title: 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: Institute of Clinical Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, PR China.
authors: Wenying
authors: W
authors: Shu
authors: Xueding
authors: X
authors: Wang
authors: Xiuyan
authors: X
authors: Yang
authors: Liuqin
authors: L
authors: Liang
authors: Jiali
authors: J
authors: Li
authors: Zhuojia
authors: Z
authors: Chen
authors: Xiao
authors: X
authors: Chen
authors: Jing
authors: J
authors: Jin
authors: Ruiming
authors: R
authors: Li
authors: Lizi
authors: L
authors: Zhao
authors: Min
authors: M
authors: Huang
mesh_terms: D000085:Acetates / Q000737:chemistry; D000097:Acetonitriles / Q000737:chemistry; D044466:Asian People; D002138:Calibration; D002681:China; D002851:Chromatography, High Pressure Liquid / Q000592:standards; D003520:Cyclophosphamide / Q000031:analogs & derivatives* / Q000097:blood* / Q000302:isolation & purification / Q000592:standards / Q000627:therapeutic use; D006801:Humans; D007166:Immunosuppressive Agents / Q000097:blood* / Q000592:standards / Q000627:therapeutic use; D008180:Lupus Erythematosus, Systemic / Q000097:blood* / Q000188:drug therapy; D052616:Solid Phase Extraction; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards
chemical_list: D000085:Acetates; D000097:Acetonitriles; D007166:Immunosuppressive Agents; C012358:4-hydroxycyclophosphamide; C007650:ethyl acetate; D003520:Cyclophosphamide; C032159:acetonitrile