Application of liquid chromatography–mass spectrometry to monitoring plasma cyclophosphamide levels in phase I trial cancer patients*

Abstract

A specific and efficient liquid chromatography-mass spectrometry (LC-MS) method was established for monitoring patient plasma cyclophosphamide levels in a phase I trial of an oral cyclophosphamide-based combination chemotherapy regimen. An Agilent 1100 Series LC-MSD system (Agilent Technologies, Avondale, PA, USA), with a single quadrupole mass detector using a positive atmospheric pressure chemical ionization (APCI) interface and single ion monitoring at m/z 261, was used. Chromatography was performed using a LUNA C8 5 microm 30 x 4.6 mm stainless steel column (Phenomenex, Torrance, CA, USA) and a mobile phase of aqueous acetonitrile pumped at a flow rate of 0.7 mL/min. High-throughput solid-phase sample extraction was performed using a Gilson ASPEC XL4 system (Gilson Medical, Middleton, WI, USA) controlled by prestored programs. The standard curve for cyclophosphamide was linear over the concentration range 0.026-1.08 microg/mL (r(2) > 0.994). Intra- and interassay accuracy and precision were 97-107 and 3-10%, respectively. The limit of detection was determined to be 0.01 microg/mL. Single ion monitoring at m/z 261 provided a high degree of specificity without interference from the matrix or other chemotherapy drugs. Automated sample processing allowed the analysis of a large number of plasma samples from a clinical trial of repeated daily oral dosing of cyclophosphamide. One hour after dosing, cyclophosphamide was detected in 98 of 106 plasma specimens at concentrations ranging between 0.03 and 4.88 microg/mL. Twenty-four hours after dosing, cyclophosphamide was detected in 72 of 77 plasma specimens at concentrations ranging between 0.06 and 3.13 microg/mL. There were no time-dependent changes in cyclophosphamide concentration during the 43 day period of repeated daily oral dosing. There was no correlation between cyclophosphamide dose and plasma concentration, despite the wide range of doses given in the clinical trial (50-125 mg/m(2)). We conclude that a solid-phase extraction LC-MS technique was validated for determining cyclophosphamide in human plasma. Interoccasion variability in the rate of oral absorption and in the clearance of systemically available drug may have contributed to the wide range of cyclophosphamide concentrations found at 1 and 24 h after tablet ingestion.

Validation of urinary excretion of cyclophosphamide as a biomarker of exposure by studying its renal clearance at high and low plasma concentrations in cancer patients

“…Thus, highly sensitive analytical methods for determination of CP in plasma and urine were developed and validated regarding LOD, precision and stability. The LODs in this study were lower compared to previous methods for determination of CP in plasma (Sadagopan et al 2001;Liu et al 2004) and urine Sannolo et al 1999;Pethran et al 2003;Kasel et al 2004). The precisions of our methods were satisfactory and the stability of CP in the samples was high.…”

Relationship of drug metabolizing enzyme genotype to plasma levels as well as myelotoxicity of cyclophosphamide in breast cancer patients

“…Plasma samples were purified by solid-phase extraction [15] using Bond Elut C18 cartridges, 1 ml (Varian). After conditioning with 1 ml acetonitrile and 1 ml water, plasma samples were loaded followed by washing with 2 ml water.…”

Determination of cyclophosphamide enantiomers in plasma by LC‐MS/MS: Application to pharmacokinetics in breast cancer and lupus nephritis patients

“…This compound is then transported to target tissues and transformed into phosphoramide mustard when entering cells, which produces cytotoxic events by alkylating nuclear DNA. 3 CPA is the most widely used alkylating agent in the treatment of hematological malignancies and of a variety of solid tumors, including leukemia, breast cancer, lung cancer, lymphomas, prostate cancer, ovarian cancer, and multiple myeloma. Although its role in the treatment of ovarian cancer and small-cell lung cancer is declining, CPA continues to be used in the treatment of advanced breast cancer as a critical component of the CMF (CPA, methotrexate and 5-fluorouracil), CEF (CPA, epirubicin and 5-fluorouracil), MVC (mitoxantrone, vinblastine and CPA), and TAC (docetaxel, doxorubicin and CPA) regimens.…”