S TABILITY OF R ESIDUES DURING S AMPLE P ROCESSING The laboratory sample processing received unproportionally little attention in the past,

5.4.2 S TABILITY OF R ESIDUES DURING S AMPLE P ROCESSING The laboratory sample processing received unproportionally little attention in the past,

though its contribution to the uncertainty and the bias of the results can be quite large. In general, supervised trial samples are transported, stored, and processed under deep- frozen conditions, whereas monitoring laboratories usually receive and process the samples at ambient temperature. The analysts were aware of the rapid decomposition of dithiocarbamates or daminozide if they were in contact with the macerated samples and eliminated the homogenization step from the method, but did not test the stability of other residues or associate the loss of residues with their potential decomposition, until some publications indicated the substantial decomposition (50% –90%) of certain com-

pounds (chlorothalonil, phthalimides, thiabendazole, dichlofluanid). 15,34 Further studies revealed that processing in the presence of dry ice (cryogenic milling) at or below

208C reduced or practically eliminated the loss of all pesticides which decomposed at ambient temperature. 35 Furthermore, cryogenic processing may provide more homoge-

neous sample matrix and reduce the uncertainty of sample processing. Notable that the decomposition of pesticides in test portions spiked after the homogenization of the sample is much smaller and does not affect the recovery of most of the compounds substantially. It may probably be attributed to the inactivation of the enzymes by the extracting solvent and the different concentration of the chemicals in the diluted extract.

The decomposition of the residues depends on the composition of the sample material and the homogenization process. When intensive and extended comminu- tion in high-speed blender is carried out to reduce the sample processing uncertainty,

a significant bias can be introduced due to the decomposition of the residues.

140 Analysis of Pesticides in Food and Environmental Samples Because the rate of decomposition may depend on the laboratory equipment, the

variety and maturity of the processed crop, and many other factors, currently there is no sufficient knowledge to extrapolate findings from one laboratory to another. Consequently the laboratories, analyzing wide range of pesticides in large number of various commodities, should apply cryogenic processing as standard procedure to reduce the chance of producing biased results. Furthermore, the laboratories should verify the suitability of their procedures as part of the method validation with testing the stability of those compounds, which are known to rapidly decompose under unfavorable conditions.

The cryogenic processing applied successfully in some laboratories 35 includes: preparation of the portion of sample to which the MRL applies (analytical sample) on receiving the fresh sample in the laboratory; placing the analytical sample into the deep-freezer within the shortest possible time; chopping, grinding the sample in the presence of sufficient amount of dry ice (about 1:1 sample=dry ice ratio) to keep the temperature below 208C (this process requires robust choppers with stainless steel bowl and lid); withdrawing the test portions needed for various extractions and confirmation of residues into appropriate unsealed containers and placing them in deep-freezer for a minimum of 16 h to allow the carbon dioxide to evaporate; weighing the mass of the test portion, adding extraction solvent and warming the test portion up to room temperature before proceeding with the extraction.

The stability of residues can be tested with a mixture of pesticides, which contains a reference compound (R) known to be stable (e.g., chlorpyrifos), at least one compound decomposing rapidly (chlorothalonil, dichlofluanid, captan) and the other compounds to be tested. The test mixture should be carefully applied on the surface of the plant material avoiding runoff. The treated sample should be kept in fume cupboard until the solvent completely evaporates. The processing under ambient temperature can now be started, while the treated sample should be placed in deep-freezer before cryogenic milling for a minimum of 16 h.

A minimum of three test portions should be withdrawn from the comminuted material, and the extract should be analyzed in duplicate. The number of test portions and the replicate analyses depend on the CV A of the analytical method and the percentage decomposition, which should be quantified with a selected probability. The result of the analysis is evaluated by comparing the measured residues to the expected ones.

The significance of the difference between the expected and survived residues (A 11 –A 0 11 ,A 12 –A 0 12 , etc.) can be calculated with the one-tail Student’s t-test for differences.

s D ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi P (d D) 2 t

calc ¼

p ffiffiffi where S d ¼

n 1 where D is the average of the differences and n is the number of measurement pairs.

Before the start of the study, one can also calculate with Equation 5.6 the minimum difference between the expected and survived residues (Table 5.3), which can be considered statistically significant, and decide on the number of test portions and replicate analysis to be performed in the study depending on the

Quality Assurance 141

TABLE 5.3 Examples for the Quantifiable Differences between the Expected and Survived Residues

Residue a n b CV A c CV qR d CV d e SD f x d g Rel. dif. % h

0.047 9.3 a Expected residue concentration.

b Number of valid residue values determined in analytical portions. c Repeatability of the recovery of the analyte (A, B, C, . . . , X). d Repeatability of the recovery of reference compound. e Relative uncertainty of the calculated difference. f Standard deviation of the difference.

g Quantifiable significant difference between the expected and survived residues is >x d . h Quantifiable relative difference.

percentage of the decomposition which should be quantified. The acceptable decrease of residues during sample processing has not been officially specified. As

a guidance value 5% –10% may be used, as it is considered acceptable difference between two standard solutions. 3