Fast quantification of cyclophosfamide and its N‐dechloroethylated metabolite 2‐dechloroethylcyclophosphamide in human plasma by UHPLC‐MS/MS

: A rapid, novel and reliable UHPLC-MS/MS method was developed and validated for simultaneous determination of cyclophosphamide (CP) and its dechloroethylated metabolite, 2-dechloroethylcyclosphamide (2-DCECP) in human plasma. The plasma samples were conducted by protein precipitation with 3-fold acetonitrile, containing 0.1% formic acid. Mass spectrometric detection was performed using electrospray positive ionization with multiple reaction monitoring mode, using tinidazole as internal standard (IS). Chromatographic separation was performed on an Agilent poroshell 120 SB-C18 column (2.1 × 75 mm, 2.7 µm) using gradient elution of acetonitrile and 0.1% formic acid at a flow rate of 0.5 mL/min, the total run time was 2.5 min. The limit of quantification (LOQ) was 20 ng/mL for both CP and 2-DCECP. Accuracies and precisions were <15% at LOQ and below 10% at quality control concentration levels. This UHPLC-MS/MS method was successfully applied for the estimation of CP and 2-DCECP in human plasma, which was also useful for clinical toxicology studies and therapeutic drug monitoring of CP.