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Biotherapeutics such as protein and peptide drugs have attracted significant attention in the medical community and pharmaceutical industry in recent years. Immunogenicity is one of the major concerns in the development and application of biotherapeutics. Although great efforts have been put forth in reducing immunogenicity, monitoring the free ('active') drug concentration and the antibody formation is critical for preclinical and clinical studies. Currently, it is still a challenging task to have a standardized test method monitoring immunogenicity when biotherapeutic compounds such as proteins and peptides are administrated. Combined with liquid chromatography/ tandem mass spectrometry detection, the equilibrium dialysis technique that is conventionally used for measuring the free and bound concentration of small organic molecules was extended to the application of measuring the free and bound concentrations of a protein drug with a relative molecular mass over 10 000 from plasma samples containing antibody. This novel approach could also be used for accurately measuring the antibody concentration when a reference standard of the antibody is available.

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]

We report here a quantitative method for the analysis of ABT-578 in human whole blood samples. Sample preparation was achieved by a semi-automated 96-well format liquid–liquid extraction (LLE) method. Aluminum/polypropylene heat seal foil was used to enclose each well of the 96-well plate for the liquid–liquid extraction. A liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method with pre-column regeneration was developed for the analysis of sample extracts. Selective reaction monitoring (SRM) of the mass transitions m/z 983–935 and m/z 931–883 was employed for the detection of ABT-578 and internal standard, respectively. The ammonium adduct ions [M + NH4]+ generated from electrospray ionization were monitored as the precursor ions. The assay was validated for a linear dynamic range of 0.20–200.75 ng/ml. The correlation coefficient (r) was between 0.9959 and 0.9971. The intra-assay CV (%) was between 1.9 and 13.5% and the inter-assay CV (%) was between 4.7 and 11.3%. The inter-assay mean accuracy was between 86.4 and 102.5% of the theoretical concentrations. [Copyright &y& Elsevier]

Here we report a strategr for rapid method development of high-throughput bioanalytical assays using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (uHPLC—MS/MS). First, a data set was established for the removal of representative phospholipids under different sample treatments to guide subsequent method development for various compounds. The recovery information of the analyte(s) of interest under different extraction conditions was then obtained during method development. With the recovery proffles and the pre-established knowledge of phospholipids removal in place, an optimal extraction condition was identified to give not only a satisfactory recovery but also a good cleanup of the sample. A rapid LC or uHPLC method was developed without the need of extensive column wash after the elution of the analyte. This strategy was demonstrated through the method development of a uHPLC—MS/MS bioanalytical assay for the quantitation of ketoconazole in human plasma with liquid—liquid extraction using a hexane and ethyl acetate solvent system. The retention time for ketoconazole through an isocradc elution was 18 s. Good accuracy and precision were obtained. Assay ruggedness was demonstrated by consistent internal standard responses and retention time for 500 sequential injections. In addition, consistent results were obtained for incurred sample reanalysis. [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: 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]

Abstract: A sensitive and selective bioanalytical assay was developed and validated for the determination of enantiomeric molindone in human plasma using high-performance liquid chromatography–tandem mass spectrometry along with supported liquid extraction procedures. The chiral separation was evaluated and optimized on macrocyclic antibiotic type chiral stationary phases (CSPs) based on teicoplanin aglycone (Chirobiotic TAG) in polar organic, polar ionic, and reversed-phase mode chromatography, respectively. Complete baseline separation was achieved on a Chirobiotic TAG column under isocratic condition in reversed-phase chromatography. The method validation was conducted using a Chirobiotic TAG column (100mm×2.1mm) over the curve range 0.100–100ng/ml for each molindone enantiomer using 0.0500ml of plasma sample. The flow rate was 0.8ml/min and the total run time was 9min. Supported liquid extraction in a 96-well plate format was used for sample preparation. Parameters including recovery, matrix effect, linearity, sensitivity, specificity, carryover, precision, accuracy, dilution integrity, and stability were evaluated. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels were RSD ≤3.4% and relative error (RE) ≤4.2% for molindone enantiomer 1, and RSD ≤2.6% and RE ≤5.0% for molindone enantiomer 2, respectively. This validated method could be used for the determination of individual molindone enantiomer in human plasma samples from clinical studies. [Copyright &y& Elsevier]