ANNEX VU.K.METHODS OF ANALYSIS TO CONTROL UNDESIRABLE SUBSTANCES IN FEED
[B. DETERMINATION OF THE LEVELS OF DIOXINS (PCDD/PCDF) AND PCBs U.K.
CHAPTER II U.K. Sample preparation and requirements for methods of analysis used in offical control of the levels of dioxins (PCDD/Fs) and dioxin-like PCBs in feed
5. Basic requirements to be met by analytical procedure for dioxins (PCDD/Fs) and dioxin-like PCBs U.K.
5.1. Low working range and limits of quantification U.K.
For PCDD/Fs, detectable quantities shall be in the upper femtogram (10 – 15 g) range because of extreme toxicity of some of these compounds. For most PCB congeners a limit of quantification in the nanogram (10 – 9 g) range is already sufficient. For the measurement of the more toxic dioxin-like PCB congeners (in particular non-ortho-substituted congeners), the lower end of the working range shall reach the low picogram (10 – 12 g) levels. For all other PCB congeners a limit of quantification in the nanogram (10 – 9 g) range is sufficient.
5.2. High selectivity (specificity) U.K.
5.2.1.A distinction is required between PCDD/Fs and dioxin-like PCBs and a multitude of other, coextracted and possibly interfering compounds present at concentrations up to several orders of magnitude higher than those of the analytes of interest. For GC-MS methods, a differentiation among various congeners is required, such as between toxic (for example, the seventeen 2,3,7,8-substituted PCDD/Fs, and twelve dioxin-like PCBs) and other congeners.U.K.
5.2.2.Bioanalytical methods shall be able to detect the target compounds as the sum of PCDD/Fs, and/or dioxin-like PCBs. Sample clean-up shall aim at removing compounds causing false non-compliant results or compounds that may decrease the response, causing false compliant results.U.K.
5.3. High accuracy (trueness and precision, bioassay apparent recovery) U.K.
5.3.1.For GC-MS methods, the determination shall provide a valid estimate of the true concentration in a sample. High accuracy is required to avoid the rejection of a sample analysis result on the basis of poor reliability of the determined TEQ level. Accuracy is expressed as trueness (difference between the mean value measured for an analyte in a certified material and its certified value, expressed as a percentage of this value) and precision (RSD R relative standard deviation calculated from results generated under reproducibility conditions).U.K.
5.3.2.For bioanalytical methods, the bioassay apparent recovery shall be determined. Bioassay apparent recovery means the BEQ level calculated from the TCDD or PCB 126 calibration curve corrected for the blank and then divided by the TEQ level determined by the confirmatory method. It aims at correcting factors like the loss of PCDD/Fs and dioxin-like compounds during the extraction and clean-up steps, co-extracted compounds increasing or decreasing the response (agonistic and antagonistic effects), the quality of the curve fit, or differences between the TEF values and the Relative Potency (REP) values. The bioassay apparent recovery is calculated from suitable reference samples with representative congener patterns around the level of interest.U.K.
5.4. Validation in the range of maximum level and general quality control measures U.K.
5.4.1.Laboratories shall demonstrate the performance of a method in the range of the maximum level, for example, 0,5x, 1x and 2x the maximum level with an acceptable coefficient of variation for repeated analysis, during the validation procedure and during routine analysis.U.K.
5.4.2.Regular blank controls and spiking experiments or analysis of control samples (preferably, if available, certified reference material) shall be performed as internal quality control measures. Quality control charts for blank controls, spiking experiments or analysis of control samples shall be recorded and checked to make sure the analytical performance is in accordance with the requirements.U.K.
5.5. Limit of quantification U.K.
5.5.1.For a bioanalytical screening method, the establishment of the limit of quantification (LOQ) is not an indispensable requirement but the method shall prove that it can differentiate between the blank and the cut-off value. When providing a BEQ level, a reporting level shall be established to deal with samples showing a response below this level. The reporting level shall be demonstrated to be different from procedure blank samples at least by a factor of three, with a response below the working range. It shall therefore be calculated from samples containing the target compounds around the required minimum level, and not from an S/N ratio or an assay blank.U.K.
5.5.2.The LOQ for a confirmatory method shall be about one fifth of the maximum level.U.K.
5.6. Analytical criteria U.K.
For reliable results from confirmatory or screening methods, the following criteria shall be met in the range of the maximum level for the TEQ or BEQ value, respectively, whether determined as total TEQ or total BEQ (as the sum of PCDD/Fs and dioxin-like PCBs) or separately for PCDD/Fs and dioxin-like PCBs:
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| Screening with bioanalytical or physico-chemical methods | Confirmatory methods |
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| False-compliant rate | < 5 % | |
| Trueness | | – 20 % to + 20 % |
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| Repeatability (RSD r ) | < 20 % | |
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| Intermediate precision (RSD R ) | < 25 % | < 15 % |
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5.7. Specific requirements for screening methods U.K.
5.7.1.Both GC-MS and bioanalytical methods may be used for screening. For GC-MS methods the requirements laid down in point 6 shall be met. For cell based bioanalytical methods specific requirements are laid down in point 7.U.K.
5.7.2.Laboratories applying screening methods for the routine control of samples shall establish a close cooperation with laboratories applying the confirmatory method.U.K.
5.7.3.Performance verification of the screening method is required during routine analysis, by analytical quality control and on-going method validation. There shall be a continuous programme for the control of compliant results.U.K.
5.7.4.Check on possible suppression of the cell response and cytotoxicity:U.K.
20 % of the sample extracts shall be measured in routine screening without and with 2,3,7,8-TCDD added corresponding to the maximum level or action threshold, to check if the response is possibly suppressed by interfering substances present in the sample extract. The measured concentration of the spiked sample shall be compared to the sum of the concentration of the unspiked extract plus the spiking concentration. If this measured concentration is more than 25 % lower than the calculated (sum) concentration, this is an indication of potential signal suppression and the respective sample shall be submitted to GC-HRMS confirmatory analysis. Results shall be monitored in quality control charts.
5.7.5.Quality control on compliant samples:U.K.
Approximately 2 to 10 % of the compliant samples, depending on sample matrix and laboratory experience, shall be confirmed by GC/HRMS.
5.7.6.Determination of false-compliant rates from quality control data:U.K.
The rate of false-compliant results from screening of samples below and above the maximum level or the action threshold shall be determined. Actual false-compliant rates shall be below 5 %. When a minimum of 20 confirmed results per matrix/matrix group is available from the quality control of compliant samples, conclusions on the false compliant rate shall be drawn from this database. The results from samples analysed in ring trials or during contamination incidents, covering a concentration range up to for example 2x the maximum level (ML), may also be included in the minimum of 20 results for evaluation of the false-compliant rate. The samples shall cover most frequent congener patterns, representing various sources.
Although screening assays shall preferentially aim to detect samples exceeding the action threshold, the criterion for determining false-compliant rates is the maximum level, taking into account the expanded measurement uncertainty of the confirmatory method.
5.7.7.Potential non-compliant samples from screening shall always be verified by a full re-analysis of the original sample by a confirmatory method of analysis. These samples may also be used to evaluate the rate of false non-compliant results. For screening methods, the rate of false non-compliant results shall be the fraction of results confirmed to be compliant from confirmatory analysis, while in previous screening the sample has been declared to be potentially non-compliant. Evaluation of the advantages of the screening method shall be based on comparison of false-non-compliant samples with the total number of samples checked. This rate shall be low enough to make the use of a screening tool advantageous.U.K.
5.7.8.Under validation conditions, bioanalytical methods shall provide a valid indication of the TEQ level, calculated and expressed as BEQ.U.K.
Also for bioanalytical methods carried out under repeated conditions, the intra-laboratory RSD r would typically be smaller than under reproducibility conditions(RSD R )]