Development and validation of an LC-MS/MS assay with multiple stage fragmentation for the quantification of alachlor...

Alachlor is one of the most widely used herbicides and can also be a carcinogenic compound. It is of great significance to establish a sensitive analytical method for the determination of alachlor in the environment and organisms. In this study, a high-performance liquid chromatography tandem mass spectrometry cubed (LC/MS³) method was developed and validated to quantify alachlor in human breast cancer cells (McF-7 cells). The cell samples were processed by simple protein precipitation with acetonitrile, then the analytes were separated on a Waters AcQuity® UPLC BEH (2.1 × 50 mm I.D, 1.7 μm) column using the gradient elution with solvent A (0.1 % formic acid) and solvent B (acetonitrile) at a flow rate of 0.5 mL/min. MS³ detection in positive ion mode was used to detect the analytes. The MRM³ transitions at m/z 270.1 → 238.0 → 162.1 and 312.2 → 238.1 → 147.2 were used to determine alachlor and butachlor, respectively. The run time for each sample was only 4 min. This method was validated for various parameters including accuracy, precision, selectivity, linearity, lower limit of quantitation (LLOQ), etc. The LC/MS³ assay was linear in the concentration range 0.5-50 ng/mL (R² ≥ 0.995). For all concentrations, the precision is < 9.49 %, and the intra-day and intra-day accuracy is < 13.05 %. Cytotoxic potential of alachlor against McF-7 cell lines was measured by MTT method after 48 h of incubation. For alachlor, half maximal inhibitory concentration (IC₅₀) on McF-7 cells was 87.95 µg/mL. This method was successfully applied to cellular pharmacokinetic study of alachlor in McF-7 cells after administration with a dose of 20 μg/mL.