Abstract: HHS Public Access Author manuscript Author Manuscript J Chromatogr B Analyt Technol Biomed Life Sci. Author manuscript; available in PMC 2019 January 01. Published in final edited form as: J Chromatogr B Analyt Technol Biomed Life Sci. 2018 January 01; 1072: 320–327. doi:10.1016/ j.jchromb.2017.11.026. Simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues using LC–ESI–MS/MS: An application for pharmacokinetic studies Yashpal S. Chhonker1,*, Richard L Sleightholm2,*, Jing Li2, David Oupický2,3, and Daryl J. Murry1,3,∆ Author Manuscript 1Department of Pharmacy Practice, University of Nebraska Medical Center, Omaha, NE 68198, United States 2Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, United States 3Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE 68198, United States Abstract Author Manuscript Hydroxychloroquine (HCQ) has been shown to disrupt autophagy and sensitize cancer cells to radiation and chemotherapeutic agents. However, the optimal delivery method, dose, and tumor concentrations required for these effects are not known. This is in part due to a lack of sensitive and reproducible analytical methods for HCQ quantitation in small animals. As such, we developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of hydroxychloroquine and its metabolites in mouse blood and tissues. The chromatographic separation and detection of analytes were achieved on a reversed phase Thermo Aquasil C18 (50 × 4.6 mm, 3μ) column, with gradient elution using 0.2 % formic acid and 0.1% formic acid in methanol as mobile phase at a flow rate of 0.5 mL/min. Simple protein precipitation was utilized for extraction of analytes from the desired matrix. Analytes were separated and quantitated using MS/MS with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode. The MS/MS response was linear over the concentration range from 1–2000 ng/mL for all analytes with a correlation coefficient (R2) of 0.998 or better. The within- and between-day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was successfully applied to a preclinical pharmacokinetic mouse study involving low volume blood and tissue samples for hydroxychloroquine and metabolites. Author Manuscript ∆ Corresponding author: Daryl J. Murry, Pharm. D., Department of Pharmacy Practice, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198-6025, Phone: 402-559-3790 (office), 402-559-2430 (lab), dj.murry@unmc.edu. *Authors contributed equally to this manuscript Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of interest There is no conflict of interest to disclose. Chhonker et al. Page 2 Author Manuscript Keywords LC-MS/MS; Hydroxychloroquine;..