Simultaneous determination of cyclophosphamide and 4-hydroxycyclophosphamide in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry – application to Chinese systemic lupus erythematosus patients

Abstract

A pharmacogenomics study of cyclophosphamide in systemic lupus erythematosus patients is being conducted in our laboratory in which the plasma concentrations of cyclophosphamide and its active metabolite 4-hydroxycyclophosphamide should be assayed rapidly and sensitively. A rapid, stable and sensitive liquid chromato-graphy/electrospray ionization tandem mass spectrometry method was developed to simultaneously determine cyclophosphamide and 4-hydroxycyclophosphamide in human plasma with ifosfomide as an internal standard. After a protein precipitation with cold acetonitrile and stabilization of 4-hydroxycyclophosphamide by ansyldrazine and extraction with ethyl acetate, separation was performed on a C18 3.5 μm 2.1 × 50 mm column with mobile phase of acetonitrile and water (50:50, v/v) with 0.1% formic acid at 200 μL/min. The chromatographic run time was 3 min. The linear calibration curves ranged from 5 to 5000 ng/mL for cyclophosphamide and 5-500 ng/mL for 4-hydroxycyclophosphamide. The recoveries of the liquid extraction were 54.5%-58.5% for cyclophosphamide and 103.5%-105.5% for 4-hydroxycyclophosphamide. The lower limit of quantification was 5 ng/mL for both analytes. The intra- and inter-day precision was <15% for quality control samples at 4000, 500, 50 ng/mL for cyclophosphamide and 4-hydroxycyclophosphamide at 400, 100, 20 ng/mL. The method was applied in this pharmacogenomics study in Chinese systemic lupus erythematosus patients treated with low-dose cyclophosphamide. The method was efficient with shorter running time and lower limit of quantification compared to previous reports and has been successfully applied in this pharmacogenomics study.