VALIDATION OF UHPLC-MS/MS METHOD AFTER MODIFIED QUECHERS SAMPLE PREPARATION FOR MONITORING OF PESTICIDE RESIDUES IN BOVINE MILK ACCORDING TO EC/2018/555
Date Issued
2022-09-22
Author(s)
Angeleska, Aleksandra
Koceva, Dushica
Stojanovska Dimzoska, Biljana
Stojovikj, Goran
Ilievska, Gordana
Abstract
The EU Regulation EC/555/2018 has foreseen implementation of monitoring program
for selected pesticide residues in bovine milk, aimed for further dietary exposure assessment.
Therefore, since 2019 the required pesticide substances were introduced into the National
Residue Control Program for animal origin food in North Macedonia. For this purpose,
UHPLC-MS/MS method was optimized and validated after modified QuEChERS (quick,
easy, cheap, effective, rugged, and safe) sample preparation. The MS/MS method was
optimized for 19 pesticide substances and metabolites: carbaryl, carbofuran, fenvalerate,
indoxacarb (carbamate pesticides), cypermethrin, deltamethrin, bifenthrin permethrin
(pyrethroids), malathion, parathion, diazinone, dichlorvos, chlorpyriphos, chlorpyriphosmethyl,
pyrimiphos-methyl (organophosphates), famoxadone, fipronil, including
metabolites fipronil sulfone, fipronil sulfide. For each compound the two most sensitive
MRM transitions were selected for further determination. Analysis was performed on
Waters UHPLC-MS/MS system consisted of H-class UHPLC and Xevo TQ-S micro triple
quadrupole detector, in ESI+ and ESI- mode. Chromatographic separation was performed
on C18 column with 100 mm length and 1.7 μm particle diameter, using gradient elution
with water and methanol, both modified with formic acid and ammonium formate (0.1
% v/v and 5 mmol/L, respectively), at flow rate of 0.45 mL/min. The optimized method
was validated according to DG SANTE Document 2017/11813. Validation parameters
were linearity, limit of quantification (LOQ), precision, accuracy, and matrix effect.
The determined LOQs from the concentration level at which S/N ratio was higher than
1:10, were in the range 1.0 – 8.3 μg/L, and were equal or below the MRL values for the
pesticides of interest. Linearity was determined in the range 1-100 μg/L, obtaining five level
calibration curves with correlation coefficients ≥0.99 for all tested analytes. Method
precision was tested at two concentration levels - 10 and 50 μg/kg), except for carbofuran,
for which the target levels were 1 and 10 μg/kg. The calculated relative standard deviation
(RSD) ranged from 6.3 to 15.5 %. The method accuracy, estimated from the recovery
at two concentration levels, was in the range 73.8 – 107.3 %. Matrix effect did not have
significant influence on the analyte signal suppression or enhancement, thus solvent standards could be used for calibration. The method performance characteristics were
satisfactory according to the SANTE document requirements (LOQ ≤ MRL, precision
≤20 %, and recovery 70-120 %). With this, it was proven that the proposed method was
suitable for analysis of the defined scope of substances, even for pyrethroids, for which gas
chromatography was considered to be a more appropriate analysis technique.
for selected pesticide residues in bovine milk, aimed for further dietary exposure assessment.
Therefore, since 2019 the required pesticide substances were introduced into the National
Residue Control Program for animal origin food in North Macedonia. For this purpose,
UHPLC-MS/MS method was optimized and validated after modified QuEChERS (quick,
easy, cheap, effective, rugged, and safe) sample preparation. The MS/MS method was
optimized for 19 pesticide substances and metabolites: carbaryl, carbofuran, fenvalerate,
indoxacarb (carbamate pesticides), cypermethrin, deltamethrin, bifenthrin permethrin
(pyrethroids), malathion, parathion, diazinone, dichlorvos, chlorpyriphos, chlorpyriphosmethyl,
pyrimiphos-methyl (organophosphates), famoxadone, fipronil, including
metabolites fipronil sulfone, fipronil sulfide. For each compound the two most sensitive
MRM transitions were selected for further determination. Analysis was performed on
Waters UHPLC-MS/MS system consisted of H-class UHPLC and Xevo TQ-S micro triple
quadrupole detector, in ESI+ and ESI- mode. Chromatographic separation was performed
on C18 column with 100 mm length and 1.7 μm particle diameter, using gradient elution
with water and methanol, both modified with formic acid and ammonium formate (0.1
% v/v and 5 mmol/L, respectively), at flow rate of 0.45 mL/min. The optimized method
was validated according to DG SANTE Document 2017/11813. Validation parameters
were linearity, limit of quantification (LOQ), precision, accuracy, and matrix effect.
The determined LOQs from the concentration level at which S/N ratio was higher than
1:10, were in the range 1.0 – 8.3 μg/L, and were equal or below the MRL values for the
pesticides of interest. Linearity was determined in the range 1-100 μg/L, obtaining five level
calibration curves with correlation coefficients ≥0.99 for all tested analytes. Method
precision was tested at two concentration levels - 10 and 50 μg/kg), except for carbofuran,
for which the target levels were 1 and 10 μg/kg. The calculated relative standard deviation
(RSD) ranged from 6.3 to 15.5 %. The method accuracy, estimated from the recovery
at two concentration levels, was in the range 73.8 – 107.3 %. Matrix effect did not have
significant influence on the analyte signal suppression or enhancement, thus solvent standards could be used for calibration. The method performance characteristics were
satisfactory according to the SANTE document requirements (LOQ ≤ MRL, precision
≤20 %, and recovery 70-120 %). With this, it was proven that the proposed method was
suitable for analysis of the defined scope of substances, even for pyrethroids, for which gas
chromatography was considered to be a more appropriate analysis technique.
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