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Abstract: In recent years, there has been increasing interest in using LC–MS/MS to measure protein drugs (biotherapeutics) in plasma/serum to support pharmacokinetic/toxicokinetic (PK/TK) studies. A number of strategies have been proposed to quantify different types of protein drug candidates in biological matrices. However, the application of LC–MS/MS in biotherapeutics is still very limited in regulated bioanalysis due to practical challenges. In this manuscript, we propose a “data-dependent” approach for the LC–MS/MS support of protein drug candidates, focusing on operational aspects. We have developed and validated a fast, simple and reliable LC–MS/MS method to quantify a protein drug candidate in mouse serum. This method can fit into our current workflow for routine small molecule LC–MS/MS assays with an overall sample preparation time of less than two hours. The method has been demonstrated to be accurate, precise and rugged, and it has been successfully applied to analyze samples from toxicology studies. The results were verified using a ligand binding assay and tested by standard-addition sample reanalysis. In addition, general recommendations for developing and validating an LC–MS/MS assay based on surrogate peptide(s) of protein drug candidates are also proposed for regulated bioanalysis. [Copyright &y& Elsevier]

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 C 2 , D 4 ]-BMS-927711 internal standard (IS) solution in 20/80 MeOH/water for 10 min, followed by sonication with 50 μL of 100 mM NH 4 OAc 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. [ABSTRACT FROM AUTHOR]

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]

Abstract: A fast, sensitive, and enantioselective LC–MS/MS bioanalytical method was developed and validated for the direct determination of individual alprenolol enantiomers in human plasma using cellobiohydrolase (CBH) chiral stationary phases (CSP) along with supported liquid extraction (SLE) procedures. Complete baseline separation of enantiomeric alprenolol was achieved within 2min in reversed phase chromatography at 0.9ml/min. SLE in a 96-well plate format was used for sample extraction. The method validation was conducted over the curve range of 0.500–500ng/ml for each alprenolol enantiomer using 0.0500ml of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤7.3% relative standard deviation (RSD) and −6.2 to 8.0% relative error (RE) for both alprenolol enantiomers. [Copyright &y& Elsevier]

Abstract: A rapid and specific LC–MS/MS based bioanalytical method was developed and validated for the determination of 18-(p-iodophenyl)octadecyl phosphocholine (CLR1401), a novel phosphocholine drug candidate, in rat plasma. The optimal chromatographic behavior of CLR1401 was achieved on a Kromasil silica column (50mm×3mm, 5μm) under hydrophilic interaction chromatography. The total LC analysis time per injection was 2.8min with a flow rate of 1.5mL/min under gradient elution. Liquid–liquid extraction in a 96-well format using ethyl acetate was developed and applied for method validation and sample analysis. The method validation was conducted over the curve range of 2.00–1000ng/mL using 0.0500mL of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed≤5.9% relative standard deviation (RSD) and −10.8 to −1.4% relative error (RE). The method was successfully applied to determine the toxicokinetics of CLR1401 in rats from three dose groups of 0.4, 4.0, and 10.0mg/kg/day via intravenous administration. [Copyright &y& Elsevier]

Abstract: Dried blood spots (DBSs) technology was evaluated in an assay for the quantitation of dextromethorphan (DM) and its metabolite, dextrorphan (DT), in human whole blood using high performance liquid chromatography with tandem mass spectrometry method (LC–MS/MS). Both the parent drug and metabolite were spiked in the blood matrix and subsequently allowed to dry on a specimen collection card. The dried blood spots were removed using a manual punch and then extracted into methyl tert-butyl ether (MTBE). The organic supernatant was transferred and evaporated and the residue was reconstituted in 20% acetonitrile. The overall method recovery of DM and DT was 87.8% and 95.4%, respectively. The assay was linear over the concentration range of 0.2–200ng/mL for both analytes. Several factors that potentially affect DBS assay quantitation were investigated, such as punch size, DBS sample punch-out location, and the volume of the blood sample pipetted on the specimen collection cards. The study determined that punch size does not affect assay quantitation accuracy. Indeed, a larger punch size increases the sensitivity due to the larger sampled blood spots. Sampling from different location on the specimen collection cards shows no significant variation for both drugs. The study also shows that acceptable results can be achieved with some variation of the sample volume, which allows a simple blood sampling procedure at the test sites. To achieve the similar lower limit of quantitation (LLOQ) as the plasma assay, several blood spots at the same concentration level were stacked together and extracted. Bioanalytical assays using the DBS technique are promising given the advantages of the method over the plasma assay. [Copyright &y& Elsevier]

Abstract: A high through-put, sensitive, and enantioselective LC–MS/MS-based bioanalytical method was developed and validated for the simultaneous determination of individual acebutolol (AC) and its active metabolite–diacetolol (DC) enantiomers in human plasma using cellobiohydrolase (CBH) chiral stationary phases (CSP). Systematic optimization of chromatographic conditions including organic content, buffer concentration, and pH of mobile phases was conducted to improve the through-put for the direct separation of both AC and DC on CBH column during method development. Complete baseline separation of enantiomeric AC and DC was achieved within 1.5min with a LC flow rate of 0.9ml/min under method validation conditions. To further improve the assay through-put, supported liquid extraction (SLE) in a 96-well plate format was used for sample extraction. The method validation was conducted over the curve range of 0.0500–50.0ng/ml for each AC and DC enantiomer using 0.100ml of plasma sample. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels showed ≤4.5% relative standard deviation (R.S.D.) and −8.7 to 5.6% relative error (R.E.) for individual AC and DC enantiomers. [Copyright &y& Elsevier]

• 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]