performance test

 sumber : COMMISSION DECISION 2002/657/EC

 

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 % (β-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

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

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: CV = 2(1 – 0,5 log C)
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

 

 

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 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 10 000 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.

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 atleastone 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