Journal of Pharmaceutical and Biomedical Analysis 174 (2019) 168–174 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba Simultaneous determination of bendamustine and -hydroxybendamustine in mice dried blood spots and its application in a mice pharmacokinetic study Neeraj Kumar Saini a , Suresh P. Sulochana a , Vinay Kiran a , Sadanand Rangnathrao Mallurwar a , Wolfgang Richter b , Nuggehally R. Srinivas c , Ramesh Mullangi a,∗ a Drug Metabolism and Pharmacokinetics, Jubilant Biosys Ltd., Industrial Suburb, Yeshwanthpur, Bangalore, 560 022, India b TUBE Pharmaceuticals GmbH, Leberstr. 20, A-1110 Wien, Austria c Jubilant Generics Limited, Noida, 201 301, India a r t i c l e i n f o Article history: Received 8 February 2019 Received in revised form 4 May 2019 Accepted 23 May 2019 Available online 28 May 2019 Keywords: Bendamustine -Hydroxybendamustine LC-MS/MS DBS Method validation Mice blood Pharmacokinetics a b s t r a c t A selective, sensitive and rapid mice dried blood spot (DBS) method has been developed and validated for the simultaneous quantification of bendamustine (BM) and -hydroxy-bendamustine (HBM) as per regulatory guidelines using an LC-MS/MS. Quality control, calibration curve and study sample DBS cards were sonicated with 5% formic acid in water before extraction with ethyl acetate enriched with internal standard (I.S.). The organic layer was evaporated and residue was reconstituted in 0.1% formic acid in acetonitrile for LC-MS/MS analysis. Chromatographic resolution of both analytes (BM and HBM) and the I.S. (loperamide) was achieved on an Atlantis dC 18 column using 0.2% formic acid:acetonitrile (25:75, v/v) as an eluant delivered at a constant flow-rate of 0.5 mL/min. The total chromatographic run time was 3.2 min. The MS/MS ion transitions monitored were m/z 358.0 → 228.0, 374.0 → 338.0 and 477.0 → 210.0 for BM, HBM and the I.S, respectively. The assay was linear in the range of 5.65–2544 ng/mL for both BM and HBM. The within-run and between-run accuracy and within-run and between-run precision were in the range of 0.96–1.00 and 1.36–9.94%, respectively for BM; 0.88–1.03 and 4.57–11.7%, respectively for HBM on mice DBS cards. Stability studies showed that both analytes were stable at room temperature for 7 days and at −80 ◦ C for 55 days on DBS cards. The validated DBS method has been applied to a pharmacokinetic study in mice. © 2019 Elsevier B.V. All rights reserved. 1. Introduction Intravenous infusion of bendamustine (BM; Fig. 1) is used to treat various leukemia’s namely multiple myeloma, lympho- cytic and non-Hodgkin’s either as a single agent drug therapy or with other chemotherapy drugs such as etoposide, methotrex- ate, vincristine, bortezomib, rituximab etc [1–4]. BM is extensively metabolized by not only rapid hydrolysis to mono- and di- hydroxy metabolites, but also from traditional Phase-I and Phase-II metabolic pathways [5]. The oxidative Phase-I metabolism of BM, mainly mediated by CYP1A2, produces two active metabolites namely -hydroxybendamustine (HBM equipotent to BM) and N- ∗ Corresponding author at: Jubilant Biosys, Industrial Suburb, Yeshwanthpur, Ban- galore, 560022, India. E-mail address: mullangi.ramesh@jubilantinnovation.com (R. Mullangi). desmethylbendamustine (NDMBM ˜ 5-fold less active than BM) [5]. By undergoing Phase-II metabolism, BM generates sulphate, cys- teine and glutathione conjugates, which are amenable for excretory mechanisms. There are numerous HPLC [6,7] and LC-MS/MS [8–10] methods reported to date for the quantification of BM alone or along with its metabolites in various biological matrices which include blood, plasma, brain homogenate, urine and feces (Table 1). Till date, there is no dried blood spot (DBS) method reported for simultaneous quantitation of BM and HBM in any species blood, let alone human blood. Given the extensive use of BM in treating certain type of can- cers and continued exploratory clinical investigations of BM along with other anti-cancer agents including targeted therapies, there is the need of a DBS method that provides an opportunity for bioan- alytical assay with small sample volumes in a debilitating patient population. In this paper, we report the development and validation of a simple, selective and high-throughput DBS LC-MS/MS method https://doi.org/10.1016/j.jpba.2019.05.052 0731-7085/© 2019 Elsevier B.V. All rights reserved.