Journal of pharmaceutical and biomedical analysis最新文献

Accurate and reliable quantification of antigen components in vaccines is critical in vaccine quality control and evaluation of immunogenic consistency. However, conventional immunoassays often suffer from limited specificity, trace-level antigen concentrations, and indirect quantification. In this study, we demonstrated an ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the determination of effective antigen components in inactivated SARS-CoV-2 vaccines. Specifically, the vaccine samples were denatured and digested with trypsin to generate tryptic peptides. Then, the signature peptides derived from the nucleocapsid protein and their stable isotope-labeled internal standards were selectively captured and separated using anti-peptide antibody-conjugated magnetic beads. The signature peptides were characterized by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry, which confirmed their amino acid sequence and multi-charged ionization states. Quantitative analysis was then performed using UHPLC-MS/MS in positive electrospray ionization mode with multiple reaction monitoring. Chromatographic separation of the signature peptides was achieved on an ACQUITY Premier Peptide BEH C₁₈ column using 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile as the mobile phases. The method was validated and exhibited excellent linearity for the signature peptides over the concentration range of 1-60 μg/L, with correlation coefficients higher than 0.999. The recovery ranged from 75.1 % to 86.3 %, with intra-day precision (RSD) of 0.8-1.0 % and inter-day precision of 1.3-3.6 %. Finally, the method was successfully applied to determine the effective antigen components in inactivated SARS-CoV-2 vaccine samples. The concentrations of the signature peptide ADETQALPQR ranged from 4.95 to 12.95 µg/L across the three vaccine batches analyzed, corresponding to 4.38-11.47 nmol/L of nucleocapsid protein. The results indicated that the method exhibited great promise for the determination of active antigenic proteins in inactivated SARS-CoV-2 vaccine samples and provided an alternative analytical platform for vaccine quality control.

CG-0255, a thiol prodrug of clopidogrel's active metabolite H4 (CG-0236), is a novel thienopyridine P2Y12 receptor antagonist under initial clinical development for the treatment of acute coronary syndromes. Unlike clopidogrel, CG-0255 is converted to the active thiol metabolite H4 (CG-0236) in a single hydrolytic step. Compared with clopidogrel, CG-0255 exhibits more efficient and consistent H4 formation in humans, which can be quantified in plasma following either intravenous or oral administration. In this study, we developed and validated a sensitive, rapid, and robust UHPLC-MS/MS method for the simultaneous quantification of CG-0255 and its derivatized active metabolite (MP-H4, CG-0261) in human plasma. After solid-phase extraction from 94.5 μL of plasma, analytes and isotope-labeled internal standards were separated on an ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) using isocratic elution with 0.1 % formic acid in water and acetonitrile (57:43, v/v) at a flow rate of 0.5 mL/min, followed by a 3.5 min column washing and re-equilibration, giving a total analytical run time of 7 min. Baseline separation of CG-0255, CG-0261, and their respective isomers was achieved. Detection was performed using positive electrospray ionization in multiple reaction monitoring mode on a Q-Trap 6500⁺ mass spectrometer. Calibration curves were linear over 0.05-25 ng/mL for both analytes, corresponding to 0.0353-17.65 ng/mL for H4 (CG-0236). Intra- and inter-day precision and accuracy were within ±15 % at all quality-control levels. The validated assay was successfully applied to two phase I clinical studies conducted at our center, characterizing the pharmacokinetics of CG-0255 following single-dose intravenous and multiple-dose oral administration. This UHPLC-MS/MS method provides a reliable platform for the quantitative evaluation of CG-0255 and its active metabolite in human plasma, and is well suited to support further global clinical development.

Central nervous system (CNS) infections require adequate drug exposure at the site of action, yet antibiotic meropenem (MER) shows limited cerebrospinal fluid (CSF) penetration and easily undergoes non-enzymatic degradation to an inactive open-ring metabolite (ORM). In this study, we developed a simple, sensitive, and fast liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of MER and ORM in human serum and CSF. Chromatographic separation was accomplished on an Agela Venusil MP C18 column, with MER-d6 and ORM-d6 as internal standards. Methanol was found to promote methanolysis, yielding a characteristic product (m/z 416.2). Therefore, acetonitrile was selected as both the organic phase and the protein-precipitation solvent. Method validation was conducted according to the ICH M10 guideline. Follow validation, the method was successfully applied to 57 serum and 16 CSF samples. ORM concentrations in human CSF were reported for the first time. This method provides a valuable tool to support MER monitoring in patients with CNS infections.

Insomnia is increasingly recognized as a disorder with complex metabolic underpinnings. We investigated the changes in fecal metabolite levels of 5-hydroxyindoleacetic acid (5-HIAA), octopamine (OA), oleoylethanolamide (OEA), and elaidic acid (EA) in patients with sleep disorders, as well as their correlations with insomnia severity. Sixty participants were divided into two groups, with thirty patients with sleep disorders hospitalized in the Department of Neurology, Zhongda Hospital, Southeast University (October 2024-March 2025) and 30 healthy controls recruited during the same period. Fecal samples were collected from all participants, and metabolite levels were analyzed via untargeted metabolomics analysis using liquid chromatography-mass spectrometry (LC-MS). The Chinese version of the Insomnia Severity Index (C-ISI) was employed to evaluate insomnia severity, and the correlations between insomnia severity and these four metabolites were subjected to statistical analysis. Both univariate and multivariate analyses revealed significant metabolic differences between groups. The experimental group showed significantly lower levels of 5-HIAA (FC = 0.947, P = 0.020) and OA (FC = 0.953, P < 0.001), but higher OEA (FC = 1.101, P < 0.001) and EA (FC = 1.026, P < 0.001). C-ISI scores correlated negatively with 5-HIAA (r = -0.380, P = 0.003) and OA (r = -0.448, P < 0.001), and positively with OEA (r = 0.500, P < 0.001) and EA (r = 0.408, P = 0.001). These fecal metabolites associate with insomnia severity and may serve as potential biomarkers for understanding its pathophysiology and developing interventions.

The demand for rapid and reliable analytical methods in the pharmaceutical industry continues to grow, with Ultra-/High-Performance Liquid Chromatography (U/HPLC) remaining the gold standard for impurity profiling, quantification of active pharmaceutical ingredients, and degradation product analysis. However, traditional HPLC methods are often constrained by pressure limitations at higher flow rates, which can hinder analytical throughput. While recent advancements in column technology have improved performance, they typically exacerbate pressure-related challenges. In this study, we evaluate a novel column technology designed to address these limitations by enabling high-resolution separations at reduced pressures and increased flow rates. Our findings demonstrate that the column, when operated in Radial Flow Stream Splitting (RFS) mode, maintains quantitative accuracy and repeatability while achieving up to a 120 % improvement in separation efficiency and a 30 % reduction in backpressure compared to conventional operation. By way of assaying over-the-counter medication, we have found no difference in the quantitative reliability of the assay when in RFS vs stock mode, despite reducing the analysis time by up to 40 %.

A streamlined early phase formulation development workflow has been developed for monoclonal antibodies (mAbs) to provide a more efficient process, driving down costs and reducing timelines, without compromising the quality and hence patient safety. The proposed novel workflow combines liquid chromatography-mass spectrometry multi-attribute method (LC-MS MAM) and sensitive ligand binding using surface plasmon resonance (SPR). By linking the two methodologies it is possible to obtain a comprehensive understanding of a mAb's critical quality attributes (CQAs) and provide a structure/function correlation. As LC-MS MAM cannot address all aspects of degradation, high throughput methods for the analysis of high molecular weight material (HMWM), and conformational and colloidal stability, were also evaluated. The workflow comprises an initial forced degradation study, to verify stability-indication and identify potential degradation routes. Secondly, optimal pH, based on conformational and colloidal stability, is determined. Finally, stabilising excipients are evaluated by design of experiment (DoE). We have verified this workflow using pembrolizumab. In an initial forced degradation study, LC-MS MAM and PD-1 ligand binding could identify the CQAs. Met105 oxidation, located in the CDR3 region, was identified as the major CQA. DoE demonstrated that 25 mM methionine inhibited Met105 oxidation and stabilised PD-1 binding. With this streamlined process, we were able to improve the stability of the protein by formulating in 20 mM histidine, 25 mM methionine, 0.02 % PS80 and 300 mM sucrose, at pH 5.5. The described workflow has the potential to decrease the demand for precious early development material as well as reduce costs and shorten timelines.

Accurate detection of point mutations in mitochondrial DNA (mtDNA) is crucial for diagnosing various mitochondrial disorders. In this study, we developed an ultra-high-performance liquid chromatography coupled with high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) method for the direct, label-free identification and localization of single-nucleotide mutations using synthetic 20- and 49-mer oligonucleotides as model fragments representing the pathogenic mtDNA point mutation (mt.3243 A>G). Three mobile phase systems, including ammonium bicarbonate (ABC), triethylamine/hexafluoroisopropanol (TEA/HFIP), and tributylamine/HFIP (TBA/HFIP), were systematically evaluated to assess their effects on oligonucleotide retention behavior and duplex stability under denaturing and non-denaturing conditions. The ABC buffer provided optimal performance for maintaining partial duplex integrity, while TEA/HFIP offered superior ionization efficiency for single-stranded analysis. Deconvoluted mass spectra revealed accurate monoisotopic mass differences between wild-type and mutant oligonucleotides, including ∼ + 16 Da for the sense strand (A>G), ∼ -15 Da for the antisense strand (T > C), and ∼ + 1 Da for the duplex, enabling confident mutation discrimination at the intact molecular level. High-resolution MS achieved excellent mass accuracy within ±3 ppm, and high-energy collision dissociation (HCD) MS/MS enabled sequence-specific fragmentation that localized the mutation site with high confidence when compared with theoretical fragments. Overall, this study establishes a reliable analytical framework for mutation detection in oligonucleotide models and highlights the potential of UHPLC-HRMS/MS as a complementary tool for targeted mtDNA fragment analysis.

Isomerization of aspartic acid (Asp) to isoaspartic acid (isoAsp) within the complementarity-determining regions (CDRs) of monoclonal antibodies (mAbs) can lead to conformational changes that decrease antigen-binding affinity. Although isomerization can significantly alter the chromatographic and electrophoretic profiles, precise localization of this modification requires a mass spectrometry-based approach, such as peptide mapping. In this work, we present a case study that investigates various analytical strategies to identify the root cause of significant changes observed in the chromatographic and electrophoretic profiles of an mAb during formulation development. LC-MS analysis of reduced mAb using high-resolution mass spectrometry, peptide mapping using trypsin digestion, and fraction collection of the newly identified peak followed by trypsin digestion suggested that isomerization occurs within the CDR of the mAb. However, due to the presence of three Asp residues within a single tryptic peptide, this modification could not be precisely localized. To overcome this limitation, we developed a sequential enzymatic digestion strategy, utilizing trypsin followed by Asp-N digestion, which enabled accurate localization and quantification of the isomerization site. The resulting data indicated that the main isoAsp signal originated from isomerization at the DS motif that increased substantially over time in the liquid formulation, while no significant change was observed in the lyophilized formulation. The level of isomerization determined through this sequential digestion method correlated well with the LC-UV quantitation data of the reduced mAb.