Utilization of In Vitro, In Vivo and In Silico Tools to Evaluate the pH-Dependent Absorption of a BCS Class II Compound and Identify a pH-Effect Mitigating Strategy.

PURPOSE: To describe a stepwise approach to evaluate the pH effect for a weakly basic drug by in vitro, in vivo and in silico techniques and identify a viable mitigation strategy that addresses the risk. METHODS: Clinical studies included assessment of the pH effect with famotidine. In vitro dissolution was evaluated in various biorelevant media and in a pH-shift test. PK studies in dogs were conducted under pentagastrin or famotidine pre-treatment and GastroPlus was employed to model human and dog PK data and simulate the performance in human. RESULTS: Clinical data indicated considerable pH dependent absorption of the drug when dosed in the presence of H2-antagonists. In vitro dissolution and in vivo dog data confirmed that the observed pH effect was due to reduced dissolution rate and lower solubility at increased gastric and intestinal pH. A salt form was identified to overcome the effect by providing fast dissolution and prolonged supersaturation. GastroPlus simulations predicted a mitigation of the pH effect by the salt. CONCLUSIONS: The drug exhibited a strong pH-effect in humans. The in vitro, in vivo and modeling approach provides a systematic workflow to evaluate the risk of a new drug and identify a strategy able to mitigate the risk.

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.

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.

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.

Improved liquid-liquid extraction with inter-well volume replacement dilution workflow and its application to quantify BMS-927711 in rat dried blood spots by UHPLC-MS/MS.

An UHPLC-MS/MS method was developed and validated to quantify BMS-927711, a drug candidate to treat migraine, in rat dried blood spots (DBS). The DBS samples were extracted using an improved liquid-liquid extraction (LLE) strategy involving in the sonication of DBS punches in 20% MeOH aqueous solution containing the internal standard, [(13)C2, D4]-BMS-927711, and then with a 100mM NH4OAc buffer solution, followed by an automated LLE with EtOAc-hexane (70:30, v/v). The presence of 20% MeOH as an organic modifier in the elution solution significantly improved the analyte elution efficiency and assay performance. A novel inter-well volume replacement dilution workflow was introduced for DBS sample dilution before LLE step. This was a simple two-step process, firstly a small portion of the DBS blank solution was discarded, and then the same volume of a concentrated DBS sample solution was spiked into the leftover blank solution to achieve a desired dilution. Chromatographic separation was achieved on an Acuity UPLC(®) BEH C18 column (2.1mm×50mm, 1.7µm) and the analyte was detected by selected reaction monitoring (SRM) with positive electrospray ionization on an AB Sciex Triple Quad 5500 mass spectrometer. The standard curve was linear from 5.00 to 5000ng/mL with assay precision ≤4.9% CV, and assay accuracy within ±3.1%Dev of the nominal values. Accurate sample dilution was achieved by using inter-well volume replacement with a precision of ≤4.2% CV and an accuracy of ±3.3% for dilution QC at 50,000ng/mL with 100-fold dilution (n=18). This robust UHPLC-MS/MS assay has been successfully applied to the non-clinical studies in rats. By using inter-well volume replacement workflow, accurate dilution was demonstrated using only one DBS blank sample for a typical dilution of <50-fold, and using only two blank DBS samples for a dilution of up to 625-fold. Moreover, this new workflow makes it easier to automate DBS sample dilution.

A rapid, accurate and robust UHPLC-MS/MS method for quantitative determination of BMS-927711, a CGRP receptor antagonist, in plasma in support of non-clinical toxicokinetic studies.

BMS-927711 is a calcitonin gene-related peptide (CGRP) receptor antagonist that is being developed for the treatment of migraine. A rapid, accurate and robust assay was developed and validated for the quantitation of BMS-927711 in rat, monkey, rabbit and mouse plasma using ultra high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). A simplified method screening strategy was utilized that included a liquid-liquid extraction (LLE) methodology and eleven LC columns (ten sub-2 µm UHPLC columns and one 2.6 µm HPLC column) for screening with emphasis on the removal of phospholipids, avoidance of metabolite interference and ruggedness of LC conditions. A stable isotope labeled [(13)C2, D4]-BMS-927711 was used as the internal standard, and 50 µL of plasma samples were used for extraction by automated LLE with methyl tert-butyl ether (MTBE) in 96-well format. Chromatographic separation was achieved with an isocratic elution and a gradient column wash on a Waters Acuity UPLC(®) BEH C18 column (2.1 mm × 50 mm, 1.7 µm) with run time of 3.7 min. Positive electrospray ionization was performed using selected reaction monitoring (SRM) with transitions of m/z 535>256 for BMS-927711 and m/z 541>256 for [(13)C2, D4]-BMS-927711. The standard curve, which ranged from 3.00 to 3000 ng/mL for BMS-927711, was fitted to a 1/x(2) weighted linear regression model. The intra-assay precision was within 5.2% CV, inter-assay precision was within 5.9% CV, and the assay accuracy was within ±5.2% deviation (%Dev) of the nominal values in all the species. The stability of an N-carbamoyl glucuronide metabolite was carefully investigated, and the conversion of this metabolite to BMS-927711 was minimal and manageable without a stabilization procedure. The method was successfully applied to multiple non-clinical toxicokinetic studies in different species in support of the investigative new drug (IND) filing.

A simplified and completely automated workflow for regulated LC-MS/MS bioanalysis using cap-piercing direct sampling and evaporation-free solid phase extraction.

Automated sample extraction for regulated bioanalysis by liquid chromatography/tandem mass spectrometry (LC-MS/MS) still presents significant challenges. A new sample preparation methodology with a simplified and completely automated workflow was developed to overcome these challenges using cap piercing for direct biofluid transfer and evaporation-free solid phase extraction (SPE). Using pierceable cap sample tubes, a robotic liquid handler was able to sample without uncapping or recapping during sample preparation. Evaporation for SPE was eliminated by using a mobile phase-compatible elution solvent followed by sample dilution prior to LC-MS/MS analysis. Presented here are three LC-MS/MS assays validated using this methodology to support three CNS drug development programs: (1) BMS-763534 and its metabolite, BMS-790318, in dog plasma; (2) BMS-694153 in monkey plasma; and (3) Pexacerfont (BMS-562086) and two metabolites, BMS-749241 and DPH-123554, in human plasma. These assays were linear from 1.00 to 1000 or 2.00 to 2000ng/mL for each analyte with excellent assay accuracy, precision and reproducibility. These assays met acceptance criteria for regulated bioanalysis and have been successfully applied to drug development study samples. The methodology described here successfully eliminated all manual intervention steps achieving fully automated sample preparation without compromising assay performance. Importantly, this methodology eliminates the potential exposure of the bioanalyst to any infectious biofluids during sample preparation.

Esterase inhibitors as ester-containing drug stabilizers and their hydrolytic products: potential contributors to the matrix effects on bioanalysis by liquid chromatography/tandem mass spectrometry.

RATIONALE: Esterase inhibitors are widely used to stabilize ester-containing drugs in biological matrices for quantitative liquid chromatography/tandem mass spectrometry (LC/MS/MS) assays. These co-existing inhibitors could cause matrix effects on bioanalysis and jeopardize the assay performance. We therefore developed an LC/MS/MS methodology to monitor the fate of inhibitors and evaluate their matrix effects, which is described in this study. METHODS: Human plasma containing 20 mM of diisopropylfluorophosphate (DFP), paraoxon, eserine, phenylmethylsulfonyl fluoride (PMSF) or 2-thenoyltrifluoroacetone (TTFA) was extracted by liquid-liquid extraction (LLE) and analyzed by an LC/MS/MS assay for BMS-068645 (a model drug) with additional pre-optimized selected reaction monitoring (SRM) transitions using positive/negative electrospray ionization (ESI) mode for each inhibitor. Hydrolytic products were characterized by product ion or neutral loss scan LC/MS/MS analysis. The matrix effect contribution from each inhibitor was evaluated by post-column infusion of BMS-068645. RESULTS: In the extracted samples by LLE, SRM chromatograms revealed the presence of paraoxon, eserine and TTFA with peak intensity of >2.50E08. Three DFP hydrolytic products, diisopropyl phosphate (DP), triisopropyl phosphate (TP) and DP dimer, and one PMSF hydrolytic product, phenymethanesulfonic acid (PMSA), were identified in the extracted samples. In post-column infusion profiles, ion suppression or enhancement was observed in the retention time regions of eserine (~10% suppression), paraoxon (~70% enhancement) and DP dimer (~20% suppression). CONCLUSIONS: The SRM transitions described here make it possible to directly monitor the inhibitors and their hydrolytic products. In combination with post-column infusion, this methodology provides a powerful tool to routinely monitor the matrix effects-causing inhibitors, so that their matrix effects on the bioanalysis can be evaluated and minimized.