21. Permitted limit means maximum residue limit, maximum level or other maximum tolerance for substances established elsewhere in Community legislation. | |
| 1.22. Precision means the closeness of agreement between independent test results obtained under stipulated (predetermined) conditions. The measure of precision usually is expressed in terms of imprecision and computed as standard deviation of the test result. Less precision is determined by a larger standard deviation (2). | |
| 1.23. Proficiency study means analysing the same sample allowing laboratories to choose their own methods, provided these methods are used under routine conditions. The study has to be performed according to ISO guide 43-1 (3) and 43-2 (4) and can be used to assess the reproducibility of methods. | |
| 1.24. Qualitative method means an analytical method which identifies a substance on the basis of its chemical, biological or physical properties. | |
| 1.25. Quantitative method means an analytical method which determines the amount or mass fraction of a substance so that it may be expressed as a numerical value of appropriate units. | |
| 1.26. Reagent blank determination means the complete analytical procedure applied without the test portion or using an equivalent amount of suitable solvent in place of the test portion. | |
| 1.27. Recovery means the percentage of the true concentration of a substance recovered during the analytical procedure. It is determined during validation, if no certified reference material is available. | |
| 1.28. Reference material means a material of which one or several properties have been confirmed by a validated method, so that it can be used to calibrate an apparatus or to verify a method of measurement. | |
| 1.29. Repeatability means precision under repeatability conditions (2). | |
| 1.30. Repeatability conditions means conditions where independent test results are obtained with the same method on identical test items in the same laboratory by the same operator using the same equipment (2). | |
| 1.31. Reproducibility means precision under reproducibility conditions (2)(4). | |
| 1.32. Reproducibility conditions means conditions where test results are obtained with the same method on identical test items in different laboratories with different operators using different equipment (2)(4). | |
| 1.33. Ruggedness means the susceptibility of an analytical method to changes in experimental conditions which can be expressed as a list of the sample materials, analytes, storage conditions, environmental and/or sample preparation conditions under which the method can be applied as presented or with specified minor modifications. For all experimental conditions which could in practice be subject to fluctuation (e.g. stability of reagents, composition of the sample, pH, temperature) any variations which could affect the analytical result should be indicated. | |
| 1.34. Sample blank determination means the complete analytical procedure applied to a test portion taken from a sample from which the analyte is absent. | |
| 1.35. Screening method means methods that are used to detect the presence of a substance or class of substances at the level of interest. These methods have the capability for a high sample throughput and are used to sift large numbers of samples for potential non-compliant results. They are specifically designed to avoid false compliant results. | |
| 1.36. Single laboratory study (in-house validation) means an analytical study involving a single laboratory using one method to analyse the same or different test materials under different conditions over justified long time intervals. | |
| 1.37. Specificity means the ability of a method to distinguish between the analyte being measured and other substances. This characteristic is predominantly a function of the measuring technique described, but can vary according to class of compound or matrix. | |
| 1.38. Standard addition means a procedure in which the test sample is divided in two (or more) test portions. One portion is analysed as such and known amounts of the standard analyte are added to the other test portions before analysis. The amount of the standard analyte added has to be between two and five times the estimated amount of the analyte in the sample. This procedure is designed to determine the content of an analyte in a sample, taking account of the recovery of the analytical procedure. | |
| 1.39. Standard analyte means an analyte of known and certified content and purity to be used as a reference in the analysis. | |
| 1.40. Substance means matter of particular or definite chemical constitution and its metabolites. | |
| 1.41. Test portion means the quantity of material drawn from the test sample on which the test or observation is carried out. | |
| 1.42. Test sample means a sample prepared from a laboratory sample and from which test portions will be taken. | |
| 1.43. Trueness means the closeness of agreement between the average value obtained from a large series of test results and an accepted reference value. Trueness is usually expressed as bias (2). | |
| 1.44. Units means those units described in ISO 31 (20) and Directive 71/354/EC (19). | |
| 1.45. Validation means the confirmation by examination and the provision of effective evidence that the particular requirements of a specific intended use are fulfilled (1). | |
| 1.46. Within-laboratory reproducibility means precision obtained in the same laboratory under stipulated (predetermined) conditions (concerning e.g. method, test materials, operators, environment) over justified long time intervals. | |
| 2. PERFORMANCE CRITERIA AND OTHER REQUIREMENTS FOR ANALYTICAL METHODS | |
| Analytical methods or combinations of methods other than those described below may only be used for screening or confirmatory purposes if it can be proven that they fulfil the relevant requirements established in this Decision. | |
| 2.1. GENERAL REQUIREMENTS | |
| 2.1.1. Handling of samples | |
| Samples shall be obtained, handled and processed in such a way that there is a maximum chance of detecting the substance. Sample handling procedures shall prevent the possibility of accidental contamination or loss of analytes. | |
| 2.1.2. Performance of tests | |
| 2.1.2.1. Recovery | |
| During the analysis of samples the recovery shall be determined in each batch of samples, if a fixed recovery correction factor is used. If the recovery is within limits, the fixed correction factor may then be used. Otherwise the recovery factor obtained for that specific batch shall be used, unless the specific recovery factor of the analyte in the sample is to be applied in which case the standard addition procedure (see 3.5) or an internal standard shall be used for the quantitative determination of an analyte in a sample. | |
| 2.1.2.2. Specificity | |
| A method shall be able to distinguish between the analyte and the other substances under the experimental conditions. An estimate to which extent this is possible has to be provided. Strategies to overcome any foreseeable interference with substances when the described measuring technique is used, e.g. homologues, analogues, metabolic products of the residue of interest have to be employed. It is of prime importance that interference, which might arise from matrix components, is investigated. | |
| 2.2. SCREENING METHODS | |
| Only those analytical techniques, for which it can be demonstrated in a documented traceable manner that they are validated and have a false compliant rate of < 5 % (â-error) at the level of interest shall be used for screening purposes in conformity with Directive 96/23/EC. In the case of a suspected non-compliant result, this result shall be confirmed by a confirmatory method. | |
| 2.3. CONFIRMATORY METHODS FOR ORGANIC RESIDUES AND CONTAMINANTS | |
| Confirmatory methods for organic residues or contaminants shall provide information on the chemical structure of the analyte. Consequently methods based only on chromatographic analysis without the use of spectrometric detection are not suitable on their own for use as confirmatory methods. However, if a single technique lacks sufficient specificity, the desired specificity shall be achieved by analytical procedures consisting of suitable combinations of clean-up, chromatographic separation(s) and spectrometric detection. | |
| The following methods or method combinations are considered suitable for the identification of organic residues or contaminants for the substance groups indicated: | |
| Table 1 | |
| Suitable confirmatory methods for organic residues or contaminants | |
| >TABLE> | |
| 2.3.1. Common performance criteria and requirements | |
| Confirmatory methods shall provide information on the chemical structure of the analyte. When more than one compound gives the same response, then the method cannot discriminate between these compounds. Methods based only on chromatographic analysis without the use of spectrometric detection are not suitable on their own for use as confirmatory methods. | |
| Where used in the method, a suitable internal standard shall be added to the test portion at the beginning of the extraction procedure. Depending on availability, either stable isotope-labelled forms of the analyte, which are particularly suited for mass-spectrometric detection, or compounds that are structurally related to the analyte shall be used. | |
| When no suitable internal standard can be used, the identification of the analyte shall be confirmed by co-chromatography. In this case only one peak shall be obtained, the enhanced peak height (or area) being equivalent to the amount of added analyte. With gas chromatography (GC) or liquid chromatography (LC), the peak width at half-maximum height shall be within the 90-110 % range of the original width, and the retention times shall be identical within a margin of 5 %. For thin layer chromatography (TLC) methods, only the spot presumed to be due to the analyte shall be intensified; a new spot shall not appear and the visual appearance shall not change. | |
| Reference or fortified material containing known amounts of analyte, at or near either the permitted limit or the decision limit (non-compliant control sample) as well as compliant control materials and reagent blanks should preferably be carried through the entire procedure simultaneously with each batch of test samples analysed. The order for injecting the extracts into the analytical instrument is as follows: reagent blank, compliant control sample, sample(s) to be confirmed, compliant control sample again and finally non-compliant control sample. Any variation from this sequence shall be justified. | |
| 2.3.2. Additional performance criteria and other requirements for quantitative methods of analysis | |
| 2.3.2.1. Trueness of quantitative methods | |
| In the case of repeated analyses of a certified reference material, the guideline ranges for the deviation of the experimentally determined recovery corrected mean mass fraction from the certified value are as follows: | |
| Table 2 | |
| Minimum trueness of quantitative methods | |
| >TABLE> | |
| When no such CRMs are available, it is acceptable that trueness of measurements is assessed through recovery of additions of known amounts of the analyte(s) to a blank matrix. Data corrected with the mean recovery are only acceptable when they fall within the ranges shown in Table 2. | |
| 2.3.2.2. Precision of quantitative methods | |
| The inter-laboratory coefficient of variation (CV) for the repeated analysis of a reference or fortified material, under reproducibility conditions, shall not exceed the level calculated by the Horwitz Equation. The equation is: | |
| >REFERENCE TO A GRAPHIC> | |
| where C is the mass fraction expressed as a power (exponent) of 10 (e.g. 1 mg/g = 10-3). Examples are shown in the table 3. | |
| Table 3 | |
| Examples for reproducibility CVs for quantitative methods at a range of analyte mass fractions | |
| >TABLE> | |
| For analyses carried out under repeatability conditions, the intra-laboratory CV would typically be between one half and two thirds of the above values. For analyses carried out under within-laboratory reproducibility conditions, the within-laboratory CV shall not be greater than the reproducibility CV. | |
| In the case of substances with an established permitted limit, the method shall achieve within-laboratory reproducibility not greater than the corresponding reproducibility CV at a concentration of 0,5 × the permitted limit. | |
| 2.3.3. Performance criteria and other requirements for mass spectrometric detection | |
| Mass spectrometric methods are suitable for consideration as confirmatory methods only following either an on-line or an off-line chromatographic separation. | |
| 2.3.3.1. Chromatographic separation | |
| For GC-MS procedures, the gas chromatographic separation shall be carried out using capillary columns. For LC-MS procedures, the chromatographic separation shall be carried out using suitable LC columns. In any case, the minimum acceptable retention time for the analyte under examination is twice the retention time corresponding to the void volume of the column. The retention time (or relative retention time) of the analyte in the test portion shall match that of the calibration standard within a specified retention time window. The retention time window shall be commensurate with the resolving power of the chromatographic system. The ratio of the chromatographic retention time of the analyte to that of the internal standard, i.e. the relative retention time of the analyte, shall correspond to that of the calibration solution at a tolerance of ± 0,5 % for GC and ± 2,5 % for LC. | |
| 2.3.3.2. Mass spectrometric detection | |
| Mass-spectrometric detection shall be carried out by employing MS-techniques such as recording of full mass spectra (full scans) or selected ion monitoring (SIM), as well as MS-MSn techniques such as Selected Reaction Monitoring (SRM), or other suitable MS or MS-MSn techniques in combination with appropriate ionisation modes. In high-resolution mass spectrometry (HRMS), the resolution shall typically be greater than 10000 for the entire mass range at 10 % valley. | |
| Full scan: When mass spectrometric determination is performed by the recording of full scan spectra, the presence of all measured diagnostic ions (the molecular ion, characteristic adducts of the molecular ion, characteristic fragment ions and isotope ions) with a relative intensity of more than 10 % in the reference spectrum of the calibration standard is obligatory. | |
| SIM: When mass spectrometric determination is performed by fragmentography, the molecular ion shall preferably be one of the selected diagnostic ions (the molecular ion, characteristic adducts of the molecular ion, characteristic fragment ions and all their isotope ions). The selected diagnostic ions should not exclusively originate from the same part of the molecule. The signal-to-noise ratio for each diagnostic ion shall be >= 3:1. | |
| Full scan and SIM: The relative intensities of the detected ions, expressed as a percentage of the intensity of the most intense ion or transition, shall correspond to those of the calibration standard, either from calibration standard solutions or from spiked samples, at comparable concentrations, measured under the same conditions, within the following tolerances: | |
| Table 4 | |
| Maximum permitted tolerances for relative ion intensities using a range of mass spectrometric techniques | |
| >TABLE> | |
| Interpretation of mass spectral data: The relative intensities of the diagnostic ions and/or precursor/product ion pairs have to be identified by comparing spectra or by integrating the signals of the single mass traces. Whenever background correction is applied, this shall be applied uniformly throughout the batch (see 2.3.1, paragraph 4) and shall be clearly indicated. | |
| Full scan: When full scan spectra are recorded in single mass spectrometry, a minimum of four ions shall be present with a relative intensity of >= 10 % of the base peak. The molecular ion shall be included if it is present in the reference spectrum with a relative intensity of >= 10 %. At least four ions shall lie within the maximum permitted tolerances for relative ion intensities (Table 5). Computer-aided library searching may be used. In this case, the comparison of mass spectral data in the test samples to that of the calibration solution has to exceed a critical match factor. This factor shall be determined during the validation process for every analyte on the basis of spectra for which the criteria described below are fulfilled. Variability in the spectra caused by the sample matrix and the detector performance shall be checked. | |
| SIM: When mass fragments are measured using other than full-scan techniques, a system of identification points shall be used to interpret the data. For the confirmation of substances listed in Group A of Annex I of Directive 96/23/EC, a minimum of 4 identification points shall be required. For the confirmation of substances listed in Group B of Annex I of Directive 96/23/EC, a minimum of 3 identification points are required. The table below shows the number of identification points that each of the basic mass spectrometric techniques can earn. However, in order to qualify for the identification points required for confirmation and the sum of identification points to be calculated: | |
| (a) a minimum of at least one ion ratio shall be measured, and | |
| (b) all relevant measured ion ratios shall meet the criteria described above, and | |
| (c) a maximum of three separate techniques can be combined to achieve the minimum number of identification points. | |
| Table 5 | |
| The relationship between a range of classes of mass fragment and identification points earned | |
| >TABLE> | |
| Footnotes: | |
| (1) Each ion may only be counted once. | |
| (2) GC-MS using electron impact ionisation is regarded as being a different technique to GC-MS using chemical ionisation. | |
| (3) Different analytes can be used to increase the number of identification points only if the derivatives employ different reaction chemistries. | |
| (4) For substances in Group A of Annex 1 to Directive 96/23/EC, if one of the following techniques are used in the analytical procedure: HPLC coupled with full-scan diode array spectrophotometry (DAD); HPLC coupled with fluorescence detection; HPLC coupled to an immunogram; two-dimensional TLC coupled to spectrometric detection; a maximum of one identification point may be contributed, providing that the relevant criteria for these techniques are fulfilled. | |
| (5) Transition products include both daughter and granddaughter products. | |
| Table 6 | |
| Examples of the number of identification points earned for a range of techniques and combinations thereof (n = an integer) | |
| >TABLE> | |
| 2.3.4. Performance criteria and other requirements for chromatography coupled to infrared detection | |
| Adequate peaks: Adequate peaks are absorption maxima in the infrared spectrum of a calibration standard fulfilling the following requirements. | |
| 2.3.4.1. Infra-red detection | |
| Absorption maximum: This shall be in the wavenumber range 4000-500 cm-1. | |
| Intensity of absorption: This shall not be less than either: | |
| (a) a specific molar absorbance of 40 with respect to peak base line; or | |
| (b) a relative absorbance of 12,5 % of the absorbance of the most intense peak in the region 4000-500 cm-1 | |
| when both are measured with respect to zero absorbance, and 5 % of the absorbance of the most intense peak in the region 4000-500 cm-1 when both are measured with respect to their peak base line. | |
| Note: | |
| Although adequate peaks according to (a) may be preferred from a theoretical point of view, those according to (b) are easier to determine in practice. | |
| The number of peaks in the infrared spectrum of the analyte whose frequencies correspond with an adequate peak in the spectrum of the calibration standard, within a margin of ± 1 cm-1 is determined. | |
| 2.3.4.2. Interpretation of infra-red spectral data | |
| Absorption shall be present in all regions of the analyte spectrum which correspond with an adequate peak in the reference spectrum of the calibration standard. A minimum of six adequate peaks is required in the infrared spectrum of the calibration standard. If there are less than six adequate peaks (7), the spectrum at issue cannot be used as a reference spectrum. The "score", i.e. the percentage of the adequate peaks found in the infrared spectrum of the analyte, shall be at least 50. Where there is no exact match for an adequate peak, the relevant region of the analyte spectrum shall be consistent with the presence of a matching peak. The procedure is only applicable to absorption peaks in the sample spectrum with an intensity of a least three times the peak to peak noise. | |
| 2.3.5. Performance criteria and other requirements for the determination of an analyte using LC with other detection techniques | |
| 2.3.5.1. Chromatographic separation | |
| An internal standard shall be used if a material suitable for this purpose is available. It shall preferably be a related standard with a retention time close to that of the analyte. The analyte shall elute at the retention time that is typical for the corresponding calibration standard under the same experimental conditions. The minimum acceptable retention time for an analyte shall be two times the retention time corresponding to the void volume of the column. The ratio of the retention time of the analyte to that of the internal standard, i.e. the relative retention time of the analyte, shall be the same as that of the calibration standard in the appropriate matrix, within a margin of ± 2,5 %. | |
| 2.3.5.2. Full-scan UV/VIS detection | |
| The performance criteria for LC methods have to be fulfilled. | |
| The absorption maxima in the spectrum of the analyte shall be at the same wavelengths as those of the calibration standard within a margin determined by the resolution of the detection system. For diode array detection, this is typically within ± 2 nm. The spectrum of the analyte above 220 nm shall, for those parts of the two spectra with a relative absorbance >= 10 %, not be visibly different from the spectrum of the calibration standard. This criterion is met when firstly the same maxima are present and secondly when the difference between the two spectra is at no point observed greater than 10 % of the absorbance of the calibration standard. In the case computer-aided library searching and matching are used, the comparison of the spectral data in the test samples to that of the calibration solution has to exceed a critical match factor. This factor shall be determined during the validation process for every analyte on the basis of spectra for which the criteria described above are fulfilled. Variability in the spectra caused by the sample matrix and the detector performance shall be checked. | |
| 2.3.5.3. Performance criteria for fluorimetric detection | |
| The performance criteria for LC methods have to be fulfilled. | |
| This applies to molecules that exhibit native fluorescence and to molecules that exhibit fluorescence after either transformation or derivatisation. The selection of the excitation and emission wavelengths in combination with the chromatographic conditions shall be done in such a way to minimise the occurrence of interfering components in blank sample extracts. | |
| The nearest peak maximum in the chromatogram shall be separated from the designated analyte peak by at least one full peak width at 10 % of the maximum height of the analyte peak. | |
| 2.3.5.4. Performance criteria for the determination of an analyte by an LC-immunogram | |
| A LC immunogram is not suitable on its own for use as a confirmatory method. | |
| Relevant criteria for LC methods have to be fulfilled. | |
| The pre-defined quality control parameters, e.g. non-specific binding, the relative binding of the control samples, the absorbance value of the blank have to be within the limits obtained during validation of the assay. | |
| The immunogram has to be constructed of at least five fractions. | |
| Each fraction shall be less than half of the peak width. | |
| The fraction with the maximum content of the analyte has to be the same for the suspect sample, the non-compliant control sample and the standard. | |
| 2.3.5.5. Determination of an analyte using LC with UV/VIS detection (single wavelength) | |
| LC with UV/VIS detection (single wavelength) is not suitable on its own for use as a confirmatory method. | |
| The nearest peak maximum in the chromatogram shall be separated from the designated analyte peak by at least one full peak width at 10 % of the maximum height of the analyte peak. | |
| 2.3.6. Performance criteria and other requirements for the determination of an analyte by 2-D TLC coupled to full-scan UV/VIS spectrometric detection | |
| Two-dimensional HPTLC and co-chromatography are mandatory. | |
| The RF values of the analyte shall agree with the RF values of the standards within ±5 %. | |
| The visual appearance of the analyte shall be indistinguishable from that of the standard. | |
| For spots of the same colour the centre of the nearest spot should be shall separated from the centre of the spot of the analyte by at least half the sum of the spot diameters. | |
| The spectrum of the analyte shall not be visually different from the spectrum of the standard, as described for full-scan UV/VIS detection. | |
| In the case computer-aided library searching and matching are used, the comparison of the spectral data in the test samples to that of the calibration solution has to exceed a critical match factor. This factor shall be determined during the validation process for every analyte on the basis of spectra for which the criteria described above are fulfilled. Variability in the spectra caused by the sample matrix and the detector performance shall be checked. | |
| 2.3.7. Performance criteria and requirements for the determination of an analyte by GC in combination with electron capture detection (ECD) | |
| An internal standard shall be used if a material suitable for this purpose is available. It shall preferably be a related substance with a retention time close to that of the analyte. The analyte shall elute at a retention time which is typical for the corresponding calibration standard under the same experimental conditions. The minimum acceptable retention time for an analyte shall be two times the retention time corresponding to the void volume of the column. The ratio of the retention time of the analyte to that of the internal standard, i.e. the relative retention time of the analyte, shall be the same as that of the calibration standard in the appropriate matrix, within a margin of ± 0,5 %. The nearest peak maximum in the chromatogram shall be separated from the designated analyte peak by at least one full peak width at 10 % of the maximum height of the analyte peak. For additional information, co-chromatography may be used. | |
| 2.4. CONFIRMATORY METHODS FOR ELEMENTS | |
| Confirmatory analyses for chemical elements shall be based on the concept of unequivocal identification and accurate as well as precise quantification by means of physical-chemical properties unique to the chemical element at hand (e.g. element characteristic wavelength of emitted or absorbed radiation, atomic mass) at the level of interest. | |
| The following methods or combinations of methods are considered suitable for the identification of chemical elements: | |
| Table 7 | |
| Suitable confirmatory methods for chemical elements | |
| >TABLE> | |
| 2.4.1. Common performance criteria and other requirements for confirmatory methods | |
| Reference or fortified material containing known amounts of analyte, at or near either the maximum permitted limit or the decision limit (non-compliant control sample) as well as compliant control materials and reagent blanks should preferably be carried through the entire procedure simultaneously with each batch of test samples analysed. The recommended order for injecting the extracts into the analytical instrument is as follows: reagent blank, compliant control sample, sample to be confirmed, compliant control sample and finally non-compliant control sample. Any variation from this shall be justified. | |
| In general, most analytical techniques require complete digestion of the organic matrix to obtain solutions prior to determination of the analyte. This can be achieved by using microwave mineralisation procedures, which minimise the risk of loss and/or contamination of the analytes of interest. Decontaminated Teflon vessels of good quality shall be used. If other wet or dry digestion methods are resorted to, documented evidence shall be available to exclude potential loss or contamination phenomena. As an alternative to digestion, separation procedures (e.g. extraction) may under certain circumstances be chosen to separate analytes from matrix components and/or to concentrate analytes in order to introduce them into the analytical equipment. | |
| As regards calibration, be it external or based on the standard addition method, care shall be taken not to exceed the working range established for the analysis. In the case of external calibration, it is mandatory that calibration standards are prepared in a solution that matches as closely as possible the composition of the sample solution. Background correction shall be also applied if required by specific analytical circumstances. | |
| 2.4.2. Additional performance criteria and other requirements for quantitative methods of analysis | |
| 2.4.2.1. Trueness of quantitative methods | |
| In the case of repeated analyses of a certified reference material for elements, the deviation of the experimentally determined mean content from the certified value shall not lie outside the limit ± 10 %. When no such CRMs are available, it is acceptable that trueness of measurements is assessed through recovery of additions of known amounts of the element to the unknown samples. Attention is drawn to the fact that, unlike the analyte, the added element is not chemically bound in the real matrix and that therefore results obtained by this approach have lesser validity than those achieved through the use of CRMs. Recovery data are only acceptable when they are within ± 10 % of the target value. | |
| 2.4.2.2. Precision of quantitative methods | |
| In the case of repeated analysis of a sample carried out under within-laboratory reproducibility conditions, the intra-laboratory coefficient of variation (CV) of the mean shall not exceed the following values: | |
| Table 8 | |
| CVs for quantitative methods at a range of element mass fractions | |
| >TABLE> | |
| 2.4.3. Specific requirements for differential pulse anodic stripping voltametry (DPASV) | |
| Complete destruction of organic matter in samples prior to DPASV determinations is of the greatest importance. No broad signals due to the presence of organic materials shall be seen in the voltamograms. Inorganic matrix constituents may influence peak heights in DPASV. Therefore, quantification has to be done by the method of standard additions. Specimens of typical voltamograms of a sample solution shall be supplied with the method. | |
| 2.4.4. Specific requirements for atomic absorption spectrometry (AAS) | |
| This technique is basically mono-elemental and requires therefore optimisation of the experimental settings depending on the particular element to be quantified. Wherever possible, results shall be checked qualitatively and quantitatively by resorting to alternative absorption lines (ideally, two different lines shall be selected). Calibration standards shall be prepared in a solution matrix that matches as closely as possible that of the sample measurement solution (e.g. acid concentration or modifier composition). To minimise blank values, all reagents shall be of the highest available purity. Depending on the mode chosen to vaporise and/or atomise the sample, various types of AAS can be distinguished. | |
| 2.4.4.1. Specific requirements for flame AAS | |
| The instrument settings shall be optimised for each element. Especially the gas composition and flow rates have to be checked. A continuum source corrector shall be used to avoid interferences caused by background absorption. In the case of unknown matrices, a check shall be made as to whether or not background correction is required. | |
| 2.4.4.2. Specific requirements for graphite furnace AAS | |
| Contamination in the laboratory often affects accuracy when working at ultra-trace levels in the graphite furnace. Therefore high purity reagents, deionised water and inert plastic ware for sample and standard handling should be used. The instrument settings for each element shall be optimised. Especially the pre-treatment- and atomisation-conditions (temperature, time) and the matrix modification have to be checked. | |
| Working under isothermal atomisation conditions (e.g. transverse 0heated graphite tube with integrated Lvov platform (8) will reduce the influence of the matrix concerning the atomisation of the analyte. In combination with matrix modification and Zeeman-background correction (9), quantification by means of a calibration curve based upon measuring of aqueous standard solutions will be allowed. | |
| 2.4.5. Specific requirements for hydride generation atomic absorption spectrometry | |
| Organic compounds containing elements such as arsenic, bismuth, germanium, lead, antimony, selenium, tin and tellurium can be very stable and require oxidative decomposition to obtain correct results for total element content. Therefore, microwave digestion or high-pressure ashing under strong oxidative conditions is recommended. The greatest care shall be devoted to the complete and reproducible conversion of the elements into their corresponding hydrides. | |
| The formation of arsenic hydride in hydrochloric acid solution with NaBH4 depends on the oxidation state of arsenic (As III: fast formation, As V: longer formation period). To avoid a loss of sensitivity for the determination of As V with flow injection technique, caused by the short reaction time in this system, As V has to be reduced to As III after the oxidative decomposition. Potassium iodide/ascorbic acid or cysteine are suitable for this purpose. Blanks, calibration solutions and sample solutions shall be treated in the same way. Working with a batch system allows determining both arsenic species without affecting accuracy. Due to the delayed formation of As V-hydride, calibration shall be performed by peak area integration. The instrument settings shall be optimised. The gas flow, which transfers the hydride to the atomisator, is especially important and shall be checked. | |
| 2.4.6. Specific requirements for cold vapour atomic absorption spectrometry | |
| Cold vapour is used only in the case of mercury. Due to volatilisation and adsorption losses of elemental mercury, special care is necessary during the whole analysis. Contamination by reagents or the environment has to be avoided carefully. | |
| Organic compounds containing mercury require oxidative decomposition to obtain correct results for total mercury content. For decomposition, sealed systems with microwave digestion or high pressure asher are to be used. Special care is required for cleaning the equipment that had contact with mercury. | |
| Working with the flow injection technique is advantageous. For lower decision limits, adsorption of elemental mercury on gold/platinum adsorber followed by thermal desorption is recommended. Contact of the adsorber or the cell with moisture will disturb the measurement and shall be avoided. | |
| 2.4.7. Specific requirements for inductively coupled plasma atomic emission spectrometry (ICP-AES) | |
| Inductively coupled plasma atomic emission spectrometry (10) is a multi-element method, which allows a simultaneous measurement of various elements. To use the ICP-AES, the samples first have to be digested to decompose organic matrices. Sealed systems with microwave digestion or high pressure ashing shall be used. For a meaningful ICP-AES analysis, the instrument calibration and element or wavelength selection play an essential role. For instrument calibration, in case of linear calibration curves, it is usually necessary to measure calibration solutions of only four concentrations, because ICP-AES calibration curves are generally linear over four to six orders of magnitude of concentration. Calibration of the ICP-AES system should normally be performed with a multi-element standard, which shall be prepared in a solution that contains the same acid concentration as the measurement solution. For the linear curve, the element concentrations shall be checked. | |
| The selection of wavelengths for measurement of the emission from the analytes is appropriate for the concentrations of the elements to be determined. When the analyte concentration falls outside of the working range of an emission line, a different emission line shall be used. At first, the most sensitive emission line (not interfered) shall be chosen, then a less sensitive line. When working at or near the detection limit, the most sensitive line for the respective analyte is usually the best choice. Spectral and background interferences are causing the major difficulties in ICP-AES. Possible interferences are e.g. simple background shift, sloping background shift, direct spectral overlap and complex background shift. Each of these interferences has its own causes and remedies. Depending on the matrices, interference corrections and optimisation of operating parameters shall be applied. Some interferences can be avoided by dilution or by adaptation of the matrices. With each batch of test samples analysed, reference and fortified material containing known amounts of the analyte(s) as well as blank material shall be treated in the same way as the test samples. For testing for a drift, the standard shall be checked e.g. after 10 samples. All reagents and the plasma gas shall be of the highest available purity. | |
| 2.4.8. Specific requirements for inductively coupled mass spectrometry (ICP-MS)(11)) | |
| The determination of trace elements of average atomic mass, such as chromium, copper and nickel may be subject to strong interference from other isobaric and polyatomic ions. This can be circumvented only when a resolution power of at least 7000-8000 is available. Difficulties associated with the MS techniques include instrumental drift, matrix effects and molecular ion interference (m/z < 80). Multiple internal standardisation covering the same mass range as the elements to be determined is required to correct instrumental drift and matrix effects. | |
| Complete decomposition of organic matter in samples prior to ICP-MS measurements is required. As in the AAS, after digestion in sealed vessels, volatile elements e.g. iodine are to be transferred to a stable oxidation state. Most severe interference results from molecular ion combinations of argon (plasma gas), hydrogen, carbon, nitrogen and oxygen (dissolution acids, plasma gas impurities and entrained atmospheric gases) and the sample matrix. Complete digestion, background measurements, appropriate choice of analytical masses sometimes associated with a lower abundance (poorer detection limit) and of dissolution acids, e.g. nitric acid, are required to avoid interferences. | |
| For the elements to be determined, interferences are to be excluded by the appropriate choice of specific analytical masses including confirmation of isotope ratios. Instrument response considering Fano-factors shall be checked for each measurement by the use of internal standards. | |
| 3. VALIDATION | |
| Validation shall demonstrate that the analytical method complies with the criteria applicable for the relevant performance characteristics. | |
| Different control purposes require different categories of methods. The following table determines which performance characteristic shall be verified for which type of method. | |
| Table 9 | |
| Classification of analytical methods by the performance characteristics that have to be determined | |
| >TABLE> | |
| S = screening methods; C = confirmatory methods; + = determination is mandatory. | |
| 3.1. VALIDATION PROCEDURES | |
| This chapter provides examples and/or references for validation procedures of analytical methods. Other approaches to demonstrate that the analytical method complies with performance criteria for the performance characteristics may be used, provided that they achieve the same level and quality of information. | |
| Validation can also be performed by conducting an interlaboratory study such as established by Codex Alimentarius, ISO or the IUPAC (12), or according to alternative methods such as single laboratory studies or in-house validation (13)(14). This part focuses on single laboratory studies (on in-house validation) using a modular approach. This approach consists of: | |
| 1. a set of common performance characteristics independent of the validation model used and | |
| 2. more specific model-dependent procedures as described in Table 10. | |
| Table 10 | |
| Model-independent and model-dependent performance parameters | |
| >TABLE> | |
| 3.1.1. Model-independent performance characteristics | |
| Irrespective of the validation approach chosen, the following performance characteristics have to be determined. To minimise the workload, a carefully designed and statistically sound approach can be used to combine experiments performed to determine different parameters. | |
| 3.1.1.1. Specificity | |
| For analytical methods, the power of discrimination between the analyte and closely related substances (isomers, metabolites, degradation products, endogenous substances, matrix constituents, etc) is important. Two approaches are necessary to check for interferences. | |
| Therefore, potentially interfering substances shall be chosen and relevant blank samples shall be analysed to detect the presence of possible interferences and to estimate the effect of the interferences: | |
| - select a range of chemically related compounds (metabolites, derivatives, etc.) or other substances likely to be encountered with the compound of interest that may be present in the samples; | |
| - analyse an appropriate number of representative blank samples (n >= 20) and check for any interferences (signals, peaks, ion traces) in the region of interest where the target analyte is expected to elute; | |
| - additionally, representative blank samples shall be fortified at a relevant concentration with substances that are likely to interfere with the identification and/or quantification of the analyte; | |
| - after analysis, investigate whether: | |
| - the presence may lead to a false identification, | |
| - the identification of the target analyte is hindered by the presence of one or more of the interferences, or | |
| - the quantification is influenced notably. | |
| 3.1.1.2. Trueness | |
| In this paragraph, the determination of trueness (one component of accuracy) is described. Trueness can only be established by means of certified reference material (CRM). A CRM be used whenever available. The procedure is described in detail in ISO 5725-4 (5). An example is given below: | |
| - analyse six replicates of the CRM in accordance with the test instructions for the method, | |
| - determine the concentration of the analyte present in each sample of the replicates, | |
| - calculate the mean, the standard deviation and the coefficient of variation (%) for these concentrations, | |
| - calculate the trueness by dividing the detected mean concentration by the certified value (measured as concentration) and multiply by 100, to express the result as a percentage. | |
| Trueness ( %) = mean recovery-corrected concentration detected × 100/certified value. | |
| If no CRM is available, instead of trueness, the recovery can be determined as described under 4.1.2.1 below. | |
| 3.1.1.3. Applicability/ruggedness (minor changes) | |
| Such studies use the deliberate introduction of minor reasonable variations by the laboratory and the observation of their consequences. | |
| The pre-investigative studies have to be carried out by selecting factors of the sample pre-treatment, clean up and analysis, which may influence the measurement results. Such factors may include the analyst, the source and the age of reagents, solvents, standards and sample extracts, the rate of heating, the temperature, the pH-value as well as many other factors that may occur in the laboratory. These factors should be modified in an order of magnitude that matches the deviations usually encountered among laboratories. | |
| - Identify possible factors that could influence the results. | |
| - Vary each factor slightly. | |
| - Conduct a ruggedness test using the approach of Youden (15)(16). (Other approved methods may be used at this point. The Youden approach, however, keeps the required time and effort to a minimum). The Youden approach is a fractional factorial design. Interactions between the different factors cannot be detected. | |
| - Where a factor is found to influence the measurement results significantly, conduct further experiments to decide on the acceptability limits of this factor. | |
| - Factors that significantly influence the results should be identified clearly in the method protocol. | |
| The basic idea is not to study one alteration at a time but to introduce several variations at once. As an example, let A, B, C, D, E, F, G denote the nominal values for seven different factors that could influence the results, if their nominal values are changed slightly. Let their alternative values be denoted by the corresponding lower case letters a, b, c, d, e, f and g. This results in 27 or 128 different possible combinations. | |
| It is possible to choose a subset of eight of these combinations that have a balance between capital and small letters (Table 11). Eight determinations have to be made, which will use a combination of the chosen factors (A-G). The results of the determinations are shown in Table 11 below as S-Z. | |
| Table 11 | |
| Experiment design for ruggedness studies (minor changes) | |
| >TABLE> | |
| For calculations see examples for ruggedness testing in 3.3. | |
| 3.1.1.4. Stability | |
| It has been observed that insufficient stability of the analyte or matrix constituents in the sample during storage or analysis may give rise to significant deviations in the outcome of the result of analysis. Furthermore, the stability of the calibration standard in solution should be checked. Usually analyte stability is well characterised under various storage conditions. Monitoring of the storage condition will form part of the normal laboratory accreditation system. When this is not known, examples are given below on how the stability can be determined. | |
| Stability of the analyte in solution: | |
| - Prepare fresh stock solutions of the analyte(s) and dilute as specified in the test instructions to yield sufficient aliquots (e.g. 40) of each selected concentration (around the minimum required performance limit for substances for which no permitted limit has been established or around the permitted limit for other substances. Prepare both solutions of the analyte used for fortification and used in the final analysis solution, and any other solution that is of interest (e.g. derivatised standards). | |
| - Measure the analyte content in the freshly prepared solution according to the test instructions. | |
| - Dispense appropriate volumes into suitable containers, label and store according to the scheme: | |
| Table 12 | |
| Scheme for the determination of analyte stability in solution | |
| >TABLE> | |
| - The storing time could be selected as one, two, three and four weeks or longer if necessary, e.g. until the first degradation phenomena are observable during identification and/or quantification. The maximum storing time and the optimum storing conditions have to be recorded. | |
| - The calculation of the concentration of the analyte(s) in each aliquot should be carried out by using the solution of the analyte freshly prepared at the time of analysis as 100 %. | |
| >REFERENCE TO A GRAPHIC> | |
| Ci= concentration at time point | |
| Cfresh= concentration of fresh solution | |
| Stability of analyte(s) in matrix | |
| - Whenever possible, incurred samples should be used. When no incurred material is available, matrix fortified with the analyte should be used. | |
| - When incurred material is available, the concentration in the material should be determined while the material is still fresh. Further aliquots of material could be taken after one, two, four and 20 weeks and the concentrations should be determined. The tissue should be stored at least minus 20 °C or lower if required. | |
| - If no incurred material is available, take some blank material and homogenise it. Divide the material into five aliquots. Fortify each aliquot with the analyte, which should preferably be prepared in a small quantity of aqueous solution. Analyse one aliquot immediately. Store the remaining aliquots at least minus 20 °C or lower if required and analyse them after one, two, four and 20 weeks. | |
| 3.1.1.5. Calibration curves | |
| When calibration curves are used for quantification: | |
| - at least five levels (including zero) should be used in the construction of the curve, | |
| - the working range of the curve should be described, | |
| - the mathematical formula of the curve and the goodness-of-fit of the data to the curve should be described, | |
| - acceptability ranges for the parameters of the curve should be described. | |
| When serial calibration based on a standard solution is necessary, acceptable ranges shall be indicated for the parameters of the calibration curve, which may vary from series to series. | |
| 3.1.2. Conventional validation procedures | |
| The calculation of the parameters in accordance with conventional methods requires the performance of several individual experiments. Each performance characteristic has to be determined for each major change (see under applicability/ruggedness above). For multi-analyte methods, several analytes can be analysed simultaneously, as long as possibly relevant interferences are ruled out previously. Several performance characteristics can be determined in a similar way. So, to minimise workload, it is advised to combine experiments as much as possible (e.g., repeatability and within-laboratory reproducibility with specificity, analysis of blank samples to determine the decision limit and testing for specificity). | |
| 3.1.2.1. Recovery | |
| If there is no CRM available, the recovery has to be determined by experiments using fortified blank matrix using, for example, the following scheme: | |
| - select 18 aliquots of a blank material and fortify six aliquots at each of 1, 1,5 and 2 times the minimum required performance limit or 0,5, 1 and 1,5 times the permitted limit, | |
| - analyse the samples and calculate the concentration present in each sample, | |
| - using the equation below, calculate the recovery for each sample, | |
| - calculate the mean recovery and CV from the six results at each level, | |
| - % Recovery = 100 × measured content/fortification level. | |
| This conventional method for the determination of recovery is a variant of the standard addition method described in 3.5, when: | |
| - the sample is considered as a blank sample instead of a sample to be analysed, | |
| - it is considered that yield(1) and recovery(2) are similar for the two test portions, | |
| - the test samples have the same masses and the test portion extracts the same volumes, | |
| - the amount of the calibration standard that is added to the second (spiked) test portion is noted xADD. (xADD = ρA.VA), | |
| - x1 is the measured value for the blank and x2 the measured value for the second (spiked) test portion, | |
| - then, % Recovery = 100 (x2 - x1)/xADD. | |
| When any of the above conditions is (or is supposed) not to be achieved, then the complete procedure for determination of the recovery by mean of the standard addition method as described in 3.5 has to be performed. | |
| 3.1.2.2. Repeatability | |
| - Prepare a set of samples of identical matrices, fortified with the analyte to yield concentrations equivalent to 1, 1,5 and 2 times the minimum required performance limit or 0,5, 1 and 1,5 times the permitted limit. | |
| - At each level the analysis should be performed with at least six replicates. | |
| - Analyse the samples. | |
| - Calculate the concentration detected in each sample. | |
| - Find the mean concentration, standard deviation and the coefficient of variation (%) of the fortified samples. | |
| - Repeat these steps on at least two other occasions. | |
| - Calculate the overall mean concentrations and CVs for the fortified samples. | |
| 3.1.2.3. Within-laboratory reproducibility | |
| - Prepare a set of samples of specified test material (identical or different matrices), fortified with the analyte(s) to yield concentrations equivalent to 1, 1,5 and 2 times the minimum required performance limit or 0,5, 1 and 1,5 times the permitted limit. | |
| - At each level the analysis should be performed with at least six replicates. | |
| - Repeat these steps on at least two other occasions with different operators and different environmental conditions, e.g. different batches of reagents, solvents etc., different room temperatures, different instruments, etc. if possible. | |
| - Analyse the samples. | |
| - Calculate the concentration detected in each sample. | |
| - Find the mean concentration, standard deviation and the coefficient of variation ( %) of the fortified samples. | |
| 3.1.2.4. Reproducibility | |
| When reproducibility has to be verified, laboratories should participate in collaborative studies according to ISO 5725-2 (5). | |
| 3.1.2.5. Decision Limit (CCα) | |
| The decision limit has to be established according to the requirements for identification or identification plus quantification as defined under "Performance criteria and other requirements for analytical methods" (part 2). | |
| In the case of substances for which no permitted limit has been established, CCα can be established: | |
| - either by the calibration curve procedure according to ISO 11843 (17) (here referred to as critical value of the net state variable). In this case blank material shall be used, which is fortified at and above the minimum required performance level in equidistant steps. Analyse the samples. After identification, plot the signal against the added concentration. The corresponding concentration at the y-intercept plus 2,33 times the standard deviation of the within-laboratory reproducibility of the intercept equals the decision limit. This is applicable to quantitative assays only (α = 1 %), | |
| - or by analysing at least 20 blank materials per matrix to be able to calculate the signal to noise ratio at the time window in which the analyte is expected. Three times the signal to noise ratio can be used as decision limit. This is applicable to quantitative and qualitative assays. | |
| In the case of substances an with established permitted limit, CCα can be established: | |
| - either by the calibration curve procedure according to ISO 11843 (17) (here referred to as critical value of the net state variable). In this case blank material shall be used, which is fortified around the permitted limit in equidistant steps. Analyse the samples. After identification, plot the signal against the added concentration. The corresponding concentration at the permitted limit plus 1,64 times the standard deviation of the within-laboratory reproducibility equals the decision limit (α = 5 %), | |
| - or by analysing at least 20 blank materials per matrix fortified with the analyte(s) at the permitted limit. The concentration at the permitted limit plus 1,64 times the corresponding standard deviation equal the decision limit (α = 5 %). | |
| See also Article 5 and point 3.2. | |
| 3.1.2.6. Detection capability (CCβ) | |
| The detection capability should be determined according to the requirements for screening, identification or identification plus quantification as defined (see part 2). | |
| In the case of substances for which no permitted limit has been established, CCβ can be established by: | |
| - the calibration curve procedure according to ISO 11843 (17) (here referred to as minimum detectable value of the net state variable). In this case representative blank material shall be used, which is fortified at and below the minimum required performance level in equidistant steps. Analyse the samples. After identification, plot the signal against the added concentration. The corresponding concentration at the decision limit plus 1,64 times the standard deviation of the within-laboratory reproducibility of the mean measured content at the decision limit equals the detection capability (β = 5 %), | |
| - analysing at least 20 blank materials per matrix fortified with the analyte(s) at the decision limit. Analyse the samples and identify the analytes. The value of the decision limit plus 1,64 times the standard deviation of the within-laboratory reproducibility of the measured content equals the detection capability (β = 5 %), | |
| - where no quantitative results are available, the detection capability can be determined by the investigation of fortified blank material at and above the decision limit. In this case the concentration level, where only <= 5 % false compliant results remain, equals the detection capability of the method. Therefore, at least 20 investigations for at least one concentration level have to be carried out in order to ensure a reliable basis for this determination. | |
| In the case of substances for which a permitted limit has been established, CCâ can be established: | |
| - either by the calibration curve procedure according to ISO 11843 (17) (here referred to as minimum detectable value of the net state variable). In this case representative blank material shall be used, which is fortified around the permitted limit in equidistant steps. Analyse the samples and identify the analyte(s). Calculate the standard deviation of the mean measured content at the decision limit. The corresponding concentration at the value of the decision limit plus 1,64 times the standard deviation of the within-laboratory reproducibility equals the detection capability (â = 5 %), | |
| - or by analysing at least 20 blank materials per matrix fortified with the analyte(s) at the decision limit. The value of the decision limit plus 1,64 times the corresponding standard deviation equals the detection capability (â = 5 %). | |
| See also section 3.2. | |
| 3.1.2.7. Ruggedness (major changes) | |
| The analytical method should be tested under different experimental conditions, which include for example, different species, different matrices or different sampling conditions. The changes introduced should be major. The importance of these changes can be evaluated, for instance, using the Youden approach (15)(16). Each performance characteristic should be determined for all major changes that have been shown to have a significant effect on the performance of the assay. | |
| 3.1.3. Validation according to alternative models | |
| When alternative validation procedures are applied, the underlying model and strategy with the respective prerequisites, assumptions and formulae shall be laid down in the validation protocol or at least references shall be given to their availability. In the following an example for an alternative approach is given. When applying e.g. the in-house validation model, the performance characteristics are determined in a manner that permits validation for major changes within the same validation procedure. This requires design of an experimental plan for validation. | |
| 3.1.3.1. Experimental plan | |
| An experimental plan has to be designed depending on the number of different species and different factors under investigation. Hence, the first step of the entire validation procedure shall consider the sample populations that will be analysed in the laboratory in the future in order to select the most important species and those factors which may influence the measurement results. Subsequently, the concentration range shall be chosen in a purpose-adapted way according to the level of interest. | |
| Example: | |
| - several analytes can be investigated simultaneously with the analytical method being validated, | |
| - two variations of the leading factor have been identified (A and B). Leading factors form the basis on which the factor levels are combined. These leading factors may include factors such as species or matrix. In this example the leading factor was varied on two levels, i.e. two different species (species A and B) were considered. In general, it is possible to vary the leading factors on more than two levels, which only increase the number of analyses to be performed, | |
| - the selected factors are to be varied on two levels (indicated as either + or -). | |
| Table 13 | |
| Examples for factors considered important for a validation procedure | |
| >TABLE> | |
| Table 14 | |
| Possible experimental plan for the above example | |
| >TABLE> | |
| As each sample (each factor level combination) has to be spiked with four different concentrations around the level of interest, and one blank sample shall be analysed for each level, 5 x 16 = 80 analyses have to be performed for the entire validation experiment. | |
| From these 80 measurement results it is possible to compute (13)(14). | |
| Recovery | |
| - repeatability per concentration level (sir), | |
| - within-laboratory reproducibility per concentration level (sir), | |
| - decision limit (CCα), | |
| - detection capability (CCβ), | |
| - power curve (β-error rate versus concentration (see 3.1.3.2), | |
| - ruggedness of major changes; ruggedness to minor changes can be determined according to paragraph 3.1.1.3, | |
| - 16 sample-related calibration curves, | |
| - one overall calibration curve, | |
| - prediction interval of the overall calibration curve, | |
| - matrix-induced deviations (smat), | |
| - run-induced deviations (srun), | |
| - effect of the individual factors on the measurement results. | |
| These performance characteristics allow the comprehensive evaluation of the performance level of the method, since not only the influence of the individual factors is investigated, but also the relevant combinations of these factors. With the help of this experiment design it is possible to decide if one or the other of the selected factors shall be excluded from the overall calibration curve, because it significantly deviates from the standard deviations of the other factors. | |
| 3.1.3.2. Power curve | |
| The power curve provides information about the detection capability of the method within the chosen concentration range. It refers to the β-error risk when applying the investigated method. The power curve allows to calculate the detection capabilities for the respective categories (screening, confirmation) or types (qualitative or quantitative) of methods for a certain β-error (e.g. 5 %). | |
| Figure 1 | |
| Power curve | |
| >PIC FILE= "L_2002221EN.003101.TIF"> | |
| Figure 1 shows an example of the graphical establishment of detection capability (CCβ) of an analytical method. This particular method has a remaining risk of taking a false decision of 5 % at a concentration of 0,50 μg/kg. At a concentration of 0,55 μg/kg the risk of taking a false compliant decision decreases to 1 %. | |
| 3.1.3.3. Reproducibility | |
| The determination of a method's reproducibility by the single laboratory studies (in-house validation) concept requires repeated participation in proficiency studies in accordance with ISO guide 43-1 (3) and 43-2 (4). The laboratories are allowed to choose their own methods, provided these methods are used under routine conditions. The standard deviation of the laboratory can be used to assess the reproducibility of the method. | |
| 3.2. GRAPHICAL REPRESENTATION OF THE DIFFERENT ANALYTICAL LIMITS | |
| Figure 2 | |
| Substances for which no permitted limit has been established | |
| >PIC FILE= "L_2002221EN.003201.TIF"> | |
| Figure 3 | |
| Substances with an established permitted limit | |
| >PIC FILE= "L_2002221EN.003301.TIF"> | |
| 3.3. CALCULATION EXAMPLE FOR RUGGEDNESS TESTING OF MINOR CHANGES ACCORDING TO THE YOUDEN APPROACH (16) | |
| Comparison of averages (A) | |
| >TABLE> | |
| >TABLE> | |
| Standard deviation of the differences Di (SDi): | |
| >PIC FILE= "L_2002221EN.003401.TIF"> | |
| When SDi is significantly larger than the standard deviation of the method carried out under within-laboratory reproducibility conditions according (see above) it is a foregone conclusion that all factors together have an effect on the result even if every single factor does not show a significant influence and that the method is not sufficiently robust against the chosen modifications. | |
| 3.4. CALCULATION EXAMPLES FOR THE IN-HOUSE VALIDATION PROCEDURE | |
| Examples and calculations for the in-house validation protocol as described under validation according to alternative models (3.1.3) (13) (14). | |
| 3.5. EXAMPLES FOR THE STANDARD ADDITION METHOD | |
| A test sample with a content T of the analyte is divided in two test portions 1 and 2 of respective masses m1 and m2. The test portion 2 is spiked with a volume VA of a solution of concentration ρA of the analyte. Two extracts of the test portions of respective volumes V1 and V2 are obtained after extraction and purification steps of the method. The recovery of the analyte is supposed to be rc. Both extracts are assayed with a measurement method of sensitivity b and give an analytical response of x1 and x2 respectively. | |
| If assumed that rc and b are the same for the analyte in the native sample and in the spiked sample, then the content T can be calculated as: | |
| >REFERENCE TO A GRAPHIC> | |
| The method will allow the determination of the recovery rc. Then, in addition with the assay described above, part of the extract of the test portion 1 (volume V3) is spiked with a known amount ρB.VB of the analyte and assayed. The analytical response is x3 and the recovery is: | |
| >REFERENCE TO A GRAPHIC> | |
| Moreover, it is possible to calculate the sensitivity b, as: | |
| >REFERENCE TO A GRAPHIC> | |
| All conditions of application and details have been described (18). | |
| 4. ABBREVIATIONS USED | |
| AAS Atomic absorption spectrometry | |
| AES Atomic emission spectrometry | |
| AOAC-I Association of Official Analytical Chemists INTERNATIONAL | |
| B bound fraction (immunoassays) | |
| CI chemical ionisation | |
| CRM Certified reference material | |
| CV coefficient of variation | |
| 2 D two dimensional | |
| DAD diode array detection | |
| DPASV differential pulse anodic stripping voltametry | |
| ECD electron capture detection | |
| EI electronic impact ionisation | |
| GC gas chromatography | |
| HPLC high performance liquid chromatography | |
| HPTLC high performance thin layer chromatography | |
| HRMS high resolution (mass spectrometry) | |
| ICP-AES inductively coupled plasma-atomic emission spectrometry | |
| ICP-MS inductively coupled plasma-mass spectrometry | |
| IR infrared | |
| ISO International Standard Organisation | |
| LC liquid chromatography | |
| LR(MS) low resolution (mass spectrometry) | |
| MRPL Minimum required performance limit | |
| MS mass spectrometry | |
| m/z mass/charge ratio | |
| RF relative migration to the solvent front (TLC) | |
| RSDL relative standard deviations of the laboratory | |
| SIM selected ion monitoring | |
| TLC thin layer chromatography | |
| UV ultra violet light | |
| VIS visible light | |
| 5. REFERENCES | |
| (1) ISO 17025: 1999 General requirement for the competence of calibration and testing laboratories. | |
| (2) ISO 3534-1: 1993 Statistical Methods for quality control - Vol. 1 vocabulary and symbols. | |
| (3) ISO Guide 43-1: 1997 Proficiency testing by interlaboratory comparisons - Part 1: Development and operation of proficiency testing schemes. | |
| (4) ISO Guide 43-2: 1997 Proficiency testing by interlaboratory comparisons - Part 2: Selection and use of proficiency testing schemes by laboratory accreditation bodies. | |
| (5) ISO 5725: 1994 Accuracy (trueness and precision) of measurement methods and results - Part 1: General principles and definitions; ISO 5725-2 Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method; Part 4: Basic methods for the determination of the trueness of a standard measurement method. | |
| (6) ISO 78-2: 1999 Chemistry - Layouts for standards - Part 2: Methods of chemical analysis. | |
| (7) W.G de Ruig and J.M Weseman "A new approach to confirmation by infrared spectrometry" J. Chemometrics 4 (1990) 61-77. | |
| (8) See e.g. May, T.W., Brumbaugh, W.G., 1982, Matrix modifier and L'vov platform for elimination of matrix interferences in the analysis of fish tissues for lead by graphite furnace atomic absorption spectrometry: Analytical Chemistry 54(7): 1032-1037 (90353). | |
| (9) Applications of Zeeman Graphite Furnace Atomic Absorption Spectrometry in the Chemical Laboratory and in Toxicology, C. Minoia, S. Caroli (Eds.), Pergamon Press (Oxford), 1992, pp. xxvi + 675. | |
| (10) Inductively Coupled Plasmas in Analytical Atomic Spectrometry, A. Montaser, D. W. Golighty (Eds.), VCH Publishers, Inc. (New York), 1992. | |
| (11) Plasma Source Mass Spectrometry Developments and Applications, G. Holland, S. D. Tanner (Eds.), The Royal Society of Chemistry, 1997, p. 329. | |
| (12) IUPAC (1995), Protocol for the design, conduct and interpretation of method-performance studies, Pure & Applied Chem, 67, 331. | |
| (13) Jülicher, B., Gowik, P. and Uhlig, S. (1998) Assessment of detection methods in trace analysis by means of a statistically based in-house validation concept. Analyst, 120, 173. | |
| (14) Gowik, P., Jülicher, B. and Uhlig, S. (1998) Multi-residue method for non-steroidal anti-inflammatory drugs in plasma using high performance liquid chromatography-photodiode-array detection. Method description and comprehensive in-house validation. J. Chromatogr., 716, 221. | |
| (15) OAC-I Peer Verified Methods, Policies and Procedures, 1993, AOAC International, 2200 Wilson Blvd., Suite 400, Arlington, Virginia 22201-3301, USA. | |
| (16) W.J. Youden; Steiner, E.H.; "Statistical Manual of the AOAC-Association of Official Analytical Chemists", AOAC-I, Washington DC: 1975, p. 35 ff. | |
| (17) ISO 11843: 1997 Capability of detection - Part 1: Terms and definitions, Part 2: Methodology in the linear calibration case Part 2: Methodology in the linear calibration case. | |
| (18) R.W. Stephany & L.A. van Ginkel: "Yield or recovery: a world of difference". Proceedings Eight Euro Food Chem, Vienna, Austria September 18-20 (1995) Federation of European Chemical Societies, Event 206. ISBN 3-900554-17X, page 2 to 9. | |
| (19) Directive 71/354/EEC of 18 October 1971 on the approximation of the laws of the Member States relating to units of measurement, OJ L 243, 29.10.1971, p. 29). | |
| (20) ISO 31-0: 1992 Quantities and units - Part 0: General principles | |
| (1) Yield: that fraction of mass of the analyte contained in the sample, which is present in the final extract. | |
| (2) Recovery (here): that fraction of mass of the analyte added to the sample, which is present in the final extract. Throughout the rest of the document it is assumed that yield and recovery are equal and therefore only the term "recovery" is used. | |
| HU.ES poglavje 3 zvezek 36 str. 493 - 521 | |
| LT.ES poglavje 3 zvezek 36 str. 493 - 521 | |
| LV.ES poglavje 3 zvezek 36 str. 493 - 521 | |
| MT.ES poglavje 3 zvezek 36 str. 493 - 521 | |
| PL.ES poglavje 3 zvezek 36 str. 493 - 521 | |
| SK.ES poglavje 3 zvezek 36 str. 493 - 521 | |
| SL.ES poglavje 3 zvezek 36 str. 493 - 521 | |
| Odločba Komisije | |
| z dne 14. avgusta 2002 | |
| o izvajanju Direktive Sveta 96/23/ES glede opravljanja analitskih metod in razlage rezultatov | |
| (notificirano pod dokumentarno številko K(2002)3044) | |
| (Besedilo velja za EGP) | |
| (2002/657/ES) | |
| KOMISIJA EVROPSKIH SKUPNOSTI JE – | |
| ob upoštevanju Pogodbe o ustanovitvi Evropske skupnosti, | |
| ob upoštevanju Direktive Sveta 96/23/ES z dne 29. aprila 1996 o ukrepih za spremljanje določenih snovi in njihovih ostankov v živih živalih in živalskih proizvodih ter razveljavitvi direktiv 85/358/EGS in 86/469/EGS ter odločb 89/187/EGS in 91/664/EGS [1] in zlasti drugega pododstavka člena 15(1) Direktive, | |
| ob upoštevanju naslednjega: | |
| (1) Prisotnost ostankov v proizvodih živalskega izvora negativno vpliva na zdravje ljudi. | |
| (2) Odločba Komisije 98/179/ES z dne 23. februarja 1998 o določitvi podrobnih pravil uradnega vzorčenja za spremljanje nekaterih snovi in njihovih ostankov v živih živalih in živalskih proizvodih [2] določa, da morajo analizo vzorcev opravljati izključno laboratoriji, ki jih je pristojni nacionalni organ pooblastil za uradno nadzorovanje ostankov. | |
| (3) Treba je zagotoviti kakovost in primerljivost rezultatov analiz, ki jih pridobijo pooblaščeni laboratoriji za uraden nadzor ostankov. To je treba doseči z uporabo sistema zagotavljanja kakovosti in specifično z uporabo metod, validiranih v skladu z enotnimi postopki in merili učinkovitosti in z zagotavljanjem sledenja splošnim standardom ali skupno dogovorjenim standardom. | |
| (4) Direktiva Sveta 93/99/EGS z dne 29. oktobra 1993 o dodatnih ukrepih glede uradnega nadzora živil in Odločba 98/179/ES [3] zahtevata, da je treba akreditirati uradne nadzorne laboratorije v skladu z ISO 17025(1)od januarja 2002. V skladu z Odločbo 98/179/ES se za pooblaščene laboratorije zahteva udeležba v mednarodno priznanem zunanjem programu za ocenjevanje nadzora kakovosti in akreditacijo. Še več, pooblaščeni laboratoriji morajo dokazati njihovo pristojnost z redno in uspešno udeležbo v ustreznih programih preskusov strokovne usposobljenosti, ki jih priznavajo ali organizirajo nacionalni referenčni laboratoriji ali referenčni laboratoriji Skupnosti. | |
| (5) Za izboljšanje koordinacije deluje mreža referenčnih laboratorijev Skupnosti, nacionalnih referenčnih laboratorijev in nacionalnih nadzornih laboratorijev na podlagi Direktive 96/23/ES. | |
| (6) Zaradi napredovanja analitske kemije od sprejetja Direktive 96/23/ES se je koncept rutinskih metod in referenčnih metod nadomestil s pristopom po merilih, v katerem se določijo merila učinkovitosti in postopki za validacijo presejevalnih in potrditvenih metod. | |
| (7) Za zagotavljanje usklajenega izvajanja Direktive 96/23/ES je treba določiti skupna merila za razlago rezultatov preskusov uradnih nadzornih laboratorijev. | |
| (8) Za zagotavljanje usklajenega izvajanja Direktive 96/23/ES je treba določiti postopno uvajanje mej najmanjše zahtevane učinkovitosti (MRPL) analitske metode za snovi, za katere ni določena nobena dovoljena meja, in zlasti za snovi, katerih uporaba ni dovoljena ali je v Skupnosti izrecno prepovedana. | |
| (9) Zaradi upoštevanja razvoja v znanstvenem in tehnološkem znanju so se prej ponovno pregledale Odločba Komisije 90/515/EGS z dne 26. septembra 1990 o določitvi referenčnih metod za odkrivanje ostankov težkih kovin in arzena [4], Odločba Komisije 93/256/EGS z dne 14. maja 1993 o določitvi metod, ki se uporabljajo za odkrivanje ostankov snovi s hormonalnim ali tirostatičnim delovanjem [5] in Odločba Komisije 93/257/EGS z dne 15. aprila 1993 o določanju referenčnih metod in seznama referenčnih laboratorijev za odkrivanje ostankov [6], kakor je bila nazadnje spremenjena z Odločbo 98/536/ES [7], in ugotovljeno je bilo, da so zastarele glede njihovega področja in določb ter jih je torej treba ustrezno razveljaviti s to odločbo. | |
| (10) Zaradi omogočanja prilagajanja metod za analiziranje uradnih vzorcev določbam te odločbe je treba določiti prehodno obdobje. | |
| (11) Ukrepi, predvideni s to odločbo, so v skladu z mnenjem Stalnega odbora za prehranjevalno verigo in zdravje živali – | |
| SPREJELA NASLEDNJO ODLOČBO: | |
| Člen 1 | |
| Vsebina in področje uporabe | |
| Ta odločba določa pravila za analitske metode, ki jih je treba uporabljati pri preskušanju uradnih vzorcev, odvzetih v skladu z drugim stavkom člena 15(1) Direktive 96/23/ES, in podrobno določa skupna merila za razlago analitskih rezultatov uradnih nadzornih laboratorijev za takšne vzorce. | |
| Ta odločba se ne uporablja za snovi, za katere so določena podrobnejša pravila v drugi zakonodaji Skupnosti. | |
| Člen 2 | |
| Opredelitev pojmov | |
| Za namene te odločbe se uporabljajo opredelitve pojmov iz Direktive 96/23/ES in iz Priloge k tej odločbi. | |
| Člen 3 | |
| Analitske metode | |
| Države članice zagotovijo, da se uradni vzorci, odvzeti v skladu z Direktivo D 96/23/ES analizirajo z uporabo metod, ki: | |
| (a) so dokumentirane v preskusnih navodilih, po možnosti v skladu z ISO 78-2(6); | |
| (b) so skladne z delom 2 Priloge k tej odločbi; | |
| (c) so validirane v skladu s postopkom, opisanim v delu 3 Priloge; | |
| (d) upoštevajo ustrezne meje najmanjše zahtevane učinkovitosti (MRPL), ki jih je treba določiti v skladu s členom 4. | |
| Člen 4 | |
| Meje najmanjše zahtevane učinkovitosti | |
| Ta odločba se ponovno pregleda, da se postopoma določijo meje najmanjše zahtevane učinkovitosti analitskih metod, ki jih je treba uporabiti za snovi, za katere se ni določila dovoljena meja. | |
| Člen 5 | |
| Kontrola kakovosti | |
| Države članice zagotovijo kakovost rezultatov analize vzorcev, odvzetih v skladu z Direktivo 96/23/ES, zlasti z nadzorom nad preskusi in/ali rezultati umeritev v skladu s poglavjem 5.9 ISO 17025 (1). | |
| Člen 6 | |
| Razlaga rezultatov | |
| 1. Rezultat analize se šteje kot neskladen, če se za analit preseže odločitvena meja potrditvene metode. | |
| 2. Če se je za snov ugotovila dovoljena meja, je odločitvena meja tista koncentracija, nad katero se da s statistično gotovostjo 1 – a sklepati, da je dovoljena meja resnično presežena. | |
| 3. Če se za snov ni ugotovila dovoljena meja, je odločitvena meja najnižji nivo koncentracije, pri kateri se da z neko metodo razločiti prisotnost posameznega analita.s statistično gotovostjo 1 –a. | |
| 4. Za snovi na seznamu v Skupini A Priloge k Direktivi 96/23/ES, je a-napaka 1 % ali manj. Za vse druge snovi je a-napaka 5 % ali manj. | |
| Člen 7 | |
| Razveljavitev | |
| Odločbe 90/515/GS, 93/256/EGS in 93/257/ES se razveljavijo. | |
| Člen 8 | |
| Prehodne določbe | |
| Metode za analizo uradnih vzorcev snovi na seznamu v skupini A Priloge I k Direktivi 96/23/ES, ki izpolnjujejo merila, določena v odločbah 90/515/EGS, 93/256/EGS in 93/257/EGS, se lahko uporabljajo do dve leti po uveljavitvi te odločbe. Metode, ki se sedaj uporabljajo za snovi na seznamu v skupini B Priloge I k Direktivi 96/23/ES morajo biti skladne s to odločbo najmanj pet let po dnevu začetka uporabe te odločbe. | |
| Člen 9 | |
| Datum začetka uporabe | |
| Ta odločba se uporablja od 1. septembra 2002. | |
| Člen 10 | |
| Naslovniki | |
| Ta odločba je naslovljena na države članice. | |
| V Bruslju, 14. avgusta 2002 | |
| Za Komisijo | |
| David Byrne | |
| Član Komisije | |
| [1] UL L 125, 23.5.1996, str. 10. | |
| [2] UL L 65, 5.3.1998, str. 31. | |
| [3] UL L 290, 24.11.1993, str. 14. | |
| [4] UL L 286, 18.10.1990, str. 33. | |
| [5] UL L 118, 14.5.1993, str. 64. | |
| [6] UL L 118, 14.5.1993, str. 75. | |
| [7] UL L 251, 11.9.1998, str. 39. | |
| -------------------------------------------------- | |
| PRILOGA | |
| MERILA UČINKOVITOSTI, DRUGE ZAHTEVE IN POSTOPKI ZA ANALITSKE METODE | |
| 1. OPREDELITEV POJMOV | |
| 1.1 Točnost pomeni čim boljše ujemanje med rezultatom preskušanja in sprejeto referenčno vrednostjo (2). Določi se s določanjem pravilnosti in natančnosti. | |
| 1.2 Napaka alfa (a) pomeni verjetnost, da je preskusni vzorec skladen, čeprav je dobljena neustrezna meritev (lažna neskladna odločitev). | |
| 1.3 Analit pomeni snov, ki jo je treba odkriti, identificirati in/ali izmeriti in derivate, ki se pojavijo med njeno analizo. | |
| 1.4 Napaka beta (b) pomeni verjetnost, da je preskusni vzorec resnično neustrezen, čeprav je bila dobljena ustrezna meritev (lažna skladna odločitev). | |
| 1.5 Odstopanje (bias) pomeni razliko med pričakovanim rezultatom preskusa in sprejeto referenčno vrednostjo (2). | |
| 1.6 Umeritveni standard pomeni merilni pripomoček, ki predstavi količino iskane snovi na način, ki veže njeno vrednost na referenčno osnovo. | |
| 1.7 Certificirani referenčni material (CRM) pomeni material, ki je imel specificirano vsebnost analita, ki mu je bila pripisana. | |
| 1.8 Vzporedna kromatografija pomeni postopek, pri katerem se izvleček pred kromatografskimi koraki razdeli na 2 dela. Prvi del se kromatografira kot tak. Drugi del se zmeša s standardnim analitom, ki ga je treba izmeriti. Nato se tudi ta mešanica kromatografira. Količina standardnega analita mora biti podobna ocenjeni količini analita v izvlečku. Ta metoda je namenjena za izboljšanje identifikacije analita, kadar se uporabljajo kromatografske metode, zlasti, kadar se ne da uporabiti noben interni standard. | |
| 1.9 Sodelovalno proučevanje pomeni analiziranje istega vzorca po isti metodi, da se določijo značilnosti učinkovitosti metode. Študija obsega naključno napako merjenja in laboratorijskega odstopanja. | |
| 1.10 Potrditvena metoda pomeni metode, ki dajejo celovite ali dodatne informacije, ki omogočajo, da se snov nedvoumno identificira in po potrebi kvantificira glede na iskani nivo. | |
| 1.11 Odločitvena meja (CCa) pomeni mejo, pri kateri in nad katero se lahko sklepa z verjetnostjo a-napake, da je vzorec neskladen. | |
| 1.12 Sposobnost odkrivanja (CCb) pomeni najmanjšo vsebnost snovi, ki se lahko zazna, prepozna in/ali izmeri v vzorcu z verjetnostjo b-napake. V primeru snovi, kjer ni določena nobena dovoljena meja, je sposobnost zaznavanja najnižja koncentracija, pri kateri lahko metoda zazna resnično kontaminirane vzorce z statistično gotovostjo 1 – b. V primeru snovi z določeno dovoljeno mejo to pomeni, da je sposobnost zaznavanja tista koncentracija, pri kateri lahko metoda zazna dovoljeno mejno koncentracijo s statistično natančnostjo 1 – b. | |
| 1.13 Ojačan vzorčni material pomeni vzorec, ki je obogaten z znano količino analita, ki ga je treba odkriti. | |
| 1.14 Medlaboratorijska študija (primerjava) pomeni organizacijo, izvedbo in ovrednotenje preskusov istega vzorca v dveh ali več laboratorijih v skladu z naprej določenimi pogoji, da se določi učinkovitost preskušanja. V skladu z namenom se lahko študija uvrsti kot sodelovalna študija ali strokovna študija. | |
| 1.15 Interni standard (IS) pomeni snov, ki ni vsebovana v vzorcu s fizikalno-kemičnimi lastnostmi, čimbolj podobnim tistim od analita, ki ga je treba identificirati in ki je dodan vsakemu vzorcu kot tudi vsakemu umeritvenemu standardu. | |
| 1.16 Laboratorijski vzorec pomeni vzorec, ki je pripravljen za pošiljanje v laboratorij in je namenjen za preiskovanje ali preskušanje. | |
| 1.17 Iskani nivo pomeni koncentracijo snovi ali analita v vzorcu, ki je pomembna za določitev skladnosti z zakonodajo. | |
| 1.18 Meja najmanjše zahtevane učinkovitosti (MRPL) pomeni najmanjšo vsebnost analita v vzorcu, ki ga je treba najmanj zaznati in potrditi. Namenjena je za uskladitev analitskega izvajanja metod za snovi, za katere se ni določila nobena dovoljena meja. | |
| 1.19 Značilnost učinkovitosti pomeni funkcionalno kakovost, ki se lahko pripiše analitski metodi. To je lahko na primer specifičnost, točnost, pravilnost, natančnost, ponovljivost, obnovljivost, izplen, sposobnost zaznavanja in robustnosti. | |
| 1.20 Merila učinkovitosti pomenijo zahteve za značilnosti učinkovitosti, v skladu s katerimi se lahko prisodi, da je analitska metoda primerna za ta namen in da daje zanesljive rezultate. | |
| 1.21 Dovoljena meja pomeni mejo največjega ostanka, največji nivo ali drugo največje dovoljeno odstopanje za snovi, določene drugod v zakonodaji Skupnosti. | |
| 1.22 Natančnost pomeni čim boljše ujemanje med neodvisnimi rezultati preskusov, dobljenimi pod nekaterimi (vnaprej določenimi)pogoji. Mera natančnosti se navadno izrazi kot netočnost in se izračuna kot standardni odmik rezultata preskusa. Manjša natančnost je določena z večjim standardnim odmikom (2). | |
| 1.23 Študija strokovne usposobljenosti pomeni analiziranje istega vzorca pri kateri laboratoriji izberejo svoje lastne metode, pod pogojem da se te metode uporabljajo pod rutinskimi pogoji. Študija mora iti izvedena v skladu z ISO smernicami 43-1 (3) in 43-2 (4)in se lahko uporablja za oceno metod ponovljivosti. | |
| 1.24 Kvalitativna metoda pomeni analitsko metodo, ki identificira snov na osnovi njenih kemičnih, bioloških ali fizikalnih lastnosti. | |
| 1.25 Kvantitativna metoda pomeni analitsko metodo, ki določa količino ali masni delež snovi, tako da se lahko izrazi kot številčna vrednost primernih enot. | |
| 1.26 Določanje slepega reagenta pomeni izvedbo celotnega analitski postopka brez dodatka za preskus uporabljenega dela preiskovanega vzorca ali ki uporablja ekvivalentno količino primernega topila namesto za preskus uporabljenega dela preiskovanega vzorca. | |
| 1.27 Izplen pomeni odstotek dejanske koncentracije snovi, ki se pridobi nazaj med analitskim postopkom. Določi se med validiranjem, če ni na voljo certificiranega referenčnega materiala. | |
| 1.28 Referenčni material pomeni material, katerega ena ali več lastnosti se je potrdila z validirano metodo, tako da se lahko uporablja za umerjanje aparata ali za preverjanje merilne metode. | |
| 1.29 Ponovljivost pomeni natančnost v pogojih ponovljivosti (2). | |
| 1.30 Pogoji ponovljivosti pomenijo pogoje, kjer se dobijo neodvisni preskusni rezultati z isto metodo na enakih preskusnih primerkih v istem laboratoriju z istim operaterjem, ki uporablja isto opremo (2). | |
| 1.31 Obnovljivost pomeni natančnost v pogojih obnovljivosti (2)(4). | |
| 1.32 Pogoji obnovljivosti pomenijo pogoje, kjer se preskusni rezultati dobijo z isto metodo na enakih preskusnih primerkih v različnih laboratorijih z različnimi operaterji, ki uporabljajo različno opremo (2)(4). | |
| 1.33 Robustnost pomeni občutljivost analitske metode za spremembe v eksperimentalnih pogojih, ki se lahko izrazijo kot seznam vzorčnega materiala, analitov, skladiščnih pogojev, okoljskih pogojev in/ali pogojev priprave vzorca, v katerih se lahko metoda uporabi, kot je predstavljena ali z manjšimi podrobno podanimi spremembami. Navedeni morajo biti vsi tisti eksperimentalni pogoji,katerih sprememba bi v praksi lahko bila predmet nestabilnosti (npr. stabilnost reagenta, sestava vzorca, pH, temperatura)in bi lahko vplivala na analitski rezultat. | |
| 1.34 Slepo določanje vzorca pomeni celoten analitski postopek, uporabljen pri preskusnem deležu, ki je vzet iz vzorca, v katerem zanesljivo ni analita. | |
| 1.35 Presejalna metoda pomeni metode, ki se uporabljajo za odkrivanje prisotnosti snovi ali vrste snovi na iskanem nivoju. Te metode imajo sposobnost za visoko vzorčno prepustnost in se uporabljajo za presejanje velikega števila vzorcev zaradi potencialnih neskladnih rezultatov. Prilagojene so na nizek delež lažno skladnih rezultatov. | |
| 1.36 Notranja laboratorijska študija (znotrajlaboratorijska validacija) pomeni analitsko študijo enega samega laboratorija, ki uporablja eno metodo za analiziranje istih ali različnih preskusnih materialov pod različnimi pogoji v utemeljenih dolgotrajnih presledkih. | |
| 1.37 Specifičnost pomeni sposobnost metode, da razlikuje med analitom, ki se meri in drugimi snovmi. Ta značilnost je v glavnem funkcija opisanih merilnih tehnik, vendar se lahko razlikuje v skladu z vrsto spojine ali matriksa. | |
| 1.38 Standardno dodajanje je postopek, pri katerem se preskusni vzorec razdeli na dva ali več preskusnih deležev. En delež se analizira kot kak, drugemu deležu preskusnega vzorca pa se pred analizo dodajo znane količine standardnega analita. Količina dodanega standardnega analita mora biti med dva do petkratno ocenjeno količino analita v vzorcu. Ta postopek je namenjen za določanje vsebnosti analita v vzorcu ob upoštevanju izplena analitskega postopka. | |
| 1.39 Standardni analit pomeni analit znane ali certificirane vsebnosti in čistoče, ki ga je treba uporabiti kot referenco pri analizi. | |
| 1.40 Snov pomeni tvarino posebne ali določene kemične sestave in njene metabolite. | |
| 1.41 Za preskus uporabljeni del vzorca pomeni količino materiala, odvzetega od preskusnega vzorca, na katerem se opravlja preskus ali opazovanje. | |
| 1.42 Vzorec pomeni vzorec, pripravljen iz laboratorijskega vzorca in od katerega bodo odvzeti za preskus uporabljeni deleži vzorca. | |
| 1.43 Pravilnost pomeni čim boljše ujemanje med povprečno vrednostjo, dobljeno iz velike serije rezultatov preskusov in sprejeto referenčno vrednostjo. Pravilnost se običajno izrazi kot odstopanje (2). | |
| 1.44 Enote pomenijo enote, opisane v ISO 31 (20) in Direktivi 71/754/ES (19). | |
| 1.45 Validacija pomeni potrditev s pregledovanjem in zagotavljanjem zadostnega dokaza, da so izpolnjene podrobne zahteve za posebej namenjeno uporabo (1). | |
| 1.46 Interna laboratorijska obnovljivost pomeni natančnost, dobljeno v istem laboratoriju pod določenimi (vnaprej določenimi) pogoji (glede npr. metode, preskusnih materialov, operaterjev, okolja) v utemeljeno dolgih časovnih presledkih. | |
| 2. MERILA UČINKOVITOSTI IN DRUGE ZAHTEVE ZA ANALITSKE METODE | |
| Analitske metode ali kombinacije metod razen tistih, ki so opisane spodaj, se lahko uporabljajo le za pregledovalne ali potrditvene namene, če se lahko dokaže, da izpolnjujejo ustrezne zahteve, določene v tej odločbi. | |
| 2.1 SPLOŠNE ZAHTEVE | |
| 2.1.1 Ravnanje z vzorci | |
| Vzorci se pridobijo, obravnavajo in obdelajo na tak način, da obstaja največja možnost za odkrivanje snovi. Postopki za ravnanje z vzorci preprečijo možnost naključne okužbe ali izgube analitov. | |
| 2.1.2 Izvajanje preskusov | |
| 2.1.2.1 Izplen | |
| Med analizo vzorcev se da izplen določiti v vsaki seriji vzorcev, če se uporabi stalni korekcijski faktor izplena. Če je izplen v mejah, se potem lahko uporablja stalni korekcijski faktor. V nasprotnem primeru se za tisto posebno serijo uporabi dobljeni faktor izplena, razen če ni treba uporabiti posebnega faktorja izplena analita v vzorcu. V tem primeru se uporabi metoda standardnega dodatka (glej 3.5) ali metoda internega standarda za kvantitativno določanje analita v vzorcu. | |
| 2.1.2.2 Specifičnost | |
| Metoda mora biti sposobna razlikovati med analitom in drugimi snovmi v eksperimentalnih pogojih. Oceno, do katere mere je to možno, je treba predvideti. Uporabiti se morajo strategije odpravljanja motenj s snovmi med uporabo opisane merilne tehnike, npr. treba je uporabiti homologne, analogne, presnovne produkte v iskanem ostanku. Prvenstveno je pomembno preiskati motnje, ki bi lahko izvirale iz sestavin matriksa. | |
| 2.2 PRESEJALNE METODE | |
| Samo tiste analitske tehnike, za katere se lahko na dokumentirano sledljiv način prikaže, da so validirane in imajo stopnjo lažno skladnih rezultatov na iskanem nivoju nižjo kot 5 % (b-napaka), se lahko uporabljajo za presejalne namene v skladu z Direktivo 96/23/ES. V primeru sumljivega neskladnega rezultata se ta rezultat potrdi s potrditveno metodo. | |
| 2.3 POTRDITVENE METODE ZA ORGANSKE SNOVI IN KONTAMINANTE | |
| Potrditvene metode za ostanke organskih snovi ali kontaminantov morajo praviloma podati informacije o kemijski zgradbi analita. Zato metode, ki temeljijo samo na kromatografski analizi brez uporabe spektrometrične detekcije, same po sebi niso primerne za uporabo kot potrditvene metode. Vendar če posamezna tehnika nima zadostne specifičnosti, se želena specifičnost doseže z analitskimi postopki, ki so sestavljeni iz primernih kombinacij čiščenja, kromatografskega ločevanja in spektrometrične detekcije. | |
| Naslednje metode ali kombinacije metod se štejejo kot primerne za identifikacijo organskih ostankov ali kontaminantov za navedene skupine snovi: | |
| Tabela 1 Primerne potrditvene metode za organske ostanke in kontaminante | |
| Merilna tehnika | Snovi Priloga 1 96/23/ES | Omejitve | | |
| LC ali GC z masno spektrometrično detekcijo | Skupini A in B | Samo če sledi ali on-line ali off-line kromatografski separaciji Samo, če se uporabijo tehnike snemanjem celotnega spektra ali z uporabo najmanj 3 (skupine B) ali 4 (skupine A) identifikacijskih točk za tehnike, ki ne beležijo celotnih masnih spektrov | | |
| LC ali GC z infrardečim zaznavanjem | Skupini A in B | Izpolnjene morajo biti posebne zahteve za absorpcijo v infrardeči spektrometriji | | |
| LC snemanje celotnega spektra z detektorjem z diodnim nizom (DAD) | Skupina B | Izpolnjene morajo biti posebne zahteve za absorpcijo v spektrometriji UV | | |
| LC- fluorescenca | Skupina B | Samo za molekule, ki kažejo primarno fluorescenco in molekule, ki kažejo fluorescenco ali po preoblikovanju ali po derivatizaciji | | |
| 2-D TLC- pregledovanje celotnega spektra UV/VIS | Skupina B | Obvezna sta dvodimenzionalni HTPLC in vzporedna kromatografija | | |
| GC Detekcija z zajetjem elektronov (ECD) | Skupina B | Samo, če se uporabita dve koloni različne polarnosti | | |
| LC imunogram | Skupina B | Samo, če se uporabita najmanj dva različna kromatografska sistema ali pa druga, neodvisna detekcijska metoda | | |
| LC-UV/VIS (ena valovna dolžina | Skupina B | Samo, če se uporabita najmanj dva različna kromatografska sistema ali pa druga, neodvisna detekcijska metod | | |
| 2.3.1 Skupna merila učinkovitosti in zahteve | |
| Potrditvena metoda praviloma zagotavlja podatke o kemijski zgradbi analita. Kadar se več kot ena sestavina enako odziva, tedaj metoda ne more razlikovati med temi sestavinami. Metode, ki temeljijo samo na kromatografski analizi brez uporabe spektrometrične detekcije, niso primerne, za uporabo kot potrditvene metode same po sebi. | |
| Kjer se uporablja metoda internega standarda, je le tega potrebno dodati preskušanemu deležu na začetku ekstrakcijskega postopka. Glede na razpoložljivost se uporabljajo ali analiti stabilne izotopsko označene oblike, ki so zlasti primerni za masno spektrometrično detekcijo ali sestavine, ki so strukturno sorodne z analitom. | |
| Kjer se ne more uporabiti noben interni standard, se identifikacija analita potrdi s vzporedno kromatografijo. V tem primeru se zazna le en kromatografski vrh, povečana višina vrha (ali ploščine pod vrhom) pa je enaka količini dodanega analita. Pri uporabi plinske kromatografije (GC) ali tekočinske kromatografije (LC) je širina vrha pri polovici največje višine v obsegu 90 – 110 % prvotne širine in retencijski časi so enaki v okviru vrednosti 5 %. V primeru tankoplastne kromatografske (TLC) metode se intenzivira samo madež, za katerega se domneva, da je nastal zaradi analita; ne sme se pojaviti nov madež, vizualni izgled madeža pa se ne sme spreminjati. | |
| Tako referenčni ali ojačani material, ki vsebuje znane količine analita na ali blizu dovoljene ali odločitvene meje (neskladen kontrolni vzorec - pozitivna kontrola), kot tudi skladni kontrolni materiali (negativna kontrola) in slepi reagenti naj po možnosti gredo skozi vse faze preskusnega postopka istočasno z vsako serijo analiziranih preskušanih vzorcev. Vrstni red za injiciranje ekstraktov v analitski instrument je naslednji: slepi reagent, skladen kontrolni vzorec, vzorec/vzorci, ki ga/jih je treba potrditi, zopet skladen kontrolni vzorec in končno neskladen kontrolni vzorec. Vsako spremembo tega zaporedja je potrebno utemelji. | |
| 2.3.2 Dodatna merila učinkovitosti in druge zahteve za kvantitativne analitske metode | |
| 2.3.2.1 Pravilnost kvantitativnih metod | |
| V primeru ponovljenih analiz potrjenega referenčnega materiala so priporočeni razponi odmika eksperimenalno določene srednje masne frakcije, korigirane z izplenom, od potrjene vrednosti naslednje: | |
| Tabela 2 Najmanjša pravilnost kvantitativnih metod | |
| Masni delež | Razpon | | |
| ≤ 1 mg/kg do 10 mg/kg | – 50 % do + 20 % | | |
| > 1 mg/kg | – 30 % do + 10 % | | |
| ≥ 10 mg/kg | – 20 % do + 10 % | | |
| Če ni na voljo nobenih takšnih CRM, je sprejemljivo, da se pravilnost meritev oceni z izplenom dodatkov znanih količin analita/analitov slepemu matriksu. Podatki, ki se korigirajo s povprečnim izplenom, so sprejemljivi samo, kadar so v razponu, prikazanem v tabeli 2. | |
| 2.3.2.2 Natančnost kvantitativnih metod | |
| Medlaboratorijski koeficient variacije (CV) za ponovljene analize referenčnega ali ojačanega materiala v pogojih obnovljivosti naj ne presega nivoja, izračunanega po Horwitzovi enačbi.Enačba je: | |
| CV = 2 | |
| kjer je C masni delež, izražen kot potenca (eksponent) 10 (npr. 1 mg/g = 10 -3). Primeri so prikazani v tabeli 3. | |
| Tabela 3 Primeri obnovljivosti CV za kvantitativne metode v razponu masnih deležev analita | |
| Masni delež | Obnovljivost CV (%) | | |
| 1 mg/kg | [1] | | |
| 10 mg/kg | [1] | | |
| 100 mg/kg | 23 | | |
| 1000 mg/kg (1mg/kg) | 16 | | |
| Za analize, izvedene v pogojih ponovljivosti, bi znotrajlaboratorijski CV bil tipično med polovico in dvema tretjinama zgornjih vrednosti. Za analize, izvedene v pogojih znotrajlaboratorijske obnovljivosti, je znotrajlaboratorijski CV višji kot CV obnovljivosti. | |
| V primeru snovi z določeno dovoljeno mejo, doseže metoda v internem laboratoriju obnovljivost, ki ni večja od ustrezne obnovljivosti CV pri koncentraciji 0,5 x dovoljena meja. | |
| 2.3.3 Merila učinkovitosti in druge zahteve za masno spektrometrično detekcijo | |
| Masne spektrometrične metode je primerno upoštevati kot potrditvene metode le kot nadaljevanje on-line ali off-line kromatografskega ločevanja. | |
| 2.3.3.1 Kromatografsko ločevanje | |
| Za postopke GC-MS se izvede plinsko kromatografsko ločevanje z uporabo kapilarnih kolon. Za postopke LC-MS postopke se kromatografsko ločevanje izvede z uporabo primernih kolon LC. V vsakem primeru je najmanjši sprejemljivi retencijski čas za preiskovani analit dvakratni retencijski čas, ki ustreza prostemu volumnu kolone. Retencijski čas (ali relativni retencijski čas) analita v preskušanem deležu ustreza času umeritvenega standarda v specificiranem okencu retencijskega časa. Okence retencijskega časa je sorazmerno z močjo ločevanja kromatografskega sistema. Razmerje kromatografkega retencijskega časa analita proti času internega standarda, t.j. relativni retencijski čas analita, ustreza tistemu od umeritvene raztopine pri toleranci ± 0,5 % za GC in ± 2,5 % za LC. | |
| 2.3.3.2 Masna spektrometrična detekcija | |
| Masna spektrometrična detekcija se izvaja z uporabo MS tehnik, kot so snemanje celotnih masnih spektrov (full scan) ali izbrano spremljanje ionov (SIM), kakor tudi tehnike MS-MSn kot je izbrano spremljanje reakcije (SMR) ali druge primerne MS ali MS-MSn tehnike v kombinaciji z ustreznimi načini ionizacije. Pri masni spektrometriji visoke ločljivosti (HRMS), je ločljivost tipično večja od 10000 za celotni masni razpon pri 10 % višini doline. | |
| Snemanje celotnega spektra (Full scan): Ko se masno spektrometrično določanje izvaja s snemanjem celotnega spektra, je obvezna prisotnost vseh merjenih diagnostičnih ionov (molekularni ion, značilni adukti molekularnega iona, karakteristični fragmentni ioni in vsi njihovi izotopni ioni) z relativno intenziteto več kot 10 % v referenčnem spektru umeritvenega standarda. | |
| SIM: Ko se masno spektrometrično določanje izvaja s fragmentografijo, je molekularni ion po možnosti eden od izbranih diagnostičnih ionov (molekularni ion, značilni adukti molekularnega iona,, značilni fragmentni ioni in vsi njihovi izotopni ioni). Izbrani diagnostični ioni naj ne bi izvirali izključno iz istega dela molekule. Razmerje signal proti šumu za vsak diagnostični ion je 3:1. | |
| Snemanje celotnega spektra in SIM: relativne intenzitete zaznanih ionov, izraženih kot odstotek intenzitete najbolj intenzivnega iona ali prehajanja, ustrezajo intenzitetam umeritvenega standarda, bodisi iz umeritvenih standardnih raztopin bodisi iz vzorcev s standardnim dodatkom, ob primerljivih koncentracijah, izmerjenih pod istimi pogoji v naslednjih tolerancah: | |
| Tabela 4 Največje dovoljene tolerance za relativne ionske intenzitete z uporabo lestvice masnih spektrometričnih tehnik | |
| Relativna intenziteta (% osnovnega vrha) | EI-CG-MS (relativno) | CI-GC-MS, GC-MSn LC-MS,LC-MSn | | |
| > 50 % | ± 10 % | ± 20 % | | |
| > 20 % do 50 % | ± 15 % | ± 25 % | | |
| > 10 % do 20 % | ± 20 % | ± 30 % | | |
| ≤10 % | ± 50 % | ± 50 % | | |
| Razlaga masnih spektralnih podatkov: Relativne intenzitete diagnostičnih ionov in/ali prekurzorjev/produktnih ionskih parov morajo biti identificirane s primerjanjem spektrov ali z integriranjem signalov posameznih masnih sledi. Kadarkoli se uporabi korekcija ozadja, se to opravi enakomerno skozi vso serijo (glej 2.3.1, odstavek 4) in se jasno označi. | |
| Snemanje celotnega spektra: Ko se celotni spektri posnemajo z masno spektrometrijo (GC-MS), morajo biti prisotni najmanj štiri ioni z relativno intenziteto 10 % osnovnega vrha. Molekularni ion se vključi, če je prisoten v referenčnem spektru z relativno intenziteto 10 %. Najmanj štirje ioni naj ležijo v največjih dovoljenih tolerancah za relativne ionske intenzitete (tabela 5). Uporablja se lahko računalniško podprta knjižnično iskanje. V tem primeru mora primerjava masnih spektralnih podatkov v preskušanem vzorcu z tistimi v umeritveni raztopini presegati kritični faktor ujemanja. Ta faktor se določi med validacijskim postopkom za vsak analit na osnovi spektrov, za katere so izpolnjena spodaj opisana merila. Preveri se variabilnost v spektrih, ki jo povzroči matriks vzorca in učinkovitost detektorja. | |
| SIM: Kadar se masni fragmenti merijo po drugih metodah razen s snemanjem celotnega spektra, se za razlago podatkov uporabi sistem identifikacijskih točk. Za potrditev snovi na seznamu v Skupini A Priloge I Direktive 96/23/ES se zahtevajo najmanj 4 identifikacijske točke. Za potrditev snovi na seznamu v Skupini B Priloge I Direktive 96/23/ES se zahtevajo najmanj 3 identifikacijske točke. Spodnja tabela prikazuje število identifikacijskih točk, ki jih lahko pridobi vsaka od osnovnih masnih spektrometričnih tehnik. Vendar, da bi izpolnili pogoje, ki se zahtevajo za potrditev in izračun vsote identifikacijskih točk: | |
| (a) se izmeri najmanj eno od ionskih razmerij in | |
| (b) vsa ustrezna izmerjena ionska razmerja izpolnjujejo zgoraj navedene pogoje in | |
| (c) se lahko kombinirajo največ tri ločene tehnike, da se doseže najmanjše število identifikacijskih točk. | |
| Tabela 5 Razmerje med razredno lestvico masnih fragmentov in dobljenimi identifikacijskimi točkami | |
| Opombe: | |
| (1) Vsak ion se šteje samo enkrat. | |
| (2) GC-MS, ki uporablja elektronsko ionizacijo, se šteje za drugačno tehniko od GC-MS, ki uporablja kemijsko ionizacijo. | |
| (3) Različni analiti se lahko uporabijo za povečanje identifikacijskih točk samo, če se za derivate uporabljajo različne reakcijske kemije. | |
| (4) Za snovi v Skupini A Priloge 1 Direktive 96/23/ES, če je uporabljena ena od naslednjih tehnik v analitskem postopku: HPLC povezano s spektrometrijo z diodnim nizom pri celotnem snemanju (full-scan diode array spectrometry, DAD); HPLC povezano s fluorescentno detekcijo; HPLC povezano z imunogramom; dvodimenzionalno TLC povezano s spektrometrično detekcijo; prispeva se lahko največ ena identifikacijska točka, če so izpolnjena ustrezna merila za te tehnike. | |
| (5) Prehodni proizvodi vključujejo naslednike in njihove podnaslednike. | |
| MS tehnika | Identifikacijske točke pridobljene na ion | | |
| Masna spektrometrija nizke ločljivosti (LR) | 1,0 | | |
| Prekurzor ion LR- MSn | 1,0 | | |
| Prehodni proizvodi LR- MSn | 1,5 | | |
| HRMS | 2,0 | | |
| Prekurzor ion HR- MSn | 2,0 | | |
| Prehodni proizvodi HR- MSn | 2,5 | | |
| Tabela 6 Primeri števila identifikacijskih točk, dobljenih za serijo tehnik in njihovih kombinacij (n = celo število) | |
| Tehnika(e) | Število ionov | Identifikacijske točke | | |
| GC-MS (EI ali CI) | N | n | | |
| GC-MS (EI in CI) | 2(EI) + 2 (ci) | 4 | | |
| GC-MS (EI ali CI) 2 derivata | 2 (derivat A) + 2 (derivat B) | 4 | | |
| LC-MS | N | n | | |
| GC-MS-MS | 1 prekurzor in 2 naslednika | 4 | | |
| LC-MS-MS | 1 prekurzor in 2 naslednika | 4 | | |
| GC-MS-MS | 2 prekurzorja iona, vsak z enim naslednikom | 5 | | |
| LC-MS-MS | 2 prekurzorja iona, vsak z 1 naslednikom | 5 | | |
| LC-MS-MS-MS | 1 prekurzor, 1 naslednik in 2 podnaslednika | 5,5 | | |
| HRMS | N | 2 n | | |
| GC-MS in LC-MS | 2 + 2 | 4 | | |
| GC-MS in HRMS | 2 + 1 | 4 | | |
| 2.3.4 Merila učinkovitosti in druge zahteve za kromatografijo povezano z infrardečo detekcijo | |
| Adekvatni vrhi: adekvatni vrhi so največje absorpcijske vrednosti v infrardečem spektru umeritvenega standarda, ki izpolnjuje naslednje zahteve. | |
| 2.3.4.1 Infrardeča detekcija | |
| Absorpcijski maksimum: je v razponu valovnega števila 4000-500 cm-1. | |
| Intenziteta absorpcije: ni manjša od bodisi: | |
| (a) specifične molske absorbance 40 glede na bazno linijo; bodisi | |
| (b) relativne absorbance 12,5 % od absorbance najintenzivnejšega vrha v predelu 4000-500 cm-1 | |
| kadar sta oba merjena glede na absorbanco nič, in 5 % absorbance najintenzivnejšega vrha v predelu 4000-500 cm-1, kadar sta oba merjena glede na njuno bazno linijo vrha. | |
| Opomba: | |
| Čeprav imajo adekvatni vrhi po (a) morda prednost s teoretičnega stališča, pa se tisti po (b) lažje določijo v praksi. | |
| Določi se število vrhov v infrardečem spektru analita, čigar frekvenca ustreza adekvatnemu vrhom v spektru umeritvenega standarda v mejah ± 1 cm-1. | |
| 2.3.4.2 Razlaga infrardečih spektralnih podatkov | |
| Absorpcija je prisotna v vseh predelih analita, ki ustrezajo adekvatnemu vrhu v referenčnem spektru umeritvenega standarda. V infrardečem spektru umeritvenega standarda se zahteva najmanj 6 adekvatnih vrhov. Če je manj kot šest adekvatnih vrhov (7), se sporni spekter ne more uporabiti kot referenčni spekter. "Rezultat", t.j. odstotek adekvatnih vrhov, ugotovljenih v infrardečem spektru analita je najmanj 50. Kjer ni točnega ujemanja za adekvatni vrh, je sovisen predel spektra analita skladen s prisotnostjo ujemajočega se vrha. Postopek velja le za absorpcijske vrhe v vzorčnem spektru z najmanj trikratno intenzivnostjo razmerja šum/vrh. | |
| 2.3.5 Merila učinkovitosti in druge zahteve za določanje analita z uporabo LC z drugimi detekcijskimi tehnikami | |
| 2.3.5.1 Kromatografsko ločevanje | |
| Interni standard se uporablja, če je na voljo material, ki je primeren za ta namen. Če je možno, je to standard, ki ima retencijski čas blizu retencijskega časa analita. Analit se eluira v retencijskem času, ki je tipičen za ustrezni umeritveni standard pod istimi eksperimentalnimi pogoji. Najmanjši sprejemljivi retencjiski čas za analit je dvakratni retencijski čas, ki ustreza prostemu volumnu kolone. Razmerje retencijskega časa analita in internega standarda, t.j. relativnega retencijskega časa analita, je enako tistemu od umeritvenega standarda v primernem matriksu v mejah ± 2,5 %. | |
| 2.3.5.2 Detekcija s snemanjem celotnega spektra (full-scan)UV/VIS | |
| Merila učinkovitosti za metode LC morajo biti izpolnjena. | |
| Maksimumi absorpcije v spektru analita so pri enaki valovni dolžini, kot jih ima umeritveni standard, v mejah, določenih s ločljivostjo detekcijskega sistema. Za detekcijo z diodnim nizom (diode array detection) je to tipično v mejah ± 2 mm. Pri valovno dolžini nad 220 nm se spekter analita, v delih, kjer je relativna absorbanca obeh spektrov ³ 10 %, od spektra umeritvenega standarda vidno ne razlikuje.To merilo je izpolnjeno, ko so prisotne, prvič največje vrednosti in drugič, ko razlika med obema spektroma na nobeni opazovani točki ni večja od 10 % absorbance umeritvenega standarda. V primeru uporabe računalniško podprtega knjižničnega iskanja in potrjevanja, mora primerjava spektralnih podatkov preskušanih vzorcev in umeritvene raztopine presegati kritični faktor ujemanja. Ta faktor se določi med postopkom validacije za vsak analit na osnovi spektrov, za katere so izpolnjena zgoraj opisana merila. Pregleda se variabilnost v spektrih, ki jo povzroči vzorčni matriks in učinkovitost detekcije. | |
| 2.3.5.3 Merila učinkovitosti fluorimetrične detekcije | |
| Merila učinkovitosti za metode LC morajo biti izpolnjena. | |
| To velja za molekule, ki so same po sebi fluorescenčne in molekule, ki kažejo fluorescenco ali po preoblikovanju ali po derivaciji. Izbira vzbujalnih in emisijskih valovnih dolžin v kombinaciji s kromatografskimi pogoji se opravi na ta način, da se pojavnost motečih komponent v ekstraktih slepega vzorca spravi na najnižjo možno raven. | |
| Najbližja najvišja točka vrha v kromatogramu se loči od označenega vrha analita za najmanj eno celotno širino vrha pri 10 % največje višine vrha analita. | |
| 2.3.5.4 Merila učinkovitosti za določitev analita po imunogramu LC | |
| Imunogram LC ni sam primeren za uporabo kot potrditvena metoda. | |
| Izpolnjena morajo biti ustrezna merila za metode LC. | |
| Predhodno določeni parametri za nadzor kakovosti, npr. nespecifična vezava, relativna vezava kontrolnih vzorcev, vrednost absorbance slepega vzorca morajo biti v mejah, dobljenih med validacijo preskusa. | |
| Imunogram mora biti sestavljen iz najmanj petih frakcij. | |
| Vsaka frakcija je manjša od polovice širine vrha. | |
| Frakcija z največjo vsebnostjo analita mora biti enaka za sumljiv vzorec, neustrezen kontrolni vzorec in standard. | |
| 2.3.5.5 Določitev analita z uporabo LC z detekcijo UV/VIS (enovalovno) | |
| LC z (enovalovno) detekcijo UV/VIS ni sama primerna za uporabo kot potrditvena metoda. | |
| Najbližja najvišja točka vrha v kromatogramu se loči od določenega vrha analita za najmanj eno celotno širino vrha pri 10 % največje višine vrha analita. | |
| 2.3.6 Merila učinkovitosti in druge zahteve za določitev analita z 2-D TLC združeno s spektrometrično detekcijo UV/VIS s snemanjem celotnega spektra | |
| Obvezna sta dvodimenzionalna HTPLC in vzporedna kromatografija. | |
| Vrednosti RF analita se ujemajo z vrednostmi standardov RF v ± 5 %. | |
| Vizualni izgled analita je enak izgledu standarda. | |
| Za madeže iste barve naj bo središče najbližjega madeža ločeno od centra madeža analita za najmanj polovico vsote premerov madeža. | |
| Spekter analita se vizualno ne razlikuje od spektra standarda, kakor je opisan za UV/VIS detekcijo s snemanjem celotnega spektra. | |
| V primeru uporabe računalniško podprtega knjižničnega iskanja in primerjanja, mora primerjava spektralnih podatkov v preskušanih vzorcih in podatkov umeritvene raztopine, presegati kritični faktor ujemanja. Ta faktor se določi med postopkom validacije za vsak analit na osnovi spektrov, za katere so izpolnjena zgoraj opisana merila. Pregleda se variabilnost v spektrih, ki jo povzroči vzorčni matriks in učinkovitost detekcije. | |
| 2.3.7 Merila učinkovitosti in zahteve za določitev analita po GC v kombinaciji z detekcijo z zajemanjem elektronov (ECD) | |
| Interni standard se uporabi, če je na voljo material, ki je primeren za ta namen. Po možnosti je to sorodna snov z retencijskim časom, ki je blizu istemu, ki ga ima analit. Analit se izloči ob retencijskem času, ki je tipičen za ustrezni umeritveni standard pod istimi eksperimentalnimi pogoji. Najmanjši sprejemljivi retencijski čas za analit je dvakratni retencijski čas, ki ustreza prostemu volumnu kolone. Razmerje med retencijskim časom analita in tistim od internega standarda, t. j. relativnim retencijskim časom analita je isti kot čas umeritvenega standarda v ustreznem matriksu v mejah ± 0,5 %. Najbližja najvišja točka vrha v kromatogramu se loči od označenega vrha analita za najmanj eno celo širino vrha pri 10 % največje višine vrha analita. Za dodatne informacije se lahko uporabi vzporedna kromatografija. | |
| 2.4 POTRDITVENE METODE ZA ELEMENTE | |
| Potrditvene analize za kemične elemente temeljijo na konceptu nedvoumne identifikacije ter točne in natančne kvantifikacije s pomočjo fizikalno-kemičnih lastnosti, ki so edinstvene za obravnavani kemični element (npr. karakteristična valovna dolžina elementa oddanega ali prejetega sevanja, atomska masa) na iskanem nivoju.. | |
| Za identifikacijo kemičnih elementov se štejejo primerne naslednje metode ali kombinacije metod: | |
| Tabela 7 Primerne potrditvene metode za kemične elemente | |
| Tehnika | Merjeni parameter | | |
| Diferenčna impulzna anodna Stripping voltametrija | Električni signal | | |
| Atomska absorpcijska spektrometrija | |
| Plamenska | Valovna dolžina absorpcije | | |
| Nastajanje hidridov | Valovna dolžina absorpcije | | |
| Hladilne pare | Valovna dolžina absorpcije | | |
| Elektrotermična atomizacija (grafitna peč) | Valovna dolžina absorpcije | | |
| Atomska emisijska spektrometrija | |
| Induktivno sklopljena plazma | Valovna dolžina sevanja | | |
| Masna spektrometrija | |
| Induktivno sklopljena plazma | Razmerje masa/naboj | | |
| 2.4.1 Splošna merila učinkovitosti in druge zahteve za potrditvene metode | |
| Tako referenčni ali ojačani material, ki vsebuje znane količine analita na ali blizu dovoljene ali odločitvene meje (neskladen kontrolni vzorec - pozitivna kontrola), kot tudi skladni kontrolni materiali (negativna kontrola) in slepi reagenti naj po možnosti gredo skozi vse faze preskusnega postopka istočasno z vsako serijo analiziranih preskušanih vzorcev. | |
| Na splošno zahteva večina analitskih tehnik popoln razkroj organskega matriksa, da se dobijo raztopine za določevanje analita. To se lahko doseže z uporabo mikrovalovnih mineralizacijskih postopkov, ki kar najbolj zmanjšajo tveganje izgube in/ali kontaminacije iskanih analitov. Uporabljajo se dekontaminirane posode iz teflona dobre kakovosti. Če se uporabijo druge mokre ali suhe metode razkroja, se mora dokumentirano izključiti morebiten pojav izgube ali kontaminacije. Kot alternativo za razkroj, se lahko pod določenimi pogoji izberejo postopki ločevanja (npr. ekstrakcija), za ločevanje analitov od sestavin matriksa in/ali njihovega koncentriranja z namenom, da se uvedejo v analitsko opremo. | |
| Kar se tiče umeritve, ali zunanje ali na osnovi metode standardnega dodatka, je treba paziti, da se ne preseže delovni obseg, ki je določen za analizo. V primeru zunanje umeritve, je obvezno, da so umeritveni standardi pripravljeni v raztopini, ki se kar najbolj ujema z zgradbo vzorčne raztopine. Uporabi se tudi korekcija ozadja, če to zahtevajo posebne analitske okoliščine. | |
| 2.4.2 Dodatna merila učinkovitosti in druge zahteve za kvantitativne analitske metode | |
| 2.4.2.1 Pravilnost kvantitativnih metod | |
| V primeru ponovljenih analiz certificiranih referenčnih materialov za elemente, odmik eksperimentalno določene srednje vsebnosti od potrjene vrednosti ni večji od ± 10 %. Če ustreznih CRM ni na voljo, je sprejemljivo, da se pravilnost meritev oceni preko izplena dodatkov znanih količin elementa neznanim vzorcem. Pozornost se posveti dejstvu, da se za razliko od analita, dodani element ne veže kemično v pravi matriks in da imajo rezultati, dobljeni po tem pristopu, manjšo veljavo kakor tisti, ki se dosežejo z uporabo CRM. Podatki o izplenu so sprejemljivi samo, če so v okviru ± 10 % ciljne vrednosti. | |
| 2.4.2.2 Natančnost kvantitativnih metod | |
| V primeru ponovljene analize vzorca, opravljene v znotraj laboratorijskih pogojih obnovljivosti, znotrajlaboratorijski koeficient variacije (CV) povprečja ne presega naslednjih vrednosti: | |
| Tabela 8 CV za kvantitativne metode pri razponu masnih deležev elementa | |
| Masni delež | CV (%) | | |
| ≥ 10 mg/kg do 100 mg/kg | 20 | | |
| > 100 mg/kg do 1000 mg/kg | 15 | | |
| ≥ 1000 mg/kg | 10 | | |
| 2.4.3 Posebne zahteve za diferenčno impulzno anodno stripping voltametrijo (DPASV) | |
| Popoln razkroj organske snovi v vzorcih pred določitvami z DPASV je najpomembnejši. Na voltamogramih ni vidnih nobenih širokih signalov kot posledica prisotnosti organskih substanc. Sestavine anorganskega matriksa lahko vplivajo na višine vrhov v DPASV. Zato je treba izvesti kvantifikacijo z metodo standardnih dodatkov. Primerki tipičnih voltamogramov vzorčne raztopine morajo biti priloženi metodi. | |
| 2.4.4 Posebne zahteve za atomsko absorpcijsko spektrometrijo (AAS) | |
| V osnovi je ta tehnika enoelementna in zato zahteva optimizacijo eksperimentalnih nastavitev, ki so odvisne od posameznega elementa, ki ga je treba kvantificirati. Kjerkoli je možno, se rezultati pregledajo kvalitativno in kvantitativno s prerazporeditvijo na alternativne absorpcijske črte (idealno se izbereta dve različni črti). Umeritveni standardi se pripravijo v raztopini matriksa, ki se čim bliže ujema z raztopino vzorčnega merjenja. (npr. koncentracija kisline ali zgradbe modifikatorjev). Da bi čimbolj zmanjšali slepe vrednosti, se uporabljajo reagenti z najvišjo dostopno čistočo. Odvisno od izbranega načina za vaporizacijo in/ali atomizacijo vzorca se lahko ločijo različni tipi AAS. | |
| 2.4.4.1 Specifične zahteve za plamensko AAS | |
| Nastavitve instrumenta so optimizirane za vsak element. Še posebej morajo biti kontrolirana sestava plinov in njihov pretok. Uporablja se kontinuiran korektor izvora, da se izognemo intereferencam, ki jih povzroča absorpcija ozadja.V primeru neznanih matriksov se preveri, ali je potrebna korekcija ozadja ali ne. | |
| 2.4.4.2 Posebne zahteve za AAS z grafitno pečjo | |
| Kontaminacija v laboratoriju pogosto vpliva na točnost ob delu v grafitnih pečeh na nivoju ultra sledi. Za ravnanje z vzorci in standardi se morajo zato uporabljati reagenti visoke čistoče, deionizirana voda in posoda iz inertne plastike. Nastavitve instrumenta se za vsak element optimizirajo. Posebno se morajo preveriti pogoji predobdelave in atomizacije (temperatura, čas) in sprememba matriksa. | |
| Delo pod pogoji izotermalne atomizacije (npr. prečna ogrevana grafitna cev z integrirano platformo Lvov (8) bo zmanjšalo vpliv matriksa, kar se tiče atomizacije analita. V kombinaciji s spremembo matriksa in Zeemanovo korekcijo ozadja (9) se dovoli tudi kvantifikacija s pomočjo umeritvene krivulje na osnovi meritev vodnih standardnih raztopin. | |
| 2.4.5 Posebne zahteve za atomsko absorpcijsko spektrometrijo tvorbe hidridov | |
| Organske spojine, ki vsebujejo elemente kot so arzen, bizmut, germanij, svinec, antimon, selen, kositer in telur, so lahko zelo stabilne, zato je potreben razkroj z oksidacijo, da se dobijo pravilni rezultati za celotno vsebnost elementa. Zato se priporoča mikrovalovni razpad ali visokotlačni sežig v močnih pogojih oksidacije. Največjo pozornost se posveti popolni in ponovljivi pretvorbi elementov v njihove ustrezne hidride. | |
| Tvorba arzenovega hidrida v solni kislini z NaBH4 je odvisna od oksidacijskega stanja arzena (As III: hitra tvorba, As V: daljši čas tvorbe). Da bi se izognili izgubi občutljivosti za določanje As V z novo pretočno injekcijsko tehniko, ki jo povzroči krajši reakcijski čas v tem sistemu, se mora As V reducirati na As III po oksidacijski razgradnji. Za ta namen so primerni kalijev jodid/askorbinska kislina ali cistein. Slepi reagenti, umeritvene raztopine in vzorčne raztopine se obravnavane na isti način. Delo s serijskim načinom omogoča določanje obeh arzenovih vrst, ne da bi to vplivalo na točnost. Zaradi zakasnele tvorbe As V hidrida, se umerjanje izvede z integracijo površine vrha.Optimizirajo se nastavitve instrumenta. Pretok plina, ki prenese hidrid v atomizer je zlasti pomemben in se preverja. | |
| 2.4.6 Posebne zahteve za atomsko absorpcijsko spektrometrijo s tehniko hladnih par | |
| Tehnika hladne pare se uporablja samo v primeru živega srebra.Zaradi hlapljivosti in absorpcijskih izgub elementarnega živega srebra, je med celotno analizo potrebna posebna previdnost. Pazljivo se je treba izogibati kontaminacije z reagenti ali okoljem. | |
| Za organske spojine, ki vsebujejo živo srebro, je potrebna oksidacijska razgradnja, da dobimo pravilne rezultate za celotno vsebnost živega srebra. Za razgradnjo se uporablja hermetični sistem z mikrovalovnim razkrojem ali visokotlačni sežigalnik. Posebna pozornost se namenja preprečevanju kontaminacije opreme s čistili ali polutanti okolja. | |
| Delo s pretočno injekcijsko tehniko je koristno. Za nižje odločitvene meje se priporoča adsorbiranje živega srebra na zlatem/platinastem adsorberju, ki mu sledi toplotna desorpcija. Stik adsorberja ali celice z vlago bo motila meritev in se ga je treba izogniti. | |
| 2.4.7 Posebne zahteve za induktivno sklopljeno plazmo atomsko emisijsko spektrometrijo (ICP-AES) | |
| Atomska emisijska spektrometrija s tehniko induktivno sklopljene plazme (10) je večelementna metoda, ki omogoča istočasno merjenje različnih elementov. Za uporabo ICP-AES je treba vzorce najprej raztopiti, da se organski matriksi razgradijo. Uporabljajo se hermetični sistemi z mikrovalovno razgradnjo ali visokotlačnim sežigom. Bistveno vlogo za ustreznost analize ICP-AES igrajo umeritev instrumenta in izbira elementa ali valovne dolžine. Za umeritev instrumenta v primeru linearnih umeritvenih krivulj je običajno potrebno izmeriti umeritvene raztopine samo štirih koncentracij, ker so umeritvene krivulje ICP-AES ponavadi linearne v obsegu nad štiri do šest velikostnih redov koncentracije.Umeritev sistema ICP-AES se naj bi normalno izvedla z večelementnim standardom, ki se pripravi v raztopini, ki ima enako koncentracijo kisline kot merilna raztopina. Za linearno umeritveno krivuljo se morajo preveriti koncentracije elementa. | |
| Izbira valovnih dolžin za merjenje emisije iz analitov je primerna za koncentracije elementov, ki jih je treba določiti. Ko koncentracija analita izpade iz delovnega področja emisijske črte, se uporabi druga emisijska črta. Najprej se izbere najbolj občutljiva emisijska črta (nemotena), nato pa najmanj občutljiva črta. Kadar delamo blizu detekcijske meje ali na njej, je najbolj občutljiva črta za ustrezni analit običajno najboljša izbira. Interfernce spektra ali ozadja povzročajo večje težave pri ICP-AES. Možne interference so npr. preprost premik ozadja, poševen premik ozadja, spektralno prekrivanje in zapleten premik ozadja. Vsaka od te motenj ima svoje vzroke in načine odpravljanja. Odvisno od matriksov se uporabijo korekcije motenj (interferenc) in optimizacija delovnih parametrov.Nekaterim intereferencam se da izogniti z razredčitvijo ali prilagoditvijo matriksov. Z vsako serijo analiziranih preskušanih vzorcev se tako referenčni in dodani material, ki vsebuje znane količine analita(tov) kakor tudi slepi vzorec, obdelujejo na isti način kakor preskušani vzorci. Za preskušanje odklona (drift), se standard preveri npr. po 10 vzorcih. Vsi reagenti in plazma plin so najvišje dostopne čistoče. | |
| 2.4.8 Posebne zahteve za induktivno sklopljeno plazmo - masno spektrometrijo (ICP-MS)(11) | |
| Določanje elementov v sledeh s povprečno atomsko maso, kot so krom, baker in nikelj, je lahko pogojeno z močnimi interferencami drugih izobaričnih in poliatomskih ionov. To se lahko prepreči samo, kadar je na voljo ločljivost najmanj 7000-8000. Težave, povezane s tehnikami MS vključujejo odklon instrumenta, vplive matriksa in molekularno ionsko interferenco (m/z < 80). Potrebna je večkratna interna standardizacija, ki obsega isti masni razpon kot elementi, ki jih je treba določiti, da se korigirajo odklon instrumenta in vplivi matriksa. | |
| Pred meritvami ICP-MS se zahteva popolna razgradnja organske snovi v vzorcih. Kot pri AAS je po razkroju v hermetičnih posodah potrebno prevesti hlapljive elemente, npr. jod, v stabilno oksidacijsko stanje. Večina resnih interferenc je posledica molekularnih ionskih kombinacij argona (plazma plin), vodika, ogljika, dušika in kisika (razgrajevalna kislina, nečistoče plazma plina in uvedeni atmosferski plini) ter vzorčnega matriksa. Da bi se izognili intereferencam se zahteva popoln razkroj, meritve ozadja, primerna izbira analitskih mas, ki so včasih povezane z manjšim prebitkom (slabša mejna vrednost detekcije) in razgrajevalne kisline npr. dušikova kislina. | |
| Za elemente, ki jih je treba določiti, se morajo motnje izključiti s primerno izbiro specifičnih analitskih mas, vključujoč potrditev izotopnih razmerij. Odziv instrumenta po Fano-faktorjih se preveri za vsako meritev z uporabo internih standardov. | |
| 3. VALIDACIJA | |
| Validacija pokaže, ali se analitska metoda sklada z merili, ki veljajo za ustrezne značilnosti učinkovitosti. | |
| Za različne nadzorne namene so potrebna različne kategorije metod. Naslednja tabela določa, katere značilnosti učinkovitosti se preverijo za vsako vrsto modela. | |
| Tabela 9 Razvrstitev analitskih metod po značilnosti učinkovitosti, ki jih je treba določiti | |
| S = presejalne metode; C = potrditvene metode; + = določitev je obvezna. | |
| | Meja detekcije CCβ | Odločitvena meja CCα | Pravilnost/izplen | Točnost | Selektivnost/Specifičnost | Uporabljivost/robustnost/stabilnost | | |
| Kvalitativne metode | S | + | – | – | – | + | + | | |
| C | + | + | – | – | + | + | | |
| Kvantitativne metode | S | + | – | – | + | + | + | | |
| C | + | + | + | + | + | + | | |
| 3.1 VALIDACIJSKI POSTOPKI | |
| To poglavje podaja primere in/ali reference za validacijske postopke analitskih metod. Lahko se uporabijo drugi pristopi, ki dokažejo, da se analitska metoda sklada z merili učinkovitosti, ki veljajo za značilnosti učinkovitosti, pod pogojem da se doseže ista raven in kakovost informacij. | |
| Validacija se lahko izvede tudi z vodenjem medlaboratorijske študije, kakor je določena s Codex Alimentarius, ISO ali IUPAC (12), ali v skladu z alternativnimi metodami, kakor so posamezne laboratorijske študije ali notranje validacije (13)(14). Ta del se osredotoči na posamezne laboratorijske študije (pri notranji validaciji), ki uporabljajo modularen pristop. Ta pristop obsega: | |
| 1. serijo skupnih značilnosti učinkovitosti, ki so neodvisne od uporabljenega validacijskega modela in | |
| 2. bolj specifične,od modela odvisne postopke, kakor je opisano v tabeli 10. | |
| Tabela 10 Delovni parametri, ki so neodvisni in odvisni od modela | |
| Validacija | | |
| Delovni parametri, neodvisni od modela | Delovni parametri, odvisni od modela | | |
| Skupne značilnosti učinkovitosti (3.1.1) | Konvencionalni validacijski pristop (3.1.2) | Interni validacijski pristop (3.1.3) | | |
| Specifičnost | Izplen | izplen | | |
| Pravilnost | Obnovljivost | Obnovljivost | | |
| Robustnost: manjše spremembe | Obnovljivost v laboratoriju | Obnovljivost v laboratoriju | | |
| Stabilnost | Obnovljivost | Obnovljivost | | |
| Odločitvena meja (CCα) | Odločitvena meja (CCα) | | |
| Sposobnost detekcije (CCβ) | Sposobnost detekcije (CCα) | | |
| Umeritvene krivulje | Umeritvene krivulje | | |
| Robustnost: večje spremembe | Robustnost | | |
| 3.1.1 Značilnosti učinkovitosti, neodvisne od modela | |
| Ne glede na izbrani validacijski pristop je treba določiti naslednje značilnosti učinkovitosti. Da se čimbolj zmanjša delovna obremenitev, se lahko uporabi skrbno oblikovan in statistično zanesljiv pristop za kombiniranih eksperimentov, izvedenih za določitev različnih parametrov. | |
| 3.1.1.1 Specifičnost | |
| Za analitske metode je pomembna sposobnost razlikovanja med analitom in snovmi, ki so tesno povezane (izomeri, metaboliti, degradacijski produkti, endogene snovi, sestavine matriksa, itd.). Za preverjanje interferenc sta potrebna dva pristopa. | |
| Zato se izberejo potencialno moteče snovi in analizirajo ustrezni slepi vzorci, da se odkrije prisotnost možnih interferenc in oceni njihov vpliv: | |
| - izberemo niz kemično sorodnih spojin (metabolitov, derivatov, itd.) ali drugih snovi, na katere je možno naleteti pri iskani spojini, ki je lahko prisotna v vzorcih; | |
| - analiziramo primerno število reprezentativnih slepih vzorcev (n = 20) in preverimo, če so motnje (signali, vrhi, sledi ionov) v predelih, kjer se pričakuje, da se bo ciljni analit eluiral; | |
| - nadalje se reprezentativni slepi vzorci ojačajo s snovmi ustrezne koncentracije, ki verjetno motijo identifikacijo in/ali kvantifikacijo analita; | |
| - po analizi raziskati, če: | |
| - lahko prisotnost vodi k napačni identifikaciji, | |
| - je identifikacija ciljnega analita ovirana zaradi prisotnosti ene ali več motenj, ali | |
| - če je opazen vpliv na kvantifikacijo. | |
| 3.1.1.2 Pravilnost | |
| V tem odstavku je podano določevanje pravilnosti (ena sestavina točnosti). Pravilnost se lahko ugotovi le s certificiranim referenčnim materialom (CRM). CMR se uporabi, kadar koli je na voljo. Postopek je podrobno opisan v ISO 5725-4 (5). Spodaj je podan primer: | |
| - analizirati šest paralelk CRM v skladu s navodili za preskušanje za metodo, | |
| - določiti koncentracijo prisotnega analita v vsaki paralelki vzorca, | |
| - izračunati povprečje, standardni odmik in koeficient variacije ( %) za te koncentracije, | |
| - izračunati pravilnost z deljenjem zaznane povprečne koncentracije s certificirano vrednostjo (izmerjeno kot koncentracija) in pomnožiti s 100, da se rezultat izrazi kot odstotek. | |
| Pravilnost ( %) = povprečna odkrita koncentracija korigirana z izplenom x 100/certificirana vrednost. | |
| Če CRM ni na voljo, se lahko namesto pravilnosti določi izplen, kakor je opisano pod 4.1.2.1 spodaj. | |
| 3.1.1.3 Uporabljivost/robustnost (manjše spremembe) | |
| Takšne študije uporabljajo premišljeno uvajanje manjših utemeljenih sprememb v laboratoriju in opazovanje njihovih posledic. | |
| Predhodne preiskovalne študije se morajo opraviti z izbiranjem faktorjev v postopkih pred-obdelave, čiščenja in analize vzorca, ki lahko vplivajo na rezultate merjenja.Takšni faktorji lahko vključujejo analitika, izvor in starost reagentov, topil, standardov in ekstraktov vzorca, stopnjo segrevanja, temperaturo, pH kot tudi čim več drugih faktorjev, ki lahko nastopijo v laboratoriju. Ti faktorji se naj bi spreminjali po velikostnem redu, ki se ujema z odmiki, na katere običajno naletimo med laboratoriji. | |
| - Identificirati možne faktorje, ki bi lahko vplivali na rezultate. | |
| - Nekoliko spremeniti vsak faktor. | |
| - Izpeljati preskus robustnosti z uporabo Youdenovega pristopa (15)(16). (Na tej točki se lahko uporabijo druge odobrene metode. Youdenov pristop pa vzame najmanj potrebnega časa in napora). Youdenov pristop je oblika ulomka zmnožkov različnih faktorjev. Interakcije med različnimi faktorji se ne dajo določiti. | |
| - Kjer se ugotovi, da nek faktor znatno vpliva na rezultate merjenja, izvesti nadaljnje eksperimente, da se določijo meje sprejemljivosti tega faktorja. | |
| - Faktorji, ki znatno vplivajo na rezultate, se naj jasno identificirajo v protokolu metode. | |
| Osnovna ideja je, da se naenkrat ne preuči le ena sprememba, temveč da se takoj uvede več sprememb. Kot primer naj A, B, C, D, E, F, G označujejo nominalne vrednosti za sedem različnih faktorjev, ki lahko vplivajo na rezultate, če se njihove nominalne vrednosti nekoliko spremenijo, Naj se njihove alternativne vrednosti označijo z ustreznimi malimi črkami a, b, c, d, e f in g. Rezultat tega je 27 ali 128 različnih možnih kombinacij. | |
| Od teh kombinacij je možno izbrati je podsestav z osmimi kombinacijami, ki imajo ravnotežje med velikimi in malimi črkami (tabela 11). Napraviti je treba osem določitev, ki bodo uporabljale kombinacijo izbranih faktorjev (A-G). Rezultati določitev so prikazani v tabeli 11 spodaj kot S-Z. | |
| Tabela 11 Eksperimentalni vzorec za študije robustnosti (manjše spremembe) | |
| Za izračune glej primere za preskušanje robustnosti v 3.3. | |
| Vrednost faktorja F | Kombinacije števila določitev | | |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | | |
| A/a | A | A | A | A | a | a | a | a | | |
| B/b | B | B | b | b | B | B | b | b | | |
| C/c | C | c | C | c | C | c | C | c | | |
| D/d | D | D | d | d | d | d | D | D | | |
| E/e | E | e | E | e | e | E | e | E | | |
| F/f | F | f | f | F | F | f | f | F | | |
| G/g | G | g | g | G | g | G | G | g | | |
| Opažen rezultat R | S | T | U | V | W | X | Y | Z | | |
| 3.1.1.4 Stabilnost | |
| Opaženo je, da lahko nezadostna stabilnost analita ali sestavin matriksa v vzorcu med skladiščenjem ali analizo povzroči precejšnje odmike v izidu rezultata analize. Nadalje se naj pregleda stabilnost umeritvenega standarda v raztopini. Običajno je stabilnost analita značilna pod različnimi skladiščnimi pogoji. Spremljanje skladiščnega stanja bo sestavljalo del normalnega laboratorijskega akreditacijskega sistema. Kadar to ni znano, so spodaj podani primeri, kako se lahko določi stabilnost. | |
| Stabilnost analita v raztopini: | |
| - Pripraviti svežo zalogo raztopin analita/analitov in raztopiti po navodilih za preskušanje, da se naredi dovolj alikvotov (npr. 40) vsake izbrane koncentracije (okrog meje najmanjše zahtevane učinkovitosti za snovi, za katere ni določena nobena dovoljena meja ali okrog dovoljene meje za druge snovi). Pripraviti obe raztopini analita, uporabljenega za dodajanje in uporabljenega za raztopino končne analize in katero koli drugo raztopino, ki je zanimiva (npr. derivatizirani standardi). | |
| - Izmeriti vsebnost analita v sveže pripravljeni raztopini v skladu s navodili za preskušanje. | |
| - Porazdeliti namenjene volumne v primerne vsebnike, označiti in shraniti v skladu s shemo: | |
| Tabela 12 Shema za določitev stabilnosti analita v raztopini | |
| | - 20 °C | + 4 °C | + 20 °C | | |
| Temno | 10 alikvotov | 10 alikvotov | 10 alikvotov | | |
| Svetlo | | | 10 alikvotov | | |
| - Za čas skladiščenja se lahko izbere en, dva, tri in štiri tedne ali po potrebi več, npr. dokler se ne opazijo prvi pojavi propadanja med identifikacijo in/ali kvantifikacijo. Najdaljši čas skladiščenja in najboljši pogoji skladiščenja se morajo zabeležiti. | |
| - Izračun koncentracije analita(-ov) v vsakem alikvotu se naj opravi z uporabo raztopine sveže pripravljenega analita ob času analize kot 100 %. | |
| Analit, ki ostane ( %) = | |
| Ci × 100/Csveža | |
| Ci = koncentracija ob časovni točki | |
| Csveža = koncentracija sveže raztopine | |
| Stabilnost analita/analitov v matriksu | |
| - Kadar koli je možno, se naj uporabijo nastali vzorci. Če ni nobenega nastalega materiala, se naj uporabi matriks, ojačan z analitom. | |
| - Če je nastali material na razpolago, se naj določi koncentracija v materialu, dokler je še svež. Nadaljnji alikvoti materiala se lahko odvzamejo po enem, dveh, štirih in 20 tednih in določijo se naj koncentracije. Tkivo se naj shrani pri najmanj minus 20 °C ali manj, če je potrebno. | |
| - Če ni na razpolago nobenega nastalega materiala, vzemite nekaj slepega materiala in ga homogenizirajte. Razdelite material na 5 alikvotov. Vsak alikvot ojačajte z analitom, ki naj bi bil po možnosti pripravljen v majhni količini vodne raztopine. En alikvot analizirajte takoj. Ostale alikvote shranite pri najmanj minus 20 °C ali manj, če je potrebno in jih analizirajte po enem, dveh, štirih in 20 tednih. | |
| 3.1.1.5 Umeritvene krivulje | |
| Kadar se za kvantifikacijo uporabljajo umeritvene krivulje: | |
| - se naj za tvorbo krivulje uporabi najmanj pet nivojev (vključno ničelni), | |
| - opiše se naj delovno območje krivulje, | |
| - opiše se naj matematična formula krivulje in ujemanje podatkov, | |
| - opišejo se naj območja sprejemljivosti za parametre krivulje. | |
| Kadar je potrebna serijska umeritev na osnovi standardne raztopine, se označijo sprejemljivi parametri umeritvene krivulje, ki se lahko razlikuje od ene serije do druge. | |
| 3.1.2 Konvencionalni validacijski postopki | |
| Izračun parametrov v skladu s konvencionalnimi metodami zahteva izvedbo več posameznih eksperimentov. Vsaka značilnost učinkovitosti se mora določiti za vsako večjo spremembo (glej pod uporabljivost/robustnost). Za večanalitske metode se lahko preskuša hkrati več analitov, če so prej po možnosti odpravljene ustrezne medsebojne motnje. Na isti način se lahko določi več značilnosti učinkovitosti. Da bi tako čimbolj zmanjšali delovno obremenitev, se priporoča čimveč kombinirati poskuse (npr. ponovljivost in obnovljivost znotraj laboratorija s specifičnostjo, analizo slepih vzorcev za določitev odločitvene mejne vrednosti in preskušanje glede specifičnosti). | |
| 3.1.2.1 Izplen | |
| Če CRM ni na razpolago, se mora izplen določiti s poskusi z ojačanim slepim matriksom z uporabo npr. naslednje sheme: | |
| - izberite 18 alikvotov slepega materiala in dodajte vsakič šestim alikvotom preiskani analit v enkratni, 1,5-kratni in dvakratni koncentraciji meje najmanjše zahtevane učinkovitosti ali polovični, enkratni in 1,5-kratni koncentraciji dovoljene meje, | |
| - analizirajte vzorce in izračunajte koncentracijo, ki je prisotna v vsakem vzorcu, | |
| - z uporabo spodnje enačbe izračunajte izplen za vsak vzorec, | |
| - izračunajte povprečen izplen in CV za šest rezultatov z vsakega nivoja, | |
| - % izplena = 100 x izmerjena vsebnost/nivo ojačanja. | |
| Ta konvencionalna metoda za določitev izplena je varianta metode standardnega dodajanja, ki je opisana pod 3.5 kadar: | |
| - se vzorec šteje kot slepi vzorec namesto vzorca, ki ga je treba analizirati, | |
| - se šteje, da sta si izkoristek [2] in izplen [3] podobna za dva preskušana deleža, | |
| - imajo preskušani vzorci iste mase in ekstrakti preskušanih deležev iste volumne, | |
| - je količina umeritvenega standarda, ki se doda drugemu (z dodatkom) preskušanemu deležu zapisana xADD. (xADD = rA.VA), | |
| - je x1 izmerjena vrednost za slepi delež in x2 izmerjena vrednost za drugi (z dodatkom) preskušani delež, | |
| - tedaj je % izplena = 100 (x2 – x1)/x ADD. | |
| Kadar kateri koli od zgornjih pogojev ne (ali se domneva, da ne) bo dosežen, tedaj je treba izvesti celoten postopek za določitev izplena po metodi standardnega dodajanja, kakor je opisano pod 3.5. | |
| 3.1.2.2 Ponovljivost | |
| - Pripravite komplet vzorcev z enakimi matriksi, ojačanimi z analitom, da se z upoštevanjem izkoristka v preskušanem deležu dobijo koncentracije, ki so ekvivalentne enkratni 1,5-kratni in dvakratni najmanjši zahtevani meji učinkovitosti ali 0,5-kratni, enkratni in 1,5-kratni dovoljeni meji. | |
| - Na vsakem nivoju se naj analiza izvede z najmanj šestimi paralelkami. | |
| - Analizirajte vzorce. | |
| - Analizirajte koncentracijo, ki ste jo odkrili v vsakem vzorcu. | |
| - Ugotovite povprečno koncentracijo, standardni odmik in koeficient variacije ( %) ojačanih vzorcev. | |
| - Ponovite te korake še najmanj dvakrat. | |
| - Izračunajte celotno povprečno koncentracijo in CV za ojačane vzorce. | |
| 3.1.2.3 Obnovljivost v laboratoriju | |
| - Pripravite komplet vzorcev z določenim preskušanim materialom (enaki ali različni matriksi), ojačanim z analitom/analiti, da se z upoštevanjem izkoristka v preskušanem deležu dobijo koncentracije, ki so ekvivalentne enkratni 1,5-kratni in dvakratni meji najmanjše zahtevane učinkovitosti ali 0,5-kratni, enkratni in 1,5-kratni dovoljeni meji. | |
| - Na vsakem nivoju se naj analiza izvede z najmanj šestimi paralelkami. | |
| - Ponovite te korake še najmanj dvakrat z drugimi operaterji in v drugačnih pogojih okolja, npr. različne serije reagentov, raztopin, itd., različnimi temperaturami okolja, različnimi instrumenti itd., če je mogoče. | |
| - Analizirajte vzorce. | |
| - Analizirajte koncentracijo, ki ste jo odkrili v vsakem vzorcu. | |
| - Ugotovite povprečno koncentracijo, standardni odmik in koeficient variacije ( %) ojačanih vzorcev. | |
| 3.1.2.4 Obnovljivost | |
| Kadar je treba preveriti obnovljivost, naj bi se laboratoriji udeležili sodelovalnih študij v skladu z ISO 5725-2(5). | |
| 3.1.2.5 Odločitvena meja (CCα) | |
| Odločitveno mejo je treba ugotoviti v skladu z zahtevami za identifikacijo ali identifikacijo plus kvantifikacijo, kakor je opredeljeno v "Merilih učinkovitosti in drugih zahtevah za analitske metode" (del 2). | |
| V primeru snovi, za katere se ni določila nobena dovoljena meja, se lahko CCa določi: | |
| - ali s postopkom umeritvene krivulje v skladu z ISO 11843 (17) (v tem dokumentu navedena kot kritična vrednost spremenljivke čistega stanja). V tem primeru se uporabi slepi material, ki se ojača na in nad najmanjšim zahtevanim nivojem delovanja v ekvidistančnih korakih. Analizirajte vzorce. Po identifikaciji zabeležite signal proti dodani koncentraciji. Ustrezna koncentracija pri y-intercept črta plus, 2,33-krat standardno odstopanje obnovljivosti znotraj laboratorija intercept je enaka odločitveni meji. To je uporabljivo samo pri kvantitativnih preskusih (α = 1 %), | |
| - ali z analiziranjem najmanj 20 slepih materialov na matriks, da se lahko izračuna razmerje signal/šum pri časovnem okencu, v katerem se pričakuje analit. Trikratno razmerje šum/signal se lahko uporabi kot odločitvena meja. To je uporabljivo pri kvantitativnih in kvalitativnih preskusih. | |
| V primeru snovi z določeno dovoljeno mejo se CCα ugotovi: | |
| - ali s postopkom umeritvene krivulje v skladu z ISO 11843 (17) (v tem dokumentu naveden kot kritična vrednost spremenljivke čistega stanja). V tem primeru se uporabi slepi material, ki se ojača okrog dovoljene meje v ekvidistančnih korakih. Analizirajte vzorce. Po identifikaciji, zabeležite signal proti dodani koncentraciji. Ustrezna koncentracija pri dovoljeni meji plus 1,64-krat standardni odmik obnovljivosti znotraj laboratorija je enaka odločitveni meji (α = 5 %.), | |
| - ali z analiziranjem najmanj 20 slepih materialov na matriks, ojačanim z analitom/analiti ob dovoljeni mejni vrednosti. Koncentracija pri dovoljeni meji plus 1,64-krat ustrezen standardni odmik je enaka odločitveni meji (α = 5 %). | |
| Glej tudi člen 5 in točko 3.2. | |
| 3.1.2.6 Sposobnost detekcije (CCβ) | |
| Sposobnost detekcije se naj določa v skladu s zahtevami za pregledovanje, identifikacijo ali identifikacijo plus kvantifikacijo, kakor je določeno (glej del 2). | |
| V primerih snovi, za katere ni določena nobena dovoljena meja, se sposobnost detekcije določi: | |
| - s postopkom umeritvene krivulje v skladu z ISO 11843 (17) (tu navedeno kot najmanjša detekcijska vrednost spremenljivke čistega stanja). V tem primeru se uporabi reprezentativni slepi material, ki se ojača na ali pod nivo najmanjšega zahtevanega delovanja v ekvidistančnih korakih. Analizirajte vzorce. Po identifikaciji, zabeležite signal proti dodani koncentraciji. Ustrezna koncentracija pri določitveni meji plus 1,64-krat standardni odmik laboratorijske obnovljivosti povprečne izmerjene vsebnosti pri odločitveni meji je enaka sposobnosti detekcije (β = 5 %), | |
| - z analiziranjem najmanj 20 slepih materialov na matriks, ojačanim z analitom/analiti na odločitveni meji. Analizirajte vzorce in identificirajte analite. Vrednost odločitvene meje plus 1,64-krat standardni odmik laboratorijske obnovljivosti izmerjene vsebnosti je enaka sposobnosti detekcije (β = 5 %), | |
| - ko ni na razpolago nobenih kvantitativnih rezultatov, se lahko sposobnost detekcije določi s preiskavo ojačanega slepega materiala pri in nad odločitveno mejo. V tem primeru je nivo koncentracije, kjer ostane le £ 5 % lažnih skladnih rezultatov, enak sposobnosti detekcije te metode. Zato je treba izvesti najmanj 20 preiskav za najmanj en nivo koncentracije, da se zagotovi zanesljiva osnova za to določitev. | |
| V primeru snovi, za katere je določena dovoljena meja, se CCb lahko določi: | |
| - ali s postopkom umeritvene krivulje v skladu z ISO 11843 (17) (tu navedeno kot najmanjša detekcijska vrednost spremenljivke čistega stanja). V tem primeru se uporabi reprezentativni slepi material, ki se ojača okrog dovoljene meje v ekvidistančnih korakih. Analizirajte vzorce in identificirajte analit(-e). Izračunajte standardni odmik s povprečno izmerjeno vsebnostjo pri odločitveni mejni vrednosti. Ustrezna koncentracija pri vrednosti odločitvene meje plus 1,64-krat standardni odmik laboratorijske obnovljivosti je enaka sposobnosti detekcije (β= 5 %), | |
| - ali z analiziranjem najmanj 20 slepih materialov na matriks, ojačanim z analitom/analiti ob odločitveni meji. Vrednost odločitvene meje plus 1,64-krat ustrezni standardni odmik je enaka sposobnosti detekcije (β = 5 %). | |
| Glej tudi oddelek 3.2. | |
| 3.1.2.7 Robustnost (večje spremembe) | |
| Analitska metoda se naj preskusi pod različnimi poskusnimi pogoji, ki vključujejo npr. različne vrste, različne matrikse ali različne pogoje vzorčenja. Uvedene spremembe naj bodo večje. Pomen teh sprememb se npr. lahko ovrednoti npr. po Youdenovem pristopu (15)(16). Vsaka značilnost učinkovitosti se naj določi za vse večje spremembe, ki so pokazale, da imajo pomemben vpliv na izvedljivost preskusa. | |
| 3.1.3 Validacija po alternativnih modelih | |
| Kadar se uporabijo postopki alternativne validacije, se v validacijskem protokolu določita osnovni model in strategija z vsakokratnimi predpogoji, predpostavkami in formulami ali pa se vsaj podajo reference o njihovi razpoložljivosti. V nadaljevanju je podan primer alternativne metode. Pri uporabi npr. internega laboratorijskega modela validacije, se določijo značilnosti učinkovitosti na način, ki dovoljuje validacijo za večje spremembe v okviru istega validacijskega postopka. Za to je potrebna priprava poskusnega načrta za validacijo. | |
| 3.1.3.1 Poskusni načrt | |
| Poskusni načrt mora biti izdelan glede na število različnih vrst in različnih faktorjev v preiskavi. Zato prvi korak v celotnem postopku validacije upošteva vzorčne populacije, ki bodo v bodoče analizirane v laboratoriju z namenom, da se izbere najpomembnejša vrsta in tisti faktorji, ki lahko vplivajo na rezultate meritev. Nato se izbere območje koncentracije, ki je prilagojeno namenu v skladu z iskanim nivojem. | |
| Primer: | |
| - istočasno se lahko pregleda več analitov z analitsko metodo, ki je validirana, | |
| - identificirani sta dve variaciji (A in B) vodilnega faktorja.Vodilni faktorji tvorijo osnovo, na kateri se kombinirajo nivoji faktorjev. Ti vodilni faktorji lahko vključujejo faktorje kot so vrsta ali matriks. V tem primeru je bil vodilni faktor spremenjen na dveh nivojih, t.j. upoštevali sta se dve različni vrsti (vrsti A in B). Na splošno je mogoče menjati vodilne faktorje na več kot dveh nivojih, kar samo poveča število analiz, ki jih je treba izvesti, | |
| - izbrane faktorje je treba menjati na dveh nivojih (označenih kot + ali -). | |
| Tabela 13 Primeri za faktorje, ki veljajo kot pomembni za validacijski postopek | |
| Spol živali | (faktor 1) | | |
| Pasma | (faktor 2) | | |
| Transportni pogoji | (faktor 3) | | |
| Pogoji shranjevanja | (faktor 4) | | |
| Svežost vzorca | (faktor 5) | | |
| Pogoji pitanja | (faktor 6) | | |
| Različni delavci z različnimi izkušnjami | (faktor 7). | | |
| Tabela 14 Možni poskusni načrt za gornji primer | |
| Vrsta | Faktor 1 | Faktor 2 | Faktor 3 | Faktor 4 | Faktor 5 | Faktor 6 | Faktor 7 | Vzorec št. | | |
| A | + | + | + | + | – | + | – | 1 | | |
| A | + | + | – | – | + | – | – | 2 | | |
| A | + | – | + | – | – | – | + | 3 | | |
| A | + | – | – | + | + | + | + | 4 | | |
| A | – | + | + | – | + | + | + | 5 | | |
| A | – | + | – | + | – | – | + | 6 | | |
| A | – | – | + | + | + | – | – | 7 | | |
| A | – | – | – | – | – | + | – | 8 | | |
| B | + | + | + | + | + | – | + | 9 | | |
| B | + | + | – | – | – | + | + | 10 | | |
| B | + | – | + | – | + | + | – | 11 | | |
| B | + | – | – | + | – | – | – | 12 | | |
| B | – | + | + | – | – | – | – | 13 | | |
| B | – | + | – | + | + | + | – | 14 | | |
| B | – | – | + | + | – | + | + | 15 | | |
| B | – | – | – | – | + | – | + | 16 | | |
| Ker je treba vsakemu vzorcu (vsaki kombinaciji faktorskih nivojev) primešati štiri različne koncentracije okrog iskanega nivoja, in se za vsak nivo analizira slepi vzorec, mora biti izvedenih 5 x 16 = 80 analiz za celotni validacijski poskus. | |
| Od teh 80 merilnih rezultatov, je možno izračunati (13) (14). | |
| Izplen | |
| - ponovljivost na nivo koncentracije (sir), | |
| - znotraj laboratorijska obnovljivost na nivo koncentracije (sir), | |
| - odločitvena meja (CCα), | |
| - sposobnost detekcije (CCβ), | |
| - potenčna krivulja (stopnja b-napake proti koncentraciji (glej 3.1.3.2), | |
| - robustnost večjih sprememb: robustnost na manjše spremembe se da določiti v skladu z odstavkom 3.1.1.3), | |
| - 16 umeritvenih krivulj, vezanih na vzorce, | |
| - ena krivulja za celotno umeritev, | |
| - razmik predvidevanja celotne umeritvene krivulje, | |
| - odmiki, povzročeni z matriksom (smat), | |
| - odmiki, povzročeni zaradi poteka (srun), | |
| - učinek posameznih faktorjev na merilne rezultate. | |
| Te značilnosti učinkovitosti omogočajo vsestransko ovrednotenje nivoja učinkovitosti metode, ker se ne preiskuje samo vpliv posameznih faktorjev, temveč tudi povezane kombinacije teh faktorjev. S pomočjo tega poskusnega modela se je možno odločiti, če se en ali drugi od izbranih faktorjev izključi iz celotne umeritvene krivulje, ker pomembno odstopa od standardnih odmikov drugih faktorjev. | |
| 3.1.3.2 Potenčna krivulja | |
| Krivulja zmogljivosti podaja informacije o detekcijski sposobnosti te metode v okviru izbranega koncentracijskega območja. Nanaša se na tveganja b-napake, pri uporabi preiskovane metode. Krivulja zmogljivosti omogoča izračun detekcijskih sposobnosti za vsako od posameznih kategorij (presejanje, potrditev) ali vrst (kvalitativnih ali kvantitativnih) metod za določeno b-napako (npr. 5 %). | |
| +++++ TIFF +++++ | |
| Slika 1 prikazuje primer grafičnega ugotavljanja sposobnosti detekcije (CCβ) analitske metode. Pri tej posebni metodi ostane tveganje napačnega odločanja 5 % pri koncentraciji 0,50 mg/kg. Pri koncentraciji 0,55 mg/kg se tveganje lažnega skladnega odločanja zniža na 1 %. | |
| 3.1.3.3 Obnovljivost | |
| Določanje obnovljivosti metode po konceptu laboratorijskih študij v enem laboratoriju (interne validacije) zahteva ponovljeno udeležbo v strokovnih študijah v skladu z ISO vodili 43-1 (3) in 43-2 (4). Laboratorijem je dovoljeno, da izberejo svoje lastne metode pod pogojem, da se te metode uporabljajo pod rutinskimi pogoji. Standardni odmik laboratorija se lahko uporabi za oceno obnovljivosti te metode. | |
| 3.2 GRAFIČNA PREDSTAVITEV RAZLIČNIH ANALITSKIH MEJ | |
| +++++ TIFF +++++ | |
| +++++ TIFF +++++ | |
| 3.3 PRIMER IZRAČUNA ZA PRESKUŠANJE ROBUSTNOSTI MANJŠIH SPREMEMB V SKLADU Z YOUDENOVIM PRISTOPOM (16) | |
| Primerjava povprečij (A) | |
| AAΣ(Ai)/4ABΣ(Bi)/4ACΣ(Ci)/4ADΣ(Di)/4AEΣ(Ei)/4AFΣ(Ei)/4AGΣ(Gi)/4AaΣ(ai)/4AbΣ(bi)/4AcΣ(ci)/4AdΣ(di)/4AeΣ(ei)/4AfΣ(fi)/4AgΣ(gi)/4 | Primerjajte povprečja velikih črk (AA do AG) s povprečji njihovih ustreznih malih črk (Aa do Ag). Če ima faktor učinek, bo razlika znatno večja, kot so razlike drugih faktorjev. Na robustno metodo naj ne bi vplivale spremembe, na katere skoraj z gotovostjo naletimo med laboratoriji. Če ni nobene izrazite razlike, se najbolj realistična mera naključne napake poda s sedmimi razlikami. | | |
| Razlike (Di) | Kvadratne razlike (Di2) | | |
| DaA – a = Σ(Ai) -Σ (ai)DbB – b = Σ(Bi) - Σ(bi)DcC – c = Σ(Ci) -Σ (ci)DdD – d = Σ(Di) -Σ (di)DeE – e = Σ(Ei) - Σ(ei)DfF – f = Σ(Fi) -Σ (fi)DgG – g = Σ(Gi) - Σ(gi) | Da2vrednost aDb2vrednost bDc2vrednost cDd2vrednost dDe2vrednost eDf2vrednost fDg2vrednost g | | |
| Primerjava povprečij (A) | |
| +++++ TIFF +++++ | |
| Kadar je SDi znatno večji od standardnega odmika metode, izvedene pod pogoji obnovljivosti znotraj laboratorija v skladu z (glej zgoraj), je očitno, da imajo vsi faktorji skupaj učinek na rezultat, četudi vsak posamezni faktor ne kaže znatnega vpliva in da metoda ni dovolj robustna za izbrane spremembe. | |
| 3.4 PRIMERI IZRAČUNA ZA INTERNI VALIDACIJSKI POSTOPEK | |
| Primeri izračunov za protokol interne validacije, kakor je opisan z validacijo v skladu z alternativnimi modeli (3.1.3) (13) (14) | |
| 3.5 PRIMERI ZA METODO STANDARDNEGA DODATKA | |
| Preskušani vzorec z vsebnostjo T analita se razdeli na dva preskušana deleža 1 in 2, vsak s svojo maso m1 in m2. Preskušanemu deležu 2 je primešan volumen VA raztopine s koncentracijo rA analita. Dva ekstrakta preskušanih deležev se dobita z odnosnima volumnoma V1 in V2 po ekstrakcijskih in očiščevalnih korakih metode. Predvideva se izplen analita rc. Oba ekstrakta se preskusita z merilno metodo z občutljivostjo b in podata analitski odziv x1 ali x2. | |
| Če se domneva, da sta rc in b ista za analit v prvotnem vzorcu in v primešanem vzorcu, tedaj se vsebnost T lahko izračuna kot: | |
| T = | |
| x | |
| · V | |
| · m | |
| - V | |
| · m | |
| Metoda bo omogočila določitev izplena rc. Nato se dodatno k zgoraj opisanim preskusom del ekstrakta preskušanega deleža 1 (volumen V3) zmeša z znano količino ρB.VB analita in preskusi. Analitski odziv je x3 in izplen je: | |
| rc = | |
| x | |
| · V | |
| · V | |
| - x | |
| · V | |
| · T · m | |
| Nadalje je možno izračunati občutljivost b kot: | |
| b = | |
| Vsi pogoji aplikacije in podrobnosti so predhodno opisane (18). | |
| 4. UPORABLJENE KRATICE | |
| AAS atomska absorpcijska spektrometrija | |
| AES atomska emisijska spektrometrija | |
| AOAC-1 Zdruenje uradnih analitskih kemikov INTERNATIONAL | |
| B vezana frakcija | |
| CI kemična ionizacija | |
| CRM certificiran referenčni material | |
| CV koeficient variacije | |
| 2 D dvodimenzionalni | |
| DAD detekcija z diodnim nizom | |
| DPASV diferenčna impulzna anodna stripping voltametrija | |
| ECD detekcija z zajetjem elektronov | |
| EI elektronska (vplivana) ionizacija | |
| GC plinska kromatografija | |
| HPLC tekočinska kromatografija visoke ločljivosti | |
| HPTLC tankoplastna kromatografija visoke ločljivosti | |
| HRMS masna spektrometrija visoke ločljivosti | |
| ICP-AES induktivno sklopljena plazma – atomska emisijska spektrometrija | |
| ICP-MS induktivno sklopljena plazma – masna spektrometrija | |
| IR infrardeči | |
| ISO Mednarodna organizacija za standardizacijo | |
| LC tekočinska kromatografija | |
| LR(MS) masna spektrometrija nizke ločljivosti | |
| MRPL meja najmanjše zahtevane učinkovitosti | |
| MS masna spektrometrija | |
| m/z razmerje masa/naboj | |
| RF relativna migracija na fronto topila | |
| RSDL relativni standardni odmiki v laboratoriju | |
| SIM monitoring/spremljanje izbranega iona | |
| TLC tankoplastna kromatografija | |
| UV ultravijolična svetloba | |
| VIS vidna svetloba | |
| 5. NAPOTILA | |
| (1) ISO 17025: 1999 Splošne zahteve za pristojnost umerjevalnih in preskuševalnih laboratorijev. | |
| (2) ISO 3543-1: 1993 Statistične metode za obvladovanje kakovosti – Zvezek 1 slovar in simboli. | |
| (3) ISO vodilo 43-1: 1997 Strokovno preskušanje z medlaboratorijskimi primerjavami – Del 1: Razvoj in izvajanje strokovnih preskuševalnih programov | |
| (4) ISO vodilo 43-2: 1997 Strokovno preskušanje z medlaboratorijskimi primerjavami – Del 2: Selekcija in uporaba strokovnih preskuševalnih programov pri organih za akreditacijo laboratorijev | |
| (5) ISO 5725: 1994 Točnost (pravilnost in natančnost) merilnih metod in rezultatov – Del 1: Splošna načela in definicije; ISO 5725-2 Del 2: Osnovna metoda za določevanje ponovljivosti in obnovljivosti standardne merilne metode; Del 4: Osnovne metode za določanje pravilnosti standardne merilne metode. | |
| (6) ISO 78-2: 1999 Kemija –Načrti za standarde – Del 2: Metode kemične analize. | |
| (7) W.G. de Ruig in J.M.Weseman: "A new approach to confirmation by infrared spectrometry" J. chemometrics 4 (1990) 61-77. | |
| (8) Glej npr. May, T.W., Brumbaugh, W.G., Matrix modifier and L'vov platform for elimination of matrix interferences in the analysis of fish tissues for lead by graphite furnace atomic absorption spectrometry: Analytical Chemistry 54(7): 1032-1037 (90353). | |
| (9) Application of Zeeman Graphite Furnace Atomic Absorption Spectrometry in the Chemical Laboratory and in Toxicology, C. Minoia, S. Caroli (Eds). Pergamon Press (Oxford), 1992, str. xxvi + 675. | |
| (10) Inductively Coupled Plasmas in analytical Atomic Spectrometry, A. Montaser, D. W. Golighty (Eds.), VCH Publishers, Inc. (New York), 1992. | |
| (11) Plasma Source Mass Spectrometry Developments and Applications, G. Holland, S. D. Tanner (Eds.), The Royal Society of Chemistry, 1997, str. 329. | |
| (12) IUPAC (1995), Protocol for he design, conduct and interpretation of method-performance studies, Pure & Applied Chem. 67, 331. | |
| (13) Jülicher, B., Gowik, P. and Uhlig, S (1998) Assessment of detection methods in trace analysis by means of a statistically based in-house validation concept. Analyst, 120, 173. | |
| (14) Gowik P., Jülicher, B., and Uhlig, S (1998) Multi-residue method for non-steroidal anti-inflammatory drugs in plasma using high performance liquid chromatography-photodiode –array detection. Method description and comprehensive in-house validation. J. Chromatogr., 716, 221. | |
| (15) OAC-I Per Verified Methods, Policies and Procedures, 1993, AOAC International, 2200 Wilson Blvd., Suite 400, Arlington, Virginia 22201-3301, USA. | |
| (16) W. J. Youden; Steiner, E. H.; "Statistical Manual of the AOAC-Association of Official Analytical Chemists", AOAC-I, Washington DC: 1975, str. 35 ff. | |
| (17) ISO 11843: 1997 Sposobnost detekcije – Del 1: Termini in definicije, Del 2: Metodologija v primeru linearne umeritve. | |
| (18) R.W. Stephany & L.A. van Ginkel: "Yield of recovery: a world of difference". Proceedings Eight Euro Food Chem, Vienna, Austria Septembre 18-20 (1995) Federation of European Chemical Societies, Event 206. ISBN 3-900554-17X, str. 2 do 9. | |
| (19) (19 Directive 71/354/EEC of 18 October 1971 on the approximation of the laws of the Member States relating to units of measurement, OJ L 243, 29. 10. 1971. str. 29). | |
| (20) ISO 31-0: 1992 Količine in enote – Del 0: Splošna načela. | |
| [1] Za masne deleže, ki so nižji od 100 mg/kg, daje uporaba Horwitzove enačbe nesprejemljivo visoke vrednosti. Zato so CV za koncentracije, nižje od 100 mg/kg, čim nižje. | |
| [2] Izkoristek: tisti delež mase analita, vsebovanega v vzorcu, ki je prisoten v končnem ekstraktu. | |
| [3] Izplen (tukaj): tisti delež mase analita, dodanega v vzorec, ki je prisoten v končnem ekstraktu.. V nadaljnjem tekstu dokumenta se dopušča, da sta izkoristek in izplen enaka in se zato uporablja le izraz "izplen". | |
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