Imatinib method validation by high performance liquid chromatography in tandem mass spectrometry in human plasma to correlate with biochemical toxicity in patients with chronic myeloid leukemia

A Wojnicz1,3, JM Moreno1,3, MH Dumas Alonso4, T Cabaleiro1,3, MF Cano-Abad1,3, F Abad Santos1,3, C Rodriguez Antona5, JL Steegmann4, A Ruiz Nuno1,3. 1Instituto Teófilo Hernando, Faculty of Medicine, Universidad Autónoma de Madrid, 28029, Spain, 2Department of Pharmacology and Therapeutics, Faculty of Medicine, Universidad Autónoma de Madrid, 28029, Spain, 3Service of Clinical Pharmacology Health Research Institute, Hospital Universitario de la Princesa, 28006, Spain, 4Service of Hematology, Health Research Institute, Hospital Universitario de la Princesa, 28006, Spain, 5Spanish National Cancer Research Centre, Madrid, 28029, Spain

Introduction: Imatinib is a selective competitive inhibitor of protein tyrosine kinase (TKI), mainly used in the treatment of chronic myeloid leukemia (CML) Philadelphia chromosome positive (Ph+) and other types of cancers. We have developed a method of liquid chromatography in tandem with mass spectrometry (LC/MS-MS) to monitor therapeutic levels of imatinib in plasma. We measure imatinib plasmatic level in the valley before the morning dose.

Hipothesis: Our goal will be to study the hypophosphatemia and another possible biochemical toxicity to correlate with TKI´s plasma levels in CML patients.

Methods: After solid phase extraction (SPE) of plasma samples, imatinib and its internal standard (IS), imatinib-D8, were eluted with Zorbax SB-C-18 at 60ºC, under isocratic conditions through a mobile phase consisting of 4 mM ammonium formate, pH: 3.2 (solution A) and acetonitrile (ACN) solution B. The flow rate was 0.8 mL/min with 55% solution A + 45% solution B. Imatinib is detected and quantified by mass spectrometry with electrospray ionization (ESI) that operates in multiple reaction monitoring (MRM) mode.

Results: The calibration curve was linear in the range 10-5000 ng/mL, being 10 ng/mL the lower limit of quantitation (LLOQ). We have also developed methods to monitor therapeutic levels of nilotinib and dasatinib in plasma. The method for imatinib has been validated according to the recommendations of the Food and Drug Administration (FDA), including tests of matrix effect (bias<10%; n=6) and recovery efficiency (> 80% and <120%; n=6). The method is precise (coefficient of variance (CV) intra-day <2%; n=15 and inter-day CV<7%; n=10), accurate (between 95-108%), sensitive and specific, showing no interference peaks and retention times in the presence of other structurally similar drugs as nilotinib or dasatinib. We have already measured the plasmatic levels of imatinib in 44 leukemic patients from Service of Hematology, Hospital Universitario de la Princesa, Madrid. Since, Berman et al. (2006) found that in some patients treated with imatinib, hypophosphatemia was developed with serum phosphate levels lower than 2.5 mg/dL (0.8 mmol/L). We will compare our results of imatinib plasma levels with hypophosphatemia frequency in CML patients.

Conclusions: Our method of imatinib determination by high performance liquid chromatography in tandem mass spectrometry is simple, with very fast recording time (1.2 min) that would assess adherence to treatment of patients and pharmacokinetic-pharmacodynamic relationships. Thereby improving the pharmacological treatment of patients, either by adjusting doses or substituting imatinib by new drugs such as dasatinib and nilotinib. These results will improve the pharmacological treatment and life quality of our patients.

References: Berman, E. et al. Altered bone and mineral metabolism in patients receiving imatinib mesylate. N Engl J Med. 2006;354(19):2006-13.