Determination of Warfarin and Coumatetralyl in Serum by HPLC Coupled with Electrospray Ionization Mass Spectrometry

Abstract

A sensitive, fast, and accurate method to determine warfarin and coumatetralyl in serum by high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC‐ESI‐MS) was developed. After warfarin and coumatetralyl in serum was extracted by ethyl acetate, a satisfactory separation was achieved on a XDB C₁₈ column, 150 mm×2.1 mm×5 µm, by using the mobile phase consisting of acetic acid‐ammonium acetate (10 mmol/L, pH=4.5) (20%) and methanol (80%) in isocratic eluent at a constant flow rate of 0.30 mL/min. The molecular ions m/z [M‐H]⁻ 307 and 291 were selected for the quantification in the selected ion monitoring (SIM) mode for warfarin and coumatetralyl, respectively. The linear range was 0.5∼100.0 µg/L for warfarin and coumatetralyl. The limit of quantification in serum was found to be 0.5 µg/L for each. The recoveries were between 91.9∼94.9% for warfarin and 90.9∼104.0% for coumatetralyl, the intra‐ and inter‐day RSDs for each were less than 10.1%. This method can determine trace warfarin and coumatetralyl in serum and it can be used to diagnose poisoned humans and other animals.

Keywords:

• Warfarin
• Coumatetralyl
• Serum
• HPLC‐ESI‐MS

Acknowledgment

This work was supported by a grant from the Medicine Science Foundation of Ningbo, Zhejiang Province, China (No. 2004056, 200543).