Investigation of the "true" extraction recovery of analytes from multiple types of tissues and its impact on tissue bioanalysis using two model compounds.

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 S1P1 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 3H-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.

UHPLC-MS/MS bioanalysis of urinary DHEA, cortisone and their hydroxylated metabolites as potential biomarkers for CYP3A-mediated drug-drug interactions.

AIM: A UHPLC-MS/MS assay was developed to quantify urinary dehydroepiandrosterone (DHEA), 7ß-hydroxy-DHEA, cortisone and 6ß-hydroxycortisone as potential biomarkers to predict CYP3A activity. RESULTS: A sensitive assay at LLOQ of 0.500 ng/ml with good accuracy and precision was developed for the four analytes in human urine. This UHPLC-MS/MS assay was optimized by eliminating nonspecific loss of the analytes in urine, ensuring complete hydrolysis of the conjugates to unconjugated forms and use of the product ions of [M+H-H2O]+ for multiple reaction monitoring detection of DHEA and 7ß-hydroxy-DHEA. CONCLUSION: This assay was successfully applied to a pilot clinical study. It is also suitable for future drug-drug interaction studies to continue evaluating the potential of these steroids as biomarkers for CYP3A inhibition and induction.

A simple, effective approach for rapid development of high-throughput and reliable LC-MS/MS bioanalytical assays.

BACKGROUND: Rapidly developing LC-MS/MS assays with high-throughput and quality are challenging yet desired. Methodology & results: A simple method development approach was reported and demonstrated with the quantitative bioanalysis of BMS-984478, a hepatitis C virus nonstructural protein 5A inhibitor. An accurate, precise and robust LC-MS/MS method for the quantitation of BMS-984478 in rat and monkey plasma was developed and validated. Incurred sample reanalysis evaluation passed with 100% of samples meeting the acceptance criteria. The validated assay was successfully applied in toxicology studies without any failed runs. CONCLUSION: The approach was successfully applied to the bioanalysis of BMS-984478 in toxicology and clinical studies. This approach was shown to be effective and reliable in speeding the development of high-throughput and reliable LC-MS/MS assays.

Bioanalysis of dried saliva spot (DSS) samples using detergent-assisted sample extraction with UHPLC-MS/MS detection.

Dried saliva spot (DSS) sampling is a non-invasive sample collection technique for bioanalysis that can be potentially implemented at the patient's home. A UHPLC-MS/MS assay was developed using detergent-assisted sample extraction to quantify BMS-927711, a drug candidate in development for the treatment of migraines, in human DSS. By implementing DSS sampling at the patients' home, the bioanalytical sample collection for pharmacokinetic evaluation can be done at the time of the acute migraine attack without the need for clinical visits. DSS samples were prepared by spotting 15 µL of liquid saliva onto regular Whatman FTA™ DMPK-C cards and verified with a UV lamp (at λ 254 nm or 365 nm) during DSS punching. The 4-mm DSS punches in a 96-well plate were sonicated with 200 µL of [(13)C2, D4]-BMS-927711 internal standard (IS) solution in 20/80 MeOH/water for 10 min, followed by sonication with 50 µL of 100 mM NH4OAc with 1.0% Triton-X-100 (as detergent) prior to liquid-liquid extraction with 600 µL EtOAc/Hexane (90:10). UHPLC-MS/MS was performed with an Aquity(®) UPLC BEH C18 Column (2.1 × 50 mm, 1.7 µm) on a Triple Quad™ 5500 mass spectrometer. The assay was linear with a concentration range from 2.00 to 1000 ng mL(-1) for BMS-927711 in human saliva. The intra- and inter-assay precision was within 8.8% CV, and the accuracy was within ±6.7% Dev of the nominal concentration values. This UHPLC-MS/MS assay has been successfully applied to determine the drug's pharmacokinetics within a clinical study. For the first time, we observed BMS-927711 exposure in human DSS, confirming the suitability of this sampling technique for migraine patients to use at home. Detergent-assisted extraction with Triton-X-100 could be very useful in DSS or other dried matrix spot (DMS) assays to overcome low or inconsistent analyte recovery issues.

Antibody-drug conjugate bioanalysis using LB-LC-MS/MS hybrid assays: strategies, methodology and correlation to ligand-binding assays.

BACKGROUND: Antibody-drug conjugates (ADCs) are complex drug constructs with multiple species in the heterogeneous mixture that contribute to their efficacy and toxicity. The bioanalysis of ADCs involves multiple assays and analytical platforms. METHODS: A series of ligand binding and LC-MS/MS (LB-LC-MS/MS) hybrid assays, through different combinations of anti-idiotype (anti-Id), anti-payload, or generic capture reagents, and cathepsin-B or trypsin enzyme digestion, were developed and evaluated for the analysis of conjugated-payload as well as for species traditionally measured by ligand-binding assays, total-antibody and conjugated-antibody. RESULTS & CONCLUSION: Hybrid assays are complementary or viable alternatives to ligand-binding assay for ADC bioanalysis and PK/PD modeling. The fit-for-purpose choice of analytes, assays and platforms and an integrated strategy from Discovery to Development for ADC PK and bioanalysis are recommended.

Recommendations on the Development of a Bioanalytical Assay for 4ß-Hydroxycholesterol, an Emerging Endogenous Biomarker of CYP3A Activity.

The availability of reliable assays for measuring 4ß-hydroxycholesterol (4ß-HC), a CYP3A metabolite of cholesterol, is an important step in qualifying this endogenous moiety as a biomarker of CYP3A activity. Liquid and gas chromatographic methods with mass spectrometric detection have been developed with varying sensitivities, with or without derivatization. Care must be taken to chromatographically resolve 4ß-HC from the multiple isobaric cholesterol oxidation products present in plasma, including 4α-hydroxycholesterol (4α-HC). Plasma concentrations of 4ß-HC are low in humans (10-60 ng/ml), lower than many other cholesterol metabolites and far less than cholesterol itself. Stability of 4ß-HC has been established for at least 12 months at -20°C in plasma samples obtained with a typical clinical workflow. Oxidation of plasma cholesterol during storage produces both 4ß-HC and 4α-HC, and 4α-HC may be used as assessment of sample quality. As 4ß-HC concentrations over time in untreated individuals have low intra-individual variability, assay precision and reproducibility are the key assay attributes in assessing CYP3A4 induction, and potentially inhibition. Assessment of CYP3A4/5 activity with 4ß-HC relies on the differences between pre- and post-dose concentrations, in which each subject acts as their own control. To reduce analytical variability, samples from a single subject should be analyzed together to facilitate interpretation of study results. As an endogenous biomarker, 4ß-HC offers the opportunity for less invasive assessment of CYP3A induction potential of new drugs during clinical development.

A validated enantioselective LC-MS/MS assay for quantification of a major chiral metabolite of an achiral 11-ß-hydroxysteroid-dehydrogenase 1 inhibitor in human plasma: Application to a clinical pharmacokinetic study.

BMS-823778 is a potent 11-ß-hydroxysteroid-dehydrogenase 1 (11ßHSD-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 0.200-200ng/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 2h on ice. The analytes were also stable in human plasma for 25h at room temperature, 34days 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.

Quantitation of a PEGylated protein in monkey serum by UHPLC-HRMS using a surrogate disulfide-containing peptide: A new approach to bioanalysis and in vivo stability evaluation of disulfide-rich protein therapeutics.

To quantify a therapeutic PEGylated protein in monkey serum as well as to monitor its potential in vivo instability and methionine oxidation, a novel ultra high performance liquid chromatography-high resolution mass spectrometric (UHPLC-HRMS) assay was developed using a surrogate disulfide-containing peptide, DCP(SS), and a confirmatory peptide, CP, a disulfide-free peptide. DCP(SS) was obtained by eliminating the step of reduction/alkylation before trypsin digestion. It contains an intact disulfide linkage between two peptide sequences that are essential for drug function but susceptible to potential in vivo cleavages. HRMS-based single ion monitoring (SIM) on a Q Exactive™ mass spectrometer was employed to improve assay specificity and sensitivity for DCP(SS) due to its poor fragmentation and low sensitivity with SRM detection. The assay has been validated for the protein drug in monkey serum using both surrogate peptides with excellent accuracy (within ±4.4%Dev) and precision (within 7.5%CV) with a lower limit of quantitation (LLOQ) at 10 ng mL(-1). The protein concentrations in monkey serum obtained from the DCP(SS)-based assay not only provided important pharmacokinetic parameters, but also confirmed in vivo stability of the peptide regions of interest by comparing drug concentrations with those obtained from the CP-based assay or from a ligand-binding assay (LBA). Furthermore, UHPLC-HRMS allowed simultaneous monitoring of the oxidized forms of both surrogate peptides to evaluate potential ex vivo/in vivo oxidation of one methionine present in each of both surrogate peptides. To the best of our knowledge, this is the first report of using a surrogate disulfide-containing peptide for LC-MS bioanalysis of a therapeutic protein.

A highly sensitive and selective LC-MS/MS method to quantify asunaprevir, an HCV NS3 protease inhibitor, in human plasma in support of pharmacokinetic studies.

Asunaprevir (BMS-650032) is a selective hepatitis C virus (HCV) NS3 protease inhibitor with potent activity against HCV genotypes 1, 4, 5 and 6. It has been developed in conjunction with direct-acting antiviral agents, in interferon- and ribavirin-free regimen, to improve existing therapies for HCV infection. To support the pharmacokinetic analyses in asunaprevir clinical studies, we have developed and validated a highly sensitive and robust LC-MS/MS method to quantify asunaprevir in human EDTA plasma with an LLOQ of 0.05ng/mL, which was a 20-fold sensitivity improvement over a previously reported assay for asunaprevir. A deuterated labeled [D9]-asunaprevir was used as the internal standard (IS). The analyte and the IS were extracted using a semi-automated liquid-liquid extraction (LLE) at pH 7 with methyl-t-butyl ether (MTBE) in a 96-well plate containing 10µL of 10% CHAPS as the surfactant to prevent non-specific binding issue. Chromatographic separation was achieved on a Genesis C8 column (2.1×50mm, 4µm) with a gradient elution using 0.1% formic acid in water as mobile phase A and a mixture of methanol: acetone: formic acid (95:5:0.1; v/v/v) as the mobile phase B. Positive electrospray ionization was performed using multiple reaction monitoring (MRM) with transitions of m/z 748→648 for asunaprevir and m/z 757→649 for [D9]-asunaprevir,and a collision energy of 30 electron Volts (eV). The assay was validated over a standard curve range from 0.05 to 50ng/mL for asunaprevir in human plasma. The intra- and inter assay precisions were within 7.1% CV, and the % deviation was within 5.5% of their nominal concentrations. This assay has been successfully applied to multiple clinical studies with excellent assay ruggedness and reproducibility.

Post-pellet-digestion precipitation and solid phase extraction: A practical and efficient workflow to extract surrogate peptides for ultra-high performance liquid chromatography--tandem mass spectrometry bioanalysis of a therapeutic antibody in the low ng/mL range.

The current LC-MS/MS approach for bioanalysis of protein therapeutics requires generating peptides from protein molecules via trypsin digestion, followed by sensitive detection of these surrogate peptides by multiple reaction monitoring (MRM). However, the presence of huge amount of matrix-related interference peptides generated from trypsin digestion often causes substantial matrix effect or isobaric interferences during LC-MS/MS analysis. Solid phase extraction (SPE) exhibits great potential in sample extraction to overcome these challenges due to its characteristic features of high selectivity, reproducibility, cost-effectiveness and potential to be automated. Here, we report an effective SPE methodology for the bioanalysis of protein therapeutics involving post-pellet-digestion precipitation and SPE cleanup prior to UHPLC-MS/MS analysis. Specifically, proteins in serum samples were first precipitated with methanol to enrich the protein analyte in the pellet prior to trypsin digestion of the pellet (pellet-digestion). The trypsin digest was further processed by a post-pellet-digestion precipitation (second precipitation) to remove matrix-related clog-causing components prior to SPE on OASIS™ MAX (Mixed-Mode Anion-Exchange) SPE plate. This methodology successfully overcame SPE clogging issue and enabled extraction of 100µL of monkey serum on SPE with significant sensitivity improvement to achieve a lower limit of quantitation (LLOQ) of 50ng/mL for a human monoclonal antibody of the IgG4 subclass. This UHPLC-MS/MS assay was validated in a concentration range of 50-5000ng/mL with intra- and inter-assay precisions of within 9.6% CV, and assay accuracy of within ±2.9% Dev of their nominal concentrations. To our best knowledge, this is the pellet digestion with SPE method for LC-MS/MS bioanalysis of a monoclonal antibody for the first time to achieve a LLOQ in the low ng/mL concentration range.

Dried blood spot analysis without dilution: Application to the LC-MS/MS determination of BMS-986001 in rat dried blood spot.

Sample dilution is one major challenge in dried blood spot (DBS) bioanalysis. To resolve this issue, we applied a no-dilution strategy for DBS analysis by using a calibration curve with very wide linear range. We developed an LC-MS/MS DBS assay with a linear range of 5 orders of magnitude (50-5000,000ng/mL) for BMS-986001, an HIV drug under development, by simultaneously monitoring two selective reaction monitoring transitions of different intensity. The assay was validated and successfully applied to the analysis of DBS samples collected in a toxicology study in rats dosed with BMS-986001. All samples were analyzed without any dilution. We also compared the concentration data generated from the DBS method and a validated plasma assay for the same study. The two sets of data agreed well with each other, demonstrating the validity of this strategy for DBS analysis. This approach provides an effective and convenient way to eliminate complicated dilution for DBS and other sample collection techniques.

Quantitative bioanalysis of antibody-conjugated payload in monkey plasma using a hybrid immuno-capture LC-MS/MS approach: Assay development, validation, and a case study.

Antibody drug conjugates (ADCs) are complex molecules composed of two pharmacologically distinct components, the cytotoxic payload and the antibody. The measurement of the payload molecules that are attached to the antibody in vivo is important for the evaluation of the safety and efficacy of ADCs, and can also provide distinct information compared to the antibody-related analytes. However, analyzing the antibody-conjugated payload is challenging and in some cases may not be feasible. The in vivo change in drug antibody ratio (DAR), due to deconjugation, biotransformation or other clearance phenomena, generates unique and additional challenges for ADC analysis in biological samples. Here, we report a novel hybrid approach with immuno-capture of the ADC, payload cleavage by specific enzyme, and LC-MS/MS of the cleaved payload to quantitatively measure the concentration of payload molecules still attached to the antibody via linker in plasma. The ADC reference material used for the calibration curve is not likely to be identical to the ADC measured in study samples due to the change in DAR distribution over the PK time course. The assay clearly demonstrated that there was no bias in the measurement of antibody-conjugated payload for ADC with varying DAR, which thus allowed accurate quantification even when the DAR distribution dynamically changes in vivo. This hybrid assay was fully validated based on a combination of requirements for both chromatographic and ligand binding methods, and was successfully applied to support a GLP safety study in monkeys.

Scientific or regulated validation: a tiered approach? Meeting report from a joint EBF/DVDMDG workshop.

Tiered approach is rapidly gaining interest in the regulated bioanalytical community. Alternative approaches to the workflows as proposed in the regulatory Guidance (US FDA, EMA) are being used in discovery and early drug development, but with a growing array of assay types and studies requiring bioanalytical support in early drug development, the bioanalytical community is discussing how to bring best value to support these studies. Recently, international industry groups like European Bioanalysis Forum and Global Bioanalysis Consortium have discussed and published on the opportunity and need to include tiered approach more systematically in the early drug development support. On the back of these discussions, the Delaware Valley Drug Metabolism Discussion Group together with the European Bioanalysis Forum organized a meeting in Langhorne (PA, USA) to discuss the hurdles and added value of tiered approach with stakeholders from the Bioanalysis, quality assurance and PK community. The discussions focused on proposing scientific validation for studies where there is currently a mixed use of regulatory and tiered approach workflows. The meeting was well attended and the presentations and panel discussions contributed to a better understanding of what the industry is proposing as future practice.

An integrated multiplatform bioanalytical strategy for antibody-drug conjugates: a novel case study.

BACKGROUND: The bioanalytical strategy for antibody-drug conjugates (ADC) includes numerous measurements integrally designed to provide comprehensive characterization of PK, PD and immunogenicity. This manuscript describes the utilization of reagents specifically tailored to an ADC with a microtubule polymerization inhibitor payload and cathepsin B cleavable linker. METHODS: The PK strategy includes the evaluation of physiological levels of total antibody, active ADC, total ADC, antibody-conjugated payload and unconjugated payload. These data are evaluated in the context of target and antidrug antibody levels to elucidate bioactive ADC. RESULTS & CONCLUSION: Herein, we discuss how this strategy has been applied and present our preliminary observations. Continuously evolving to meet pipeline demands, the integrated bioanalytical data will provide critical insights into the exposure-response relationship.

Challenges and solutions in the bioanalysis of BMS-986094 and its metabolites including a highly polar, active nucleoside triphosphate in plasma and tissues using LC-MS/MS.

BMS-986094, a nucleotide polymerase inhibitor of the hepatitis C virus, was withdrawn from clinical trials because of a serious safety issue. To investigate a potential association between drug/metabolite exposure and toxicity in evaluations conducted after the termination of the BMS-986094 development program, it was essential to determine the levels of BMS-986094 and its major metabolites INX-08032, INX-08144 and INX-09054 in circulation and the active nucleoside triphosphate INX-09114 in target and non-target tissues. However, there were many challenges in the bioanalysis of these compounds. The chromatography challenge for the extremely polar nucleoside triphosphate was solved by applying mixed-mode chromatography which combined anion exchange and reversed-phase interactions. The LC conditions provided adequate retention and good peak shape of the analyte and showed good robustness. A strategy using simultaneous extraction but separate LC analysis of the prodrug BMS-986094 and its major circulating metabolites was used to overcome a carryover issue of the hydrophobic prodrug while still achieving good chromatography of the polar metabolites. In addition, the nucleotide analytes were not stable in the presence of endogenous enzymes. Low pH and low temperature were required for blood collection and plasma sample processing. However, the use of phosphatase inhibitor and immediate homogenization and extraction were critical for the quantitative analysis of the active triphosphate, INX-09114, in tissue samples. To alleviate the bioanalytical complexity caused by multiple analytes, different matrices, and various species, a fit-for-purpose approach to assay validation was implemented based on the needs of drug safety assessment in non-clinical (GLP or non-GLP) studies. The assay for INX-08032 was fully validated in plasma of toxicology species. The lower limit of quantification was 1.00ng/mL and the linear curve range was 1.00-500.00ng/mL using a weighted (1/x(2)) linear regression model. Intra-assay and inter-assay precision (CV, %) ranged from 2.3% to 5.5% and accuracy within ±2.2% from nominal. INX-08032 was found to be stable in acidified mouse plasma for at least 24h in wet ice bath, 125 days at -70°C and following at least three freeze-thaw cycles. No endogenous components in plasma were found to interfere with the measurement. The extraction recovery was between 90% and 95%. The assays for BMS-986094, INX-08144, INX-09054 and INX-09114 were qualified with wider acceptance criteria for accuracy and precision. Analyte stability was also evaluated to guide sample collection, storage, and processing. These assays were successfully applied to an investigative toxicokinetic and tissue metabolite profiling study described in the article.

Utilizing Internal Standard Responses to Assess Risk on Reporting Bioanalytical Results from Hemolyzed Samples.

Bioanalytical analysis of toxicokinetic and pharmacokinetic samples is an integral part of small molecule drugs development and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the technique of choice. One important consideration is the matrix effect, in which ionization of the analytes of interest is affected by the presence of co-eluting interfering components present in the sample matrix. Hemolysis, which results in additional endogenous components being released from the lysed red blood cells, may cause additional matrix interferences. The effects of the degree of hemolysis on the accuracy and precision of the method and the reported sample concentrations from hemolyzed study samples have drawn increasing attention in recent years, especially in cases where the sample concentrations are critical for pharmacokinetic calculation. Currently, there is no established procedure to objectively assess the risk of reporting potentially inaccurate bioanalytical results from hemolyzed study samples. In this work, we evaluated the effect of different degrees of hemolysis on the internal standard peak area, accuracy, and precision of the analyses of BMS-906024 and its metabolite, BMS-911557, in human plasma by LC-MS/MS. In addition, we proposed the strategy of using the peak area of the stable isotope-labeled internal standard (SIL-IS) from the LC-MS/MS measurement as the surrogate marker for risk assessment. Samples with peak areas outside of the pre-defined acceptance criteria, e.g., less than 50% or more than 150% of the average IS response in study samples, plasma standards, and QC samples when SIL-IS is used, are flagged out for further investigation.

A UHPLC-MS/MS bioanalytical assay for the determination of BMS-911543, a JAK2 inhibitor, in human plasma.

Herein we report a rapid, accurate and robust UHPLC-MS/MS assay for the quantitation of BMS-911453, a Janus kinase 2 inhibitor under clinical development for the treatment of myeloproliferative disorders, in human plasma. A systematic method development approach was used to optimize the mass spectrometry, chromatography, and sample extraction conditions, and to minimize potential bioanalytical risks. The validated method utilizes stable-isotope labeled (13)C4-BMS-911543 as the internal standard. Liquid-liquid extraction was used for sample preparation. Chromatographic separation was achieved within 2min on a Zorbax Extend-C18 column with an isocratic elution. BMS-911543 and its internal standard were detected by positive ion electrospray tandem mass spectrometry. The assay range was from 1 to 500ng/mL, and the standard curve was fitted with 1/x(2) weighted linear regression. The intra-assay precision was within 5.0% CV and the inter-assay precision was within 2.6% CV. The inter-assay mean accuracy, expressed as percents of theoretical, was between 99.8% and 102.3%. The assay has high recovery (∼80%) and minimal matrix effect (0.95-1.00). BMS-911543 was stable in human plasma for at least 24h at room temperature, 90 days at -20°C, and following three freeze-thaw cycles. The validated method was successfully applied to sample analysis in clinical studies.

"Center punch" and "whole spot" bioanalysis of apixaban in human dried blood spot samples by UHPLC-MS/MS.

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.

Selective reaction monitoring of negative electrospray ionization acetate adduct ions for the bioanalysis of dapagliflozin in clinical studies.

Dapagliflozin (Farxiga), alone, or in the fixed dose combination with metformin (Xigduo), is an orally active, highly selective, reversible inhibitor of sodium-glucose cotransporter type 2 (SGLT2) that is marketed in United States, Europe, and many other countries for the treatment of type 2 diabetes mellitus. Here we report a liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical assay of dapagliflozin in human plasma. A lower limit of quantitation (LLOQ) at 0.2 ng/mL with 50 µL of plasma was obtained, which reflects a 5-fold improvement of the overall assay sensitivity in comparison to the previous most sensitive assay using the same mass spectrometry instrumentation. In this new assay, acetate adduct ions in negative electrospray ionization mode were used as the precursor ions for selective reaction monitoring (SRM) detection. Sample preparation procedures and LC conditions were further developed to enhance the column life span and achieve the separation of dapagliflozin from potential interferences, especially its epimers. The assay also quantifies dapagliflozin's major systemic circulating glucuronide metabolite, BMS-801576, concentrations in human plasma. The assay was successfully transferred to contract research organizations (CROs), validated, and implemented for the sample analysis of pediatric and other critical clinical studies. This assay can be widely used for bioanalytical support of future clinical studies for the newly approved drug Farxiga or any combination therapy containing dapagliflozin.