Commission Decisionof 12 August 2002implementing 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)

2.3. U.K. 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

Measuring technique	Substances Annex 1 96/23/EC	Limitations
LC or GC with mass-spectrometric detection	Groups A and B	Only if following either an on-line or an off-line chromatographic separation
		Only if full scan techniques are used or using atleast3 (group B) or 4 (group A) identification points for techniques that do not record the full mass spectra
LC or GC with IR spectrometric detection	Groups A and B	Specific requirements for absorption in IR spectrometry have to be met
LC-full-scan DAD	Group B	Specific requirements for absorption in UV spectrometry have to be met
LC -fluorescence	Group B	Only for molecules that exhibit native fluorescence and to molecules that exhibit fluorescence after either transformation or derivatisation
2-D TLC - full-scan UV/VIS	Group B	Two-dimensional HPTLC and co-chromatography are mandatory
GC-Elektron capture detection	Group B	Only if two columns of different polarity are used
LC-immunogram	Group B	Only if at least two different chromatographic systems or a second, independent detection method are used
LC-UV/VIS (single wavelength)	Group B	Only if at least two different chromatographic systems or second, independent detection method are used.

2.3.1 Common performance criteria and requirements U.K.

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 U.K.

2.3.2.1 Trueness of quantitative methods U.K.

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

Mass fraction	Range
≤ 1 μg/kg	– 50 % to + 20 %
> 1 μg/kg to 10 μg/kg	– 30 % to + 10 %
≥ 10 μg/kg	– 20 % to + 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 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 U.K.

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:

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

Mass fraction	Reproducibility CV(%)
(*) For mass fractions lower than 100 μg/kg the application of the Horwitz Equation gives unacceptable high values. Therefore, the CVs for concentrations lower than 100 μg/kg shall be as low as possible.
1 μg/kg	(*)
10 μg/kg	(*)
100 μg/kg	23
1 000 μg/kg (1 mg/kg)	16

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 U.K.

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 U.K.

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 U.K.

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

Relative intensity (% of base peak)	EI-GC-MS (relative)	CI-GC-MS, GC-MSn LC-MS, LC-MSn (relative)
> 50 %	± 10 %	± 20 %
> 20 % to 50 %	± 15 %	± 25 %
>10 % to 20 %	± 20 %	± 30 %
≤ 10 %	± 50 %	± 50 %

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

MS technique	Identification points earned per ion
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.
Low resolution mass spectrometry (LR)	1,0
LR-MSn precursor ion	1,0
LR-MSn transition products	1,5
HRMS	2,0
HR- MSn precursor ion	2,0
HR-MSn transition products	2,5

Table 6

Examples of the number of identification points earned for a range of techniques and combinations thereof (n = an integer)

Technique(s)	Number of ions	Identification points
GC-MS (EI or CI)	N	n
GC-MS (EI and CI)	2 (EI) + 2 (CI)	4
GC-MS (EI or CI) 2 derivatives	2 (Derivative A) + 2 (Derivative B)	4
LC-MS	N	n
GC-MS-MS	1 precursor and 2 daughters	4
LC-MS-MS	1 precursor and 2 daughters	4
GC-MS-MS	2 precursor ions, each with 1 daughter	5
LC-MS-MS	2 precursor ions, each with 1 daughter	5
LC-MS-MS-MS	1 precursor, 1 daughter and 2 granddaughters	5,5
HRMS	N	2 n
GC-MS and LC-MS	2 + 2	4
GC-MS and HRMS	2 + 1	4

2.3.4 Performance criteria and other requirements for chromatography coupled to infrared detection U.K.

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 U.K.

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 U.K.

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 U.K.

2.3.5.1 Chromatographic separation U.K.

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 U.K.

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 U.K.

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 U.K.

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) U.K.

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 U.K.

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) U.K.

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.