ANNEX IVSupplementary provisions
ANNEX IV SAMPLE PREPARATION AND REQUIREMENTS FOR METHODS OF ANALYSIS USED IN CONTROL OF THE LEVELS OF NON-DIOXIN-LIKE PCBS IN CERTAIN FOODSTUFFS The requirements set out in this Annex shall be applied where foodstuffs are analysed for the official control of the levels of non-dioxin-like PCBs and as regards sample preparation and analytical requirements for other regulatory purposes, including the controls performed by the food business operator to ensure compliance with the provisions in Article 4 of Regulation (EC) No 852/2004. The provisions on sample preparation provided for in point 3 of Annex III of this Regulation shall also be applicable for the control of the levels of non-dioxin-like PCBs in food. 1. Applicable detection methods Gas Chromatography/Electron Capture Detection (GC-ECD), GC-LRMS, GC-MS/MS, GC-HRMS or equivalent methods. 2. Identification and confirmation of analytes of interest: — Relative retention time in relation to internal standards or reference standards (acceptable deviation of +/– 0,25 %). — Gas chromatographic separation of the non-dioxin-like PCBs (from interfering substances, especially co-eluting PCBs, in particular if levels of samples are in the range of legal limits and non-compliance is to be confirmed ( 1 ) ). — For GC-MS techniques: — Monitoring of at least the following number of molecular ions or characteristic ions from the molecular cluster: — two specific ions for HRMS, — three specific ions for LRMS, — two specific precursor ions, each with one specific corresponding transition product ion for MS-MS. — Maximum permitted tolerances for abundance ratios for selected mass fragments: Relative deviation of abundance ratio of selected mass fragments from theoretical abundance or calibration standard for target ion (most abundant ion monitored) and qualifier ion(s): ± 15 %. — For GC-ECD: Confirmation of results exceeding the maximum level with two GC columns with stationary phases of different polarity. 3. Demonstration of performance of method: Validation in the range of the maximum level (0,5 to 2 times the maximum level) with an acceptable coefficient of variation for repeated analysis (see requirements for intermediate precision in point 8). 4. Limit of quantification: The sum of the LOQs ( 2 ) of non-dioxin-like PCBs shall not be higher than one-third of the maximum level ( 3 ) . 5. Quality control: Regular blank controls, analysis of spiked samples, quality control samples, participation in interlaboratory studies on relevant matrices. 6. Control of recoveries: — Use of suitable internal standards with physico-chemical properties comparable to analytes of interest. — Addition of internal standards: — Addition to products (before extraction and clean-up process), — Addition also possible to extracted fat (before clean-up process), if maximum level is expressed on fat basis. — Requirements for methods using all six isotope-labelled non-dioxin-like PCB congeners: — Correction of results for recoveries of internal standards, — Generally acceptable recoveries of isotope-labelled internal standards are between 60 and 120 %, — Lower or higher recoveries for individual congeners with a contribution to the sum of non-dioxin-like PCBs below 10 % are acceptable. — Requirements for methods using not all six isotope-labelled internal standards or other internal standards: — Control of recovery of internal standard(s) for every sample, — Acceptable recoveries of internal standard(s) between 60 and 120 %, — Correction of results for recoveries of internal standards. — The recoveries of unlabelled congeners shall be checked by spiked samples or quality control samples with concentrations in the range of the maximum level. Acceptable recoveries for these congeners are between 60 and 120 %. 7. Requirements for laboratories In accordance with the provisions of Regulation (EC) No 882/2004, laboratories shall be accredited by a recognised body operating in accordance with ISO Guide 58 to ensure that they are applying analytical quality assurance. Laboratories shall be accredited following the EN ISO/IEC 17025 standard. In addition, the principles as described in Technical Guidelines for the estimation of measurement uncertainty and limits of quantification for PCB analysis shall be followed when applicable ( 4 ) . 8. Performance characteristics: Criteria for the sum of non-dioxin-like PCBs at the maximum level Isotope dilution mass spectrometry ( *1 ) Other techniques Trueness – 20 to + 20 % – 30 to + 30 % Intermediate precision (RSD R ) ≤ 15 % ≤ 20 % Difference between upper and lower bound calculation ≤ 20 % ≤ 20 % 9. Reporting of results — The analytical results shall contain the levels of the individual non-dioxin-like PCB congeners and the sum of non-dioxin-like PCBs, reported as lower-bound, upper-bound and medium-bound, in order to include a maximum of information in the reporting of the results and thereby enabling the interpretation of the results according to specific requirements. — The report shall also include the method used for the extraction of PCBs and lipids. The lipid content of the sample shall be determined and reported for food matrices with maximum levels expressed on fat basis and with an expected fat concentration in the range of 0-2 % (in correspondence to existing legislation). For other samples, the determination of the lipid content is optional. — The recoveries of the individual internal standards must be made available in case the recoveries are outside the range mentioned in point 6, in case the maximum level is exceeded and in other cases upon request. — As the expanded measurement uncertainty is to be taken into account when deciding about the compliance of a sample, that parameter shall also be made available. Thus, analytical results shall be reported as x +/– U whereby x is the analytical result and U is the expanded measurement uncertainty using a coverage factor of 2 which gives a level of confidence of approximately 95 %. — The results shall be expressed in the same units and with the same number of significant figures as the maximum levels laid down in Regulation (EC) No 1881/2006. ( 1 ) Congeners often found to co-elute are, e.g. PCB 28/31, PCB 52/69 and PCB 138/163/164. For GC-MS also possible interferences from fragments of higher chlorinated congeners have to be considered. ( 2 ) The principles as described in the ‘Guidance Document on the Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food’ [link to website] shall be followed when applicable. ( 3 ) It is highly recommendable to have a lower contribution of the reagent blank level to the level of a contaminant in a sample. It is in the responsibility of the laboratory to control the variation of blank levels, in particular, if the blank levels are subtracted. ( 4 ) ‘Guidance Document on Measurement Uncertainty for Laboratories performing PCDD/F and PCB Analysis using Isotope Dilution Mass Spectrometry’ [link to website], ‘Guidance Document on the Estimation of LOD and LOQ for Measurements in the Field of Contaminants in Feed and Food’ [link to website]. ( *1 ) Use of all six 13 C-labelled analogues as internal standards required