Simultaneous determination of trace diphacinone and chlorophacinone in biological samples by high performance liquid chromatography coupled with ion trap mass spectrometry

Abstract

A rapid qualitative and quantitative method for the simultaneous determination of trace diphacinone and chlorophacinone in biological samples has been established. The method mainly serves for the emergent poisoning detection. The whole blood was treated with methanol-acetonitrile (50/50, v/v) and the urine was cleaned-up by Waters Oasis HLB SPE cartridges. The samples were separated on an Extend C18 column (150 mm x 4.6 mm, 5 microm) by using the mobile phase consisted of ammonium acetate-acetic acid (0.02 mol/L, pH 5.5) - methanol (15/85, v/v). The determination was performed by high performance liquid chromatography coupled with ion trap mass spectrometry (HPLC-IT-MS) using a negative electrospray ionization interface in the multiple reaction monitoring (MRM) mode. The transitions of m/z 339 --> 167 for diphacinone and m/z 373 --> 201 for chlorophacinone were selected for the quantificantions. For the whole blood samples, the calibration curves were linear within the ranges of 1.0 - 200.0 microg/L and 0.5 - 100.0 microg/L; the limits of quantification were 1.0 microg/L and 0.5 microg/L; the spike recoveries were 90.1% - 92.2% and 87.6% - 93.4%, the intra-day relative standard deviations (RSDs) were less than 6.8% and 7.4%, and the inter-day RSDs were less than 9.9% and 10.9% for diphacinone and chlorophacinone, respectively. For the urine samples, the calibration curves were linear within the ranges of 0.2 - 40.0 microg/L and 0.1 - 20.0 microg/L; the limits of quantification were 0.2 microg/L and 0.1 microg/L; the spike recoveries were 90.1% -94.5% and 90.0% -98.0%, the intra-day RSDs were less than 6.1% and 7.3%, and the inter-day RSDs were less than 8.9% and 11.2% for diphacinone and chlorophacinone, respectively. This method is simple and sensitive for the satisfactory determination of trace diphacinone and chlorophacinone residues in poisoned patients for the clinical diagnosis.