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

BACKGROUND:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

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.

• Authors: Wenying Shu, Xueding Wang, Xiuyan Yang, Liuqin Liang, Jiali Li, Zhuojia Chen, Xiao Chen, Jing Jin, Ruiming Li, Lizi Zhao, Min Huang
• Journal: Clinical chemistry and laboratory medicine (Clin Chem Lab Med) Vol. 49 Issue 12 Pg. 2029-37 (Sep 09 2011) ISSN: 1437-4331 [Electronic] Germany
• PMID: 21902573 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Acetates
• Acetonitriles
• Immunosuppressive Agents
• 4-hydroxycyclophosphamide
• ethyl acetate
• Cyclophosphamide
• acetonitrile

Topics

• Acetates (chemistry)
• Acetonitriles (chemistry)
• Asian People
• Calibration
• China
• Chromatography, High Pressure Liquid (standards)
• Cyclophosphamide (analogs & derivatives, blood, isolation & purification, standards, therapeutic use)
• Humans
• Immunosuppressive Agents (blood, standards, therapeutic use)
• Lupus Erythematosus, Systemic (blood, drug therapy)
• Solid Phase Extraction
• Spectrometry, Mass, Electrospray Ionization (standards)