Residue Analytical Methods

When crops are treated with pesticides very small amounts may remain in or on the crop after harvesting or storage. In general, the levels in crops are very low or in many cases not detectable, even with sophisticated analytical methodology. In order to determine any pesticide residues present in foodstuffs analytical methods capable of accurately measuring extremely small amounts of pesticides are required. There are several key parameters for ensuring a robust method, the definitions of these parameters are given in an OECD document - Guidance Document on Pesticide Residue Analytical Methods¹:

Recovery - Recovery is the amount measured as a percentage of the amount of analyte(s) (active substance and relevant metabolites) originally added to a sample of the appropriate matrix, which contains either no detectable level of the analyte or a known detectable level. Recovery experiments provide information on both precision and trueness (bias), and thereby the accuracy of the method.

Selectivity (Specificity) - Selectivity refers to the extent to which the method can be used to determine particular analytes in mixtures or matrices without interferences from other components of similar behaviour. Some regulatory authorities use the term specificity to refer to selectivity.

Calibration - Calibration refers to the ability of a detection system to produce an acceptable, well defined, correlation between the instrumental response and the concentration of the analyte in the sample. The analyte concentration to be measured should be within the defined dynamic range of the instrument.

Repeatability - Repeatability refers to the closeness of agreement between mutually independent test results obtained with the same method on identical test material, in the same laboratory by the same operator using the same equipment within short intervals of time. The repeatability (within-run effect) includes contributions from any part of the procedure that varies within a run, including contributions from normal gravimetric and volumetric errors, heterogeneity of the test material, and other procedural errors during the analysis.

Reproducibility - Reproducibility refers to the closeness of agreement between independent results obtained with the same method on identical test material obtained but under different conditions. Within-laboratory or intra-laboratory reproducibility or single-laboratory reproducibility (run effect) contributes to day-to-day variations in the analytical system due to changes of analyst, batches of reagents, recalibration of instruments and laboratory environment (e.g. temperature changes). Between-laboratory or interlaboratory or multiple-laboratory reproducibility (laboratory effect) contributes to additional variations such as variations in calibration standards, differences between local interpretations of a protocol, differences in equipment or reagent source, or environmental factors, such as differences in average climatic conditions.

Limit of Detection (LOD)* - The limit of detection of an analytical procedure is the lowest amount of an analyte in a sample that can be detected but not necessarily quantified as an exact value. At the limit of detection, a positive identification can be achieved with reasonable and/or previously determined confidence in a defined matrix using a specific analytical method. The LOD is typically not required. However, if needed for a refined assessment (or some other purpose), an explanation of how the LOD was derived should be provided.

Limit of quantitation (LOQ)* - Limit of quantitation (LOQ), defined from a regulatory perspective as the lowest concentration tested at which an unambiguous identification of the analyte can be proven and at which an acceptable mean recovery with an acceptable relative standard deviation (RSD) is obtained, also referred to as the limit of determination (LOD) or Lowest Limit of Method Validation (LLMV). The LOQ should be low enough to achieve the intended purpose of the method. From an analytical perspective, 6-10 times the standard deviation of the noise provides an estimate of the LOQ, which is then verified by the fortification experiments.

Where appropriate the OECD guidance document gives the number of analyses required and acceptable values for the above parameters to demonstrate a suitable method. Similarly, in Europe a guidance document is available which also defines acceptable values for these parameters as shown below^2,3,4. In all cases validation data should be obtained for representative sample matrices to be analysed. Where a previously validated method has been adopted, validation data must be obtained for representative additional matrices.

The analytical calibration should extend over a range appropriate to the lowest and highest nominal concentration of the analyte ± at least 20%. Either duplicate determinations at three or more concentrations or single determinations at 5 or more concentrations must be made. The equation of the calibration line and the correlation coefficient (r) must be reported and a typical calibration plot submitted equation of the calibration line and the correlation coefficient (r) must be reported and a typical calibration plot submitted.

The repeatability should be expressed as relative standard deviation (rsd, %) and the number of samples (n) given. The overall rsd for every commodity, as well as the rsd for each fortification level must be experimentally determined and reported. In general the rsd should be ≤ 20% per commodity and level. In certain justified cases, e.g. determination of residues in soil lower than 0.01 mg/kg, higher variability may be accepted. Outliers identified through an appropriate method may be discarded. Where outliers have been discarded, that fact must be clearly indicated. A proper explanation as to the reason for the occurrence of individual outliers must be given. A maximum of one outlier may be discarded at each fortification level. Where more than one outlier has been identified at one fortification level, additional validation samples must be included.

A suitable limit of quantification (LOQ) must be reported including the individual and mean recovery in representative and/or difficult matrices. The mean recovery at each fortification level and for each commodity should be in the range of 70-110% (in certain justified cases recoveries outside of this range will be accepted). If necessary, recoveries should be corrected by blank values. Uncorrected recoveries must also be given. Blank values must be reported. They must be determined from the matrix used in fortification experiments and should not be higher than 30% of the LOQ.

Similar information is given in guidelines issued by the Australian authorities (APVMA)⁵ and the US Environmental protection Agency⁶.

Information on sampling, analysis and  reporting requirements specifically for environmental samples (soil, pore water, groundwater, inland surface water, sediment, seawater, precipitation water and air) is given in an IUPAC paper⁷.

            Residue Methods

Methods for the analysis of pesticides in food stuffs are available from various sources. The US Environmental Protection Agency has made methods available, in TIF image format, via the Web. These cover a large number of pesticides in a variety of foodstuffs⁸.  The US Food and Drug Administration has compiled residue methods in Pesticide Analytical Manuals (PAM I and PAM II). PAM I gives detailed information on several multi-residue methods for both fatty and non-fatty foodstuffs⁹ , PAM II has methods for individual pesticides but although the index is on-line¹⁰ the actual methods are not yet available electronically.

Another useful source of residue methods is the Manual of Pesticide Residue Analysis which contains, although this is not available on-line, however, some of the methods can be found in the Official analytical methods for residues of plant protection products and pesticides, L 00.00 15¹¹and L 00.00 16¹².

A multi-residue method based on LC-MS/MS has been described for 300 pesticides in drinking water¹³.

A relatively new multi-residue method has been readily accepted by many pesticide residue analysts, the so called QuEChERS method¹⁴ (Quick, Easy, Cheap, Effective, Rugged, Safe). This method, which covers a wide pesticide range (polar, pH-dependent compounds), claims to be rapid (8 samples in less than 30 min), simple (no laborious steps, minimal sources of errors) and cheap (ca. 1 € per sample for the sample preparation). The solvent consumption is low (10 mL acetonitrile; GC- and LC-amenable) and practically no glassware is needed¹⁵. A method for residues in low fat products is available on-line¹⁶.

References

1. OECD, Series on Testing and Assessment Number 72; Series on Pesticides Number 39 - Guidance Document on Pesticide Residue Analytical Methods, ENV/JM/MONO(2007)17, 13^th August 2007

            http://www.olis.oecd.org/olis/2007doc.nsf/LinkTo/NT00002F06/$FILE/JT03230940.PDF

2. European Commission, Directorate General Health and Consumer Protection, SANCO/825/00 rev.7

            20/06/, 17^th March 2004 - Guidance document on residue analytical methods

            http://ec.europa.eu/food/plant/protection/resources/guide_doc_825-00_rev7_en.pdf

3.     European Commission, Directorate General Health and Consumer Protection, SANCO/2007/3131(Supersedes Document No. SANCO/10232/2006) - Method Validation and Quality Control Procedures for Pesticide Residues Analysis in Food and Feed, 31^st October 2007

            http://ec.europa.eu/food/plant/protection/resources/qualcontrol_en.pdf

4. European Commission, Directorate General Health and Consumer Protection,  SANCO/3029/99 rev.4,

            Residues: Guidance for generating and reporting methods of analysis in support of  pre-registration        data requirements for Annex II (part A, Section 4) and Annex III (part A, Section 5) of Directive             91/414,             11^th July 2000

            http://ec.europa.eu/food/plant/protection/evaluation/guidance/wrkdoc12_en.pdf

5.     Australian Pesticides and Veterinary Medicines Authority, Residue Guideline No. 19, June 2000

            http://www.apvma.gov.au/publications/guidelines/rgl_19.php

6. US Environmental Protection Agency, Residue Chemistry Test Guidelines, OPPTS 860.1340, Residue Analytical Method, EPA 712–C–95–174, August 1995

            http://fedbbs.access.gpo.gov/library/epa_860/860-1340.pdf

7. H. Egli, M. Dassenakis, H. Garelick, R. van Grieken, W. J. G. M. Peijnenburg, L. Klasinc, W. Kördel, N. Priest, and T. Tavares,  Minimum Requirements for Reporting Analytical Data for Environmental Samples, (2003) Pure Appl. Chem., 75 (8) 1097–1106

http://media.iupac.org/publications/pac/2003/pdf/7508x1097.pdf

8.     US Environmental Protection Agency, Pesticides: Analytical Methods and Procedures, Index of Residue Analytical Methods (RAM)

            http://www.epa.gov/oppbead1/methods/ram12b.htm

9.     US Food and Drug Administration, Pesticide Analytical Manual, Volume I, Multi-residue Methods, 3^rd Edition, June 1994

            http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/PesticideAnalysisManualPAM/default.     htm

10. US Food and Drug Administration, Pesticide Analytical Manual, Volume II

            http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/PesticideAnalysisManualPAM/ucm11       3710.htm

11.  BfR, German Federal Institute for Risk Assessment, Official analytical methods for residues of plant protection products and pesticides (L 00.00 15)

            http://www.bfr.bund.de/cd/1637

12.  BfR, German Federal Institute for Risk Assessment, Official analytical methods for residues of plant protection products and pesticides (L 00.00 16)

            http://www.bfr.bund.de/cd/1652

13. K. Greulich and L. Alder, BfR, German Federal Institute for Risk Assessment, Fast multi residue screening of 300 pesticides in drinking water, Report BfR-IX-2005, May 2006

            http://www.bfr.bund.de/cd/5832

14.  QuEChERS.com

            http://www.quechers.com/default.htm

15.  M. Anastassiades, E. Scherbaum and D. Bertsch, Chemisches und Veterinäruntersuchungsamt Stuttgart; Validation of a Simple and Rapid Multiresidue Method (QuEChERS) and its Implementation in Routine Pesticide Analysis, MGPR Symposium,  May 2003, Aix en Provence, France

            http://www.quechers.com/default.htm

16. QuEChERS, A Mini-Multiresidue Method for the Analysis of Pesticide Residues in Low-Fat Products

            http://www.quechers.com/docs/quechers_en_oct2005.pdf

Last modified 7^th April 2010