Validation of a novel procedure for quantification of the formation of phosphoramide mustard by individuals treated with cyclophosphamide

Cancer Chemother Pharmacol. 2014 Sep;74(3):549-58. doi: 10.1007/s00280-014-2524-7. Epub 2014 Jul 23.


Purpose: Use of the patient's body surface area (mg m(-2)) as a basis for dosing does not take individual variation in metabolic capacity and rate of clearance into account. Here, we evaluated a novel approach for individual monitoring of short-lived cytotoxic agents formed from cytostatic drugs such as cyclophosphamide (CP).

Methods: The accumulated blood dose of the cytotoxic active agent phosphoramide mustard (PAM) formed from CP was measured as a reaction product with hemoglobin (Hb adduct). This adduct, N-[2-(2-oxazolidonyl)ethyl]-valyl Hb (OzVal-Hb), was detached from Hb with the adduct FIRE procedure™, and the formed analyte was quantified using LC-MS/MS. This dose biomarker for PAM and the analytical procedure was evaluated in accordance with the guidelines on bioanalytical method validation formulated by the European Medicine Agency. The evaluated method was applied to quantify blood dose levels of PAM in female breast cancer patients (n = 12) before and after three cycles of polychemotherapy regimes containing CP.

Results: OzVal-Hb, a specific and stable biomarker, could be measured with great sensitivity (lower limit of quantification = 33 pmol g(-1) Hb), high accuracy (within ±20 %) and good repeatability (CV < 20 %). The inter-individual variability in the blood level of this adduct in women with breast cancer (n = 12) who received three doses of CP in combination with one or two other cytostatic drugs was 250 % following the first dose and approximately 150 % after each subsequent dose.

Conclusions: Measurement of the biomarker OzVal-Hb can be used to quantify the short-lived cytotoxic agent PAM in a single blood sample drawn several days after therapy. This procedure may aid in individualizing doses of CP, thereby improving efficacy while both reducing the risk of and increasing the predictability of side-effects.

Publication types

  • Randomized Controlled Trial
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Antineoplastic Combined Chemotherapy Protocols / pharmacology
  • Antineoplastic Combined Chemotherapy Protocols / therapeutic use
  • Biomarkers
  • Biomarkers, Pharmacological / blood
  • Blood Chemical Analysis / methods*
  • Breast Neoplasms / drug therapy
  • Calibration
  • Cyclophosphamide / pharmacology*
  • Female
  • Hemoglobins / chemistry
  • Hemoglobins / metabolism
  • Humans
  • Limit of Detection
  • Phosphoramide Mustards / blood*
  • Tandem Mass Spectrometry / methods


  • Biomarkers
  • Biomarkers, Pharmacological
  • Hemoglobins
  • Phosphoramide Mustards
  • phosphoramide mustard
  • Cyclophosphamide