THE DEVELOPMENT OF THE METHOD FOR THE DETERMINATION OF SULFONAMIDES AND TRIMETOPRIM RESIDUAL QUANTITIES IN MILK SAMPLES BY UPLC-MS/MS METHOD AND ITS VERIFICATION THROUGH INTERLABORATORY TESTING
This manuscript presents the results of development and adoption of the confirmatory UPLC- MS/MS method for determination of sulfonamides and trimethoprim residues in whole and powdered cow's milk samples. Sample preparation includes a recovery with water (in the case of milk powder), separation of milk fat by centrifugation at 4 °C, proteins precipitation with acetonitrile using ultrasonic intensification, extraction of analytes with a two-component mixture of organic solvents from weak acidic phosphate buffer solution, concentration of obtained extracts, degreasing and reconstitution with the mobile phase. The use of tandem-mass spectrometric detector in multiple reaction monitoring mode provides high specificity for all ten sulfonamides, which molecules contain the same structural elements, and trimethoprim, as three ion transitions were used for each of the target analytes. The approach of matrix matched calibration on the milk matrix was applied, and it allowed us to disregard taking into consideration the absolute extraction of analytes during sample preparation, as well as the influence of sample matrix components on analytes ionization. The main advantages of the developed technique are simplicity, rapidity and cost effective sample preparation, fast procedure of sample analysis (the time of chromatographic separation is 2.0 min), high selectivity, and sensitivity at the level of ≤ 0.5 μg/kg (as compared with the maximum residues level of sulfonamides in dairy products, viz. 100 μg/kg). According to the European Commission Decision 2002/657/EC, the values of CCα and CCβ for sulfonamides and trimethoprim in milk were calculated. The capability of the method was confirmed on the basis of the established validation parameters, using spiked milk samples and "added – found" approach. The method was tested during the analysis of spiked samples of whole and powdered cow's milk (CV ~ 20%). The developed technique was verified by interlaboratory comparative testing of milk powder samples for sulfonamides content. The UPLC-MS/MS method for the determination of sulfonamides and trimethoprim residues in whole and milk powder samples can be used both for routine laboratory analysis of dairy products and for the examination of suspicious or questionable samples.
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