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BMS-823778 is a potent 11-β-hydroxysteroid-dehydrogenase 1 (11βHSD-1) inhibitor and a potential therapeutic agent for type 2 diabetes mellitus (T2DM). A high performance liquid chromatography-tandem mass spectrometry (LC–MS/MS) assay was developed and validated to enable reliable separation and quantification of both enantiomers of a chiral hydroxy metabolite (BMT-094817) in human plasma. Following liquid–liquid extraction in a 96-well plate format, chromatographic separation of the metabolite enantiomers was achieved by isocratic elution on a Chiralpak IA-3 column. Chromatographic conditions were optimized to ensure separation of both metabolite enantiomers. Metabolite enantiomers and stable isotope-labeled (SIL) internal standards were detected by positive ion electrospray tandem mass spectrometry. The LC–MS/MS assay was validated over a concentration range of 0.200–200 ng/mL. Intra- and inter-assay precision values for replicate quality control samples were less than 9.9% for both enantiomers during the assay validation. Mean quality control accuracy values were within ±7.3%. Assay recoveries were high (>75%) and consistent across the assay range. The metabolite enantiomers were stable in human blood for 2 h on ice. The analytes were also stable in human plasma for 25 h at room temperature, 34 days at −20 °C and −70 °C, and following five freeze-thaw cycles. No interconversion of the metabolite enantiomers was detected under any bioanalytical stress conditions, from blood collection/processing through extracted sample storage. The validated assay was successfully applied to the quantification of both metabolite enantiomers in human plasma in support of a human pharmacokinetic study. [ABSTRACT FROM AUTHOR]

RATIONALE Sensitivity is one major challenge limiting the application of liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods for bioanalysis of proteins. A novel selective peptide derivatization (SPD) strategy was proposed to improve assay sensitivity. The main concept of the SPD strategy is to selectively derivatize surrogate peptides of the target protein in the digests, while not derivatizing the abundant background peptides, thereby improving the separation of target peptides during sample extraction and chromatography, and increasing the sensitivity. Additional benefits may help improve sensitivity include (1) increased ionization efficiency; (2) improved fragmentation pattern; and (3) increased sample extraction recovery of target peptides. METHODS Feasibility assessment of the SPD strategy was conducted using BMS-986012, a monoclonal antibody, as the model protein, and with malondialdehyde (MDA) to selectively derivatize the arginine-containing surrogate peptide SLIY in tryptic-digested monkey serum samples. RESULTS The decreased polarity and basicity of the MDA-derivatized peptide SLIY (MDA-SLIY) helped improve its separation from the background peptides during solid-phase extraction (SPE) and chromatography. The recovery of MDA-SLIY was 36.1-44.2%, which was ~3-fold higher than the recovery of peptide SLIY (11.9-16.1%). There was no significant ion suppression for MDA-SLIY. Overall, SPD improved the sensitivity ~5-fold. SPD methodology was then successfully applied to the development of a sensitive LC/MS/MS assay for BMS-986012 in monkey serum. CONCLUSIONS This work demonstrates the feasibility of the SPD strategy for sensitivity enhancement. SPD can provide a simple, cost-efficient, and antibody-free sample preparation approach to improve sensitivity. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Apixaban (Eliquis™) was developed by Bristol-Myers Squibb (BMS) and Pfizer to use as an antithrombotic/anticoagulant agent and has been recently approved for the prevention of stroke and systemic embolism in patients with nonvalvular atrial fibrillation. A clinical study of apixaban, sponsored by BMS and Pfizer, included a pilot exploratory portion to evaluate the potential for future drug concentration monitoring using dried blood spot (DBS) sample collection. For DBS sample collection, a fixed blood volume was dispensed onto a DBS card by either regular volumetric pipette (venous blood collection) or capillary dispenser (finger prick blood collection). A 96-well semi-automated liquid–liquid extraction sample preparation procedure was developed to provide clean extracts for UHPLC–MS/MS quantitation. Assays using both partial-spot center punch and whole spot punch were developed and validated. The linear dynamic ranges for all the analyses were from 0.5 to 500 ng/mL. The coefficient of determination ( r 2 ) values was >0.9944 for all the validation runs. For the center punch approach, the intra-assay precision (%CV) was within 4.4% and inter-assay precision was within 2.6%. The assay accuracy, expressed as %Dev., was within ±5.4% of the nominal concentrations. One accuracy and precision run was performed using the whole spot approach, the intra-assay precision (%CV) was within 7.1% and the accuracy was within ±8.0% of the nominal concentrations. In contrast to the center punch approach, the whole spot approach eliminated the effect of hematocrit and high lipids on the analysis of apixaban in human DBS when an accurate sample blood volume was collected on DBS cards. [ABSTRACT FROM AUTHOR]

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]

LC-MS/MS has been widely applied to the quantitative analysis of tissue samples. However, one key remaining issue is that the extraction recovery of analyte from spiked tissue calibration standard and quality control samples (QCs) may not accurately represent the “true” recovery of analyte from incurred tissue samples. This may affect the accuracy of LC-MS/MS tissue bioanalysis. Here, we investigated whether the recovery determined using tissue QCs by LC-MS/MS can accurately represent the “true” recovery from incurred tissue samples using two model compounds: BMS-986104, a S1P 1 receptor modulator drug candidate, and its phosphate metabolite, BMS-986104-P. We first developed a novel acid and surfactant assisted protein precipitation method for the extraction of BMS-986104 and BMS-986104-P from rat tissues, and determined their recoveries using tissue QCs by LC-MS/MS. We then used radioactive incurred samples from rats dosed with 3 H-labeled BMS-986104 to determine the absolute total radioactivity recovery in six different tissues. The recoveries determined using tissue QCs and incurred samples matched with each other very well. The results demonstrated that, in this assay, tissue QCs accurately represented the incurred tissue samples to determine the “true” recovery, and LC-MS/MS assay was accurate for tissue bioanalysis. Another aspect we investigated is how the tissue QCs should be prepared to better represent the incurred tissue samples. We compared two different QC preparation methods (analyte spiked in tissue homogenates or in intact tissues) and demonstrated that the two methods had no significant difference when a good sample preparation was in place. The developed assay showed excellent accuracy and precision, and was successfully applied to the quantitative determination of BMS-986104 and BMS-986104-P in tissues in a rat toxicology study. [ABSTRACT FROM AUTHOR]

• LC/MS quantification of leukotoxin/leukotoxin diol in mouse plasma. • Streamlined sample prep under 10 min. • Reverse Phased UPLC < 4 min per injection. • PD biomarker movement demonstrated using 25 µL plasma per sample. LC/MS quantification of leukotoxin (LTX) and leukotoxin diol (LTXdiol) in plasma has been previously reported, however large sample volumes are required for achieving stated assay Lower Limit of Quantification (LLOQ). Reported here is a fit-for-purpose LC/MS method that reduces plasma volume from 700 to 25 µL and omits pre-concentration steps. These improvements make for a method with increased utility in mouse studies offering limited sample volumes. Additionally, omitting pre-concentration steps streamlines sample processing, which can now be completed in under 10 min. This method can be used to quickly answer if the ratio of LTX to LTXdiol changes with the dose of the therapeutic drug so this could be used as a potential biomarker for correlating PK/PD effects. No extensive assay characterization was performed before application to an exploratory in-life study. Basal levels of LTX and LTXdiol in plasma were quantified by LC-MRM across 10 individual mice, and the average signal-to-noise was 36 for LTX and 3039 for LTXdiol, with CVs of 29.4% and 15.2%, respectively. Addition of LTX and LTXdiol reference standard at 5, 25, and 75 ng/mL into pooled mouse plasma was quantifiable within 30% relative error using a surrogate matrix calibration curve ranging from 0.8 to 200 ng/mL. The average ratio of LTX to LTXdiol across the 10 mice was 0.32, consistent with previous reports. Finally, the method was applied to a mouse PK/PD study to monitor LTX/LTXdiol kinetics after a single oral dose of a soluble epoxide hydrolase inhibitor. The mean plasma ratio of LTX to LTXdiol increased up to 10-fold by 3 h post-dose followed by a decrease to near pre-dose levels by 24 h, consistent with transient inhibition of sEH-mediated conversion of LTX to LTXdiol. The method improvements described here will make subsequent quantification of LTX and LTXdiol in mouse studies significantly easier. [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]