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Metabolite interferences represent a major risk of inaccurate quantification when using LC–MS/MS bioanalytical assays. During LC–MS/MS bioanalysis of BMS-919194, a phosphate ester prodrug, in plasma samples from rat and monkey GLP toxicology studies, an unknown peak was detected in the MRM channel of the prodrug. This peak was not observed in previous discovery toxicology studies, in which a fast gradient LC–MS/MS method was used. We found out that this unknown peak would co-elute with the prodrug peak when the discovery method was used, therefore, causing significant overestimation of the exposure of the prodrug in the discovery toxicology studies. To understand the nature of this interfering peak and its impact to bioanalytical assay, we further investigated its formation and identification. The interfering compound and the prodrug were found to be isobaric and to have the same major product ions in electrospray ionization positive mode, thus, could not be differentiated using a triple quadrupole mass spectrometer. By using high-resolution mass spectrometry (HRMS), the interfering metabolite was successfully identified to be an isobaric sulfate metabolite of BMS-919194. To the best of our knowledge, this is the first report that a phosphate prodrug was metabolized in vivo to an isobaric sulfate metabolite, and this metabolite caused significant interference to the analysis of the prodrug. This work demonstrated the presence of the interference risk from isobaric sulfate metabolites to the bioanalysis of phosphate prodrugs in real samples. It is critical to evaluate and mitigate potential metabolite interferences during method development, therefore, minimize the related bioanalytical risks and ensure assay quality. Our work also showed the unique advantages of HRMS in identifying potential metabolite interference during LC–MS/MS bioanalysis. [ABSTRACT FROM AUTHOR]

BMS-986104 is a S1P 1 R modulator drug candidate under development and has been evaluated in Phase I clinical trials. BMS-986104 functions as a prodrug and undergoes enzymatic transformations in vivo to form the pharmacologically active phosphate drug, BMS-986104-P. Here, we report approaches to overcome the stability, solubility and extraction challenges in developing a sensitive, accurate and rugged LC–MS/MS method for the simultaneous quantification of the phosphate drug and its prodrug in blood lysate. An effective stabilization strategy using a cocktail of phosphatase and kinase inhibitors was developed to ensure the stability of both analytes during sample collection, storage, and processing. A combination of surfactant (CHAPS) and weak base (Tris) was found to be able to effectively improve the solubilization of the phosphate drug. The blood lysate samples were extracted by protein precipitation followed by solid-phase extraction using an Oasis HLB 96-well SPE plate. The method achieved acceptable matrix effect and recovery for the two analytes that have very different chemical properties. Stable-isotope labeled D6-BMS-986104 and D13-BMS-986104-P were utilized as the internal standards. Chromatographic separation was achieved using isocratic conditions on an Acquity UPLC BEH C18 analytical column. The two analytes and their internal standards were detected by positive ion electrospray tandem mass spectrometry. The calibration curves, which ranged from 2.00 to 2000 ng/mL for BMS-986104 and 4.00 to 4000 ng/mL for BMS-986104-P, were fitted to a 1/x2 weighted linear regression model. The intra-assay precision was within ±5.0% CV, inter-assay precision was within ±4.9% CV, and the assay accuracy was within ±5.8% of the nominal values for both analytes in rat blood lysate. The method was validated and successfully applied to support multiple pre-clinical toxicity studies. [ABSTRACT FROM AUTHOR]

Highlights: [•] An inter-well volume replacement workflow was introduced for DBS sample dilution. [•] An improved liquid–liquid extraction was used to improve analyte elution from DBS. [•] These strategies were applied to quantify BMS-927711, a drug candidate, in rat DBS. [•] Excellent assay robustness, precision and accuracy were achieved. [•] It has been applied to non-clinical toxicokinetic studies in rats. [Copyright &y& Elsevier]

Abstract: We have developed and validated an accurate, sensitive, and robust LC–MS/MS method that determines the concentration of CRx-102 (the combination of prednisolone and dipyridamole) in human plasma. In this method, prednisolone, dipyridamole, and the combined internal standards (IS) prednisolone-d6 (IS for prednisolone) and dipyridamole-d20 (IS for dipyridamole) were extracted from 100μL human EDTA plasma using methylbutyl ether. Calibration curves were linear over a concentration range of 0.4–200ng/mL for prednisolone and 5–3000ng/mL for dipyridamole. The analytes were quantitatively determined using tandem mass spectrometry operated in positive electrospray ionization in a multiple reaction monitoring (MRM) mode. This validated method has been used successfully in clinical pharmacokinetic studies of CRx-102 in healthy volunteers. [Copyright &y& Elsevier]

Highlights • A convenient and cost-effective strategy to overcome interference in LC–MS analysis. • This strategy minimizes the number of stable isotope labels used for avoiding interference. • A LC–MS/MS assay utilizing this strategy was developed for BMS-986205. • The developed assay was successfully applied to a dog absolute bioavailability study. • The strategy can also be applied to the selection of internal standards. Abstract 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 [13C 7, 15N]-BMS-986205 to its SIL-IS, [13C 7, 15N, 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 37Cl 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 [13C 7 , 15N]-BMS-986205 in dog plasma using [13C 7, 15N, 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. [ABSTRACT FROM AUTHOR]

• First application of in-sample calibration curve (ISCC) methodology in regulated bioanalysis. • Proposed method validation procedures/processes for ISCC–LC–MS/MS methods. • Successfully developed and validated an ISCC−LC–MS/MS method for BMS-984478. • Discussed the great potential of ISCC methodology for various quantitative applications. Traditionally, for a liquid chromatography tandem mass spectrometry (LC–MS/MS) bioanalytical assay, an external calibration curve is required to achieve accurate quantitation of an analyte. Recently, a novel in-sample calibration curves (ISCC) methodology that can achieve quick and accurate LC–MS/MS bioanalysis without the use of an external calibration curve was reported. The ISCC methodology utilizes the presence of multiple naturally occurring isotopologues of a stable isotopically labeled analyte to construct an in-sample calibration curve for the quantification. This methodology has great potential in many applications, for example biomarker measurement, quantitative proteomics and clinical diagnosis. Here, we assessed the feasibility of applying this ISCC–LC–MS/MS methodology in regulated bioanalysis using BMS-984478, a drug candidate, as the model compound. We also proposed method validation procedures/processes for this new approach for industry peers' consideration and feedback. A LC–MS/MS method using the ISCC strategy was successfully developed and validated for the quantitative analysis of BMS-984478 in human plasma over the range of 1.33–993.42 ng/mL. The validated ISCC-LC–MS/MS method was compared with a previously validated method using the conventional external calibration curve approach, and the two methods showed equivalent performance. Critical considerations and practical approaches in method development, validation and sample analysis were also discussed. Our work demonstrated that the ISCC–LC–MS/MS methodology is a promising approach for regulated LC–MS/MS bioanalysis. ISCC–LC–MS/MS methodology has its unique advantages and has great potential to be widely applied for various quantitative applications, and may even change the landscape of quantitative analysis. [ABSTRACT FROM AUTHOR]