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Rationale: The in‐sample calibration curve (ISCC) approach of quantification utilizes the response of isotopologue ions from spiked‐in stable isotope labeled internal standard (SIL‐IS) to build a standard curve. The quantitative analysis of the study sample is achieved based on the response of selected monoisotopic analyte ion against the calibration curve. Although this methodology has been demonstrated to be feasible by unit and high‐resolution mass spectrometers, quantitation on high‐resolution mass spectrometer with product ions has not been tested. We tested the feasibility of this approach using product ions on an high‐resolution mass spectrometer equipped with an Orbitrap detector. Methods: Using a proteomics workflow for sample preparation, two surrogate peptides were quantified from a complex matrix of protein digest from human peripheral blood mononuclear cells (hPBMCs). SIL‐IS was spiked in at different levels to construct calibration curves in a traditional manner. ISCCs were prepared using extracted ion chromatograms from isotopically resolved mass spectra and compared with traditional calibration curves. Results: A linear response was observed with ISCC approach for at least two to three orders of magnitude in MS1 as well as targeted MS2 (tMS2). From protein digests, isobaric interferences were observed for endogenous peptides on the MS1 level; this was circumvented with product‐ion‐based quantitation where for one peptide, %CV for endogenous levels was more than 20% with ISCC but higher with the traditional calibration curve approach. For the second peptide, endogenous levels could not be determined in the traditional approach as calibrant levels did not bracket the lower end, and with the ISCC approach, isotopologues at abundances lower than the endogenous level allowed for quantitative assessments. Conclusions: ISCC demonstrated improved precision across surrogate peptides from endogenous protein digests. In samples where endogenous analyte concentrations were low in abundance, ISCC rescued what would have been a non‐reportable result in a traditional bioanalytical assay as calibrant levels were not prepared at adequately low levels to bracket unknowns. ISCC using high‐resolution mass spectrometer is feasible and ideal compared to unit resolution mass spectrometers. High‐resolution mass spectrometer allows for isotopic resolution for analytes with > + 2 charge state and provides flexibility in quantification using multiple product ions. ISCC using high‐resolution mass spectrometer allows for simultaneous assaying of low abundance isotopologues, the signal acquisition of which is not constrained by limits in data acquisition or calibrant preparation as with other approaches but rather limited by platform sensitivity. In contrast to unit resolution mass spectrometers, these features offered by high‐resolution mass spectrometer could be especially useful for the drug discovery assay support where there is less lead time for assay development than for the assays to support the drug development studies. [ABSTRACT FROM AUTHOR]

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.

Currently, mass spectrometry-based protein bioanalysis is primarily achieved through monitoring the representative peptide(s) resulting from analyte protein digestion. However, this approach is often incapable of differentiating the measurement of protein analyte from its post-translational modifications (PTMs) and/or potential biotransformation (BTX) products. This disadvantage can be overcome by direct measurement of the intact protein analytes. Selected reaction monitoring (SRM) on triple quadrupole mass spectrometers has been used for the direct measurement of intact protein. However, the fragmentation efficiency though the SRM process could be limited in many cases, especially for high molecular weight proteins. In this study, we present a new strategy of intact protein bioanalysis by high-resolution (HR) full scan mass spectrometry using human lysozyme as a model protein. An HR linear ion-trap/Orbitrap mass spectrometer was used for detection. A composite of isotopic peaks from one or multiple charge states can be isolated from the background and used to improve the signal-to-noise ratio. The acquired data were processed by summing extracted ion chromatograms (EIC) of the 10 most intense isotopic ions of octuply protonated lysozyme. Quantitation of the plasma lysozyme was conducted by utilizing high resolving power and an EIC window fitting to the protein molecular weight. An assay with a linear dynamic range from 0.5 to 500 μg/mL was developed with good accuracy and precision. The assay was successfully employed for monitoring the level of endogenous lysozyme and a potential PTM in human plasma. The current instrumentation limitations and potential advantages of this approach for the bioanalysis of large proteins are discussed. [ABSTRACT FROM AUTHOR]

Abstract: The determination of protein concentrations in plasma samples often provides essential information in biomedical research, clinical diagnostics, and pharmaceutical discovery and development. Binding assays such as ELISA determine meaningful free analyte concentrations by using specific antigen or antibody reagents. Concurrently, mass spectrometric technology is becoming a promising complementary method to traditional binding assays. Mass spectrometric assays generally provide measurements of the total protein analyte concentration. However, it was found that antibodies may bind strongly with the protein analyte such that total concentrations cannot be determined. Thus, a sample preparation process was developed which included a novel “denaturing” step to dissociate binding between antibodies and the protein analyte prior to solid phase extraction of plasma samples and LC–MS/MS analysis. In so doing, the total protein analyte concentrations can be obtained. This sample preparation process was further studied by LC–MS analysis with a full mass range scan. It was found that the protein of interest and other plasma peptides were pre-concentrated, while plasma albumin was depleted in the extracts. This capability of the sample preparation process could provide additional advantages in proteomic research for biomarker discovery and validation. The performance of the assay with the novel denaturing step was further evaluated. The linear dynamic range was between 100.9ng/mL and 53920.0ng/mL with a coefficient of determination (r 2) ranging from 0.9979 and 0.9997. For LLOQ and ULOQ samples, the inter-assay CV was 12.6% and 2.7% and inter-assay mean accuracies were 103.7% and 99.5% of theoretical concentrations, respectively. For QC samples, the inter-assay CV was between 2.1% and 4.9%, and inter-assay mean accuracies were between 104.1% and 110.0% of theoretical concentrations. [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]

Highlights: [•] An inter-well volume replacement workflow was introduced for DBS sample dilution. [•] An improved liquid–liquid extraction was used to improve analyte elution from DBS. [•] These strategies were applied to quantify BMS-927711, a drug candidate, in rat DBS. [•] Excellent assay robustness, precision and accuracy were achieved. [•] It has been applied to non-clinical toxicokinetic studies in rats. [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: 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]