Simultaneous detection of trace diphacinone and chlorophacinone in biological sample by high-performance liquid chromatography coupled with mass spectrometry

Objective: To establish a method for the simultaneous detection of diphacinone and chlorophacinone in biological sample by high-performance liquid chromatography coupled with mass spectrometry(LC-MS).Methods:After the whole blood was extracted by methanol+acetonitrile(50+50,v/v) and urine was cleaned and enriched by Waters OasisHLB SPE cartridges,the samples were separated on an Extend C18 column(150 mm×4.6 mm i.d.,5 μm)by using the mobile phase consisting of ammonium acetate-acetate acid(0.02 mol/L,pH 5.5)+methanol(15+85,v/v).Detection was carried out by LC-MS using a negative electrospray ionization interface in the selected ion monitoring(SIM) mode.Quantified ions were selected for their molecular ions,m/z 339.0-for diphacinone,m/z 373.0-for chlorophacinone.Results:Calibration curves of the whole blood were linear within the range of 2.0~500.0 μg/L and 1.0~200.0 μg/L,and the limits of quantification were 2.0 μg/L and 1.0 μg/L for diphacinone and chlorophacinone,respectively.The extraction recoveries of the whole blood were 92.1%~94.5% and 93.0%~94.2%,and the RSD were less than 7.5% and 6.5% for diphacinone and chlorophacinone,respectively.Calibration curves of urine were linear within the range of 0.5~100.0 μg/L and 0.2~40.0 μg/L,and the limits of quantification were 0.5 μg/L and 0.2 μg/L for diphacinone and chlorophacinone,respectively.The extraction recoveries of urine were 95.2%~97.0% and 92.0%~96.5%,and the RSDs were less than 6.0% and 7.5% for diphacinone and chlorophacinone,respectively.Conclusion:This method is simple,sensitive,accurate and of good specificity for the satisfactory determination of trace diphacinone and chlorophacinone residues in poisoned patients.