Drug Res (Stuttg) 2017; 67(09): 497-508
DOI: 10.1055/s-0043-108124
Original Article
© Georg Thieme Verlag KG Stuttgart · New York

Sensitive LC-MS/MS Method for the Simultaneous Determination of Bendamustine and its Active Metabolite, γ-Hydroxybendamustine in Small Volume Mice and Dog Plasma and its Application to a Pharmacokinetic Study in Mice and Dogs

Devaraj V. Chandrashekar
1   Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Yeshwanthpur, Bangalore, India
,
Ponnayyan S Suresh
1   Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Yeshwanthpur, Bangalore, India
,
Rajnish Kumar
1   Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Yeshwanthpur, Bangalore, India
,
Ravi Kanth Bhamidipati
1   Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Yeshwanthpur, Bangalore, India
,
Ramesh Mullangi
1   Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd, Yeshwanthpur, Bangalore, India
,
Wolfgang Richter
2   TUBE Pharmaceuticals GmbH, Wien, Austria
,
Nuggehally R Srinivas
3   Suramus Bio, Drug Development, J. P. Nagar, Bangalore, India
› Author Affiliations
Further Information

Publication History

received 13 January 2017

accepted 29 March 2017

Publication Date:
30 May 2017 (online)

Abstract

A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the simultaneous quantification of bendamustine (BM) and γ-hydroxybendamustine (HBM) in small volume (20 µL) mice and dog plasma using phenacetin as an internal standard (IS) as per regulatory guidelines. Both the analytes and IS were extracted from mice and dog plasma using a liquid-liquid extraction method. Chromatography was achieved on Atlantis dC18 column using an isocratic mobile phase (0.2% formic acid:acetonitrile, 25:75) at a flow rate of 0.40 mL/min. The total chromatographic run time was 3.0 min and the elution of BM, HBM and IS occurred at ~1.2, 1.2 and 2.0 min, respectively. A linear response function was established 0.11–518 ng/mL for both the analytes in mice and dog plasma. The intra- and inter-day accuracy and precisions were in the range of 3.46–12.9 and 3.63–8.23%; 1.15–9.00 and 7.86–9.49% for BM and HBM, respectively in mice plasma and 2.15–6.49 and 1.73–13.1%; 4.35–13.9 and 4.33–10.5% for BM and HBM, respectively in dog plasma. This novel method has been applied to a pharmacokinetic study in mice and dogs.

 
  • References

  • 1 Kahl C, Herold M, Höffkes HG. et al. Bendamustine, methotrexate, mitoxantrone and prednisolone (BMMP) for the treatment of relapsed or refractory high-grade non-Hodgkin’s lymphoma. Onkologie 1997; 20: 406-408
  • 2 Fowler N, Kahl BS, Lee P. et al. Bortezomib, bendamustine, and rituximab in patients with relapsed or refractory follicular lymphoma: the phase II VERTICAL study. J Clin Oncol 2011; 29: 3389-3395
  • 3 Tardy MP, Gastaud L, Ojeda-Uribe M. et al. Z-BeEAM (Ibritumomab tiuxetan, Bendamustine, Etoposide, Cytarabine, Melphalan) before autologous stem cell transplantation is safe and efficient for refractory large B-cell lymphoma. Experimental Hematology & Oncology 2015; 4: 18
  • 4 von Minckwitz G, Chernozemsky I, Sirakova L. et al. Bendamustine prolongs progression-free survival in metastatic breast cancer (MBC): A phase III prospective, randomized, multicenter trial of bendamustine hydrochloride, methotrexate and 5-fluorouracil (BMF) versus cyclophosphamide, methotrexate and 5-fluorouracil (CMF) as first-line treatment of MBC. Anticancer Drugs 2005; 16: 871-877
  • 5 Dubbelman AC, Tibben M, Rosing H. et al. Development and validation of LC-MS/MS assays for the quantification of bendamustine and its metabolites in human plasma and urine. J Chromatogr B 2012; 893-894: 92-100
  • 6 He L, Grecula JC, Ling Y. et al. Development and validation of sensitive liquid chromatography/tandem mass spectrometry method for quantification of bendamustine in mouse brain tissue. J Chromatogr B 2012; 905: 141-144
  • 7 Xie F, Cheng Z, Cheng H. et al. Simultaneous determination of bendamustine and its active metabolite, gamma-hydroxy-bendamustine in human plasma and urine using HPLC-fluorescence detector: Application to a pharmacokinetic study in Chinese cancer patients. J Chromatogr B 2015; 960: 98-104
  • 8 Plenis A, Frolow A, Rekowska N. et al. Determination of bendamustine in human plasma and urine by LC-FL methods: Application in a drug monitoring. Chromatographia 2016; 79: 861-873
  • 9 US DHHS, FDA, CDER, CVM. Guidance for Industry: Bioanalytical method validation. U.S. department of health and human services, food and drug administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM). 2001. Rockville, MD, USA:
  • 10 Bonfiglio R, King RC, Olah TV. et al. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun Mass Spectrom 1999; 13: 1175-1185
  • 11 European Medicines Agency. Guideline on validation of bioanalytical methods. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf