Validation of methods for pesticide residue analysis in milk and milk products asper FSSAI regulation

Abstract

Accurate analysis of 55 pesticides as per the regulatory requirement of FSSAI for milk and milk products is a challenging task as it requires performing six different types of extractions and detection techniques. Further, before putting to use methods has to be thoroughly validated or checked for its fitness of purpose as per the National or International guidelines to ensure the accuracy of test results. The objective of this study was to develop a validated test method for the determination of55 pesticides in milk and milk products as per FSSAI regulation. QuEChERS based simultaneous extraction of multi class pesticides involves different steps i.e. mixing of reagents, separation of phases, extraction of analytes, clean up followed by reconstitution in suitable solvents. For analysis of few pesticides of different chemical nature, certain analytical steps which are saponification, derivatisation, digestion of analyte to release CS2, extractions using specific solvents, purification usingspecific cartridges at specific pH, temperature and flow conditions are required to be performed. For detection of pesticide residues, mass spectrometry either with gas or liquid chromatography is one of the best approach to meet method performance and validation criteria. In this study, the optimised extraction protocols were investigated for parameters like Linearity, Matrix Effect (ME),Limit of Quantification (LOQ), Specificity, Trueness, Precision, Ion Ratio and Retention Time (RT) to validate the fitness for purpose of the methods. The calibration curves for all the pesticides were linear over the tested range as the concentration of every analyte at each calibration level fall within the residual limit of ±20 %. The LOQ for most of pesticides is established at 5μg/kg, whereas it is 10 μg/kg for Bifenthrin, Cypermethrin,Dichlorvos, Etofenprox, Phorate, Glufosinate Ammonium, and 25μg/kg for Dithiocarbamtes as CS2. The Trueness and Precision of the methods were evaluated by analysing control samples spiked at LOQ and 2 to 10x the RL/LOQ in 6 replicates as per SANTE/11321/2021 guideline. Results for Trueness and Precision meet validation criteria of recovery 70 to 120 % and % RSDr < 20% respectively for all targeted pesticides. The methods fulfilled all other the requirements of SANTE/11321/2021 guidelines and can be extended for routine analysis of pesticide residues in milk and milk products.