Journal of Pharmaceutical and Biomedical Analysis 165 (2019) 198–206 Contents lists available at ScienceDirect Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba A convenient strategy to overcome interference in LC-MS/MS analysis: Application in a microdose absolute bioavailability study Long Yuan a,∗ , Christine Huang b , Peggy Liu-Kreyche b , Kimberly Voronin c , R. Marcus Fancher b , Alban Allentoff c , Naiyu Zheng a , Ramaswamy Iyer b , Li Zhu d , Renuka Pillutla a , Qin C. Ji a a Bioanalytical Sciences, Bristol-Myers Squibb, Princeton, NJ, 08543, USA b Pharmaceutical Candidate Optimization, Bristol-Myers Squibb, Princeton, NJ, 08543, USA c Radiochemistry, Bristol-Myers Squibb, Princeton, NJ, 08543, USA d Clinical Pharmacology & Pharmacometrics, Bristol-Myers Squibb, Princeton, NJ, 08543, USA a r t i c l e i n f o Article history: Received 26 October 2018 Received in revised form 5 December 2018 Accepted 8 December 2018 Keywords: Interference Isotopic contribution Stable isotopically labelled (SIL) compounds Microdose absolute bioavailability study Bioanalysis Quantitative LC-MS/MS a b s t r a c t Stable isotope labeled (SIL) compounds have been commonly used as internal standards (IS) to ensure the accuracy and quality of liquid chromatography–mass spectrometry (LC–MS) bioanalytical assays. Recently, the application of SIL drugs and LC–MS assays to microdose absolute bioavailability (BA) studies has gained increasing attention. This approach can provide significant cost and time saving, and higher data quality compared to the accelerator mass spectrometry (AMS)-based method, since it avoids the use of radioactive drug, high-cost AMS instrumentation and complex measurement processes. It also eliminates potential metabolite interference with AMS-based assay. However, one major challenge in the application of this approach is the potential interference between the unlabeled drug, the microdose SIL drug, and the SIL-IS during LC–MS analysis. Here we report a convenient and cost-effective strategy to overcome the interference by monitoring the isotopic ion (instead of the commonly used monoisotopic ion) of the interfered compound in MS analysis. For the BMS-986205 absolute BA case study presented, significant interference was observed from the microdose IV drug [ 13 C 7, 15 N]-BMS-986205 to its SIL-IS, [ 13 C 7, 15 N, D 3 ]-BMS-986205, since the difference of nominal molecular mass between the two compounds is only 3 mu, and there is a Cl atom in the molecules. By applying this strategy (monitoring the 37 Cl ion for the analysis of the IS), a 90-fold reduction of interference was achieved, which allowed the use of a synthetically accessible SIL compound and enabled the fast progress of the absolute BA study. This strategy minimizes the number of stable isotope labels used for avoiding interference, which greatly reduces the difficulty in synthesizing the SIL compounds and generates significant time and cost savings. In addition, this strategy can also be used to reduce the MS response of the analyte, therefore, avoiding the detector saturation issue of LC–MS/MS assay for high concentration BMS-986205. A LC–MS/MS assay utilizing this strategy was successfully developed for the simultaneous analysis of BMS-986205 and [ 13 C 7 , 15 N]-BMS- 986205 in dog plasma using [ 13 C 7, 15 N, D 3 ]-BMS-986205 as the IS. The assay was successfully applied to a microdose absolute BA study of BMS-986205 in dogs. The assay was also validated in human plasma and used to support a human absolute BA study. The same strategy can also be applied to other compounds, including those not containing Cl or other elements with abundant isotopes, or other applications (e.g. selection of internal standard), and the applications were presented. © 2018 Published by Elsevier B.V. 1. Introduction Stable isotopically labeled (SIL) compounds are non-radioactive analogs of the unlabeled compounds, in which one or more atoms are substituted by the corresponding stable isotopes, e.g., 13 C, 2 H ∗ Corresponding author. E-mail address: long.yuan@bms.com (L. Yuan). (deuterium or D), 15 N and 18 O. The physicochemical properties of SIL compounds are very similar, and often considered “identical”, to the unlabeled compounds, which leads to very similar behav- ior and performance between SIL compounds and their unlabeled version in vitro and in vivo. The most common application of SIL compounds, which utilizes their in vitro similarity, is their use as internal standards (IS) for liquid chromatography–mass spectrom- etry (LC–MS) based quantitative assays. A SIL-IS performs much better than a structural analog IS on tracking and compensating for https://doi.org/10.1016/j.jpba.2018.12.014 0731-7085/© 2018 Published by Elsevier B.V.