Analysis of bacitracin and its related substances by liquid chromatography tandem mass spectrometry

A suitable liquid chromatography quadrupole time-of-flight mass spectrometric (LC–Q-TOF–MS) method was developed for separation and characterization of related substances in bacitracin test drug. The separation was performed on LiChrospher RP-18 column using methanol as mobile phase A and 0.2% ammonium acetate buffer solution as mobile phase B in gradient elution. A total of 12 related substances were detected through high resolution mass spectrometric determination in a positive electrospray ionization mode. They were identified as co-existing active components and degradation products of bacitracin through the analysis and elucidation of both the protonated parents and the product ions of all the related substances and their fragmentation pathways were also proposed

Profiling and Preparation of Metabolites from Pyragrel in Human Urine by Online Solid-Phase Extraction Coupled with High Performance Liquid Chromatography Tandem Mass Spectrometry Followed by a Macroporous Resin-Based Purification Approach

Pyragrel, a new anticoagulant drug, is derived from the molecular combination of ligustrazine and ferulic acid. Pyragrel showed significant inhibitory activity against platelet aggregation induced by adenosine diphosphate (ADP), and had been approved for a phase I clinical trial by CFDA. To characterize the metabolites of Pyragrel in human urine after intravenous administration, a reliable online solid-phase extraction couple with high performance liquid chromatography tandem mass spectrometry (online SPE-HPLC-MSn) method was conceived and applied. Five metabolites were detected and tentatively identified, which suggested that the major metabolic pathways of Pyragrel in human were double-bond reduction, double-bond oxidation, and then followed by glucuronide conjugation. Two main metabolites were then prepared using β-glucuronide hydrolysis and macroporous resin purification approach followed by preparative high-performance liquid chromatography (PHPLC) method, with their structures confirmed on the basis of nuclear magnetic resonance (NMR) data. This study provided information for the further study of the metabolism and excretion of Pyragrel

Development of an ELISA Assay for the Determination of SARS-CoV-2 Protein Subunit Vaccine Antigen Content

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein subunit vaccine is one of the mainstream technology platforms for the development of COVID-19 vaccines, and most R&D units use the receptor-binding domain (RBD) or spike (S) protein as the main target antigen. The complexity of vaccine design, sequence, and expression systems makes it urgent to establish common antigen assays to facilitate vaccine development. In this study, we report the development of a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the antigen content of SARS-CoV-2 protein subunit vaccines based on the United States Pharmacopeia and ICH (international conference on harmonization) Q14 and Q2 (R2) requirements. A monoclonal antibody (mAb), 20D8, was identified as the detection antibody based on its high RBD binding activity (EC50 = 8.4 ng/mL), broad-spectrum anti-variant neutralizing activity (EC50: 2.7–9.8 ng/mL for pseudovirus and EC50: 9.6–127 ng/mL for authentic virus), good in vivo protection, and a recognized linear RBD epitope (369–379 aa). A porcine anti-RBD polyclonal antibody was selected as the coating antibody. Assay performance met the requirements of the analytical target profile with an accuracy and precision of ≥90% and adequate specificity. Within the specification range of 70–143%, the method capability index was >0.96; the misjudgment probability was <0.39%. The method successfully detected SARS-CoV-2 protein subunit vaccine antigens (RBD or S protein sequences in Alpha, Beta, Gamma, or Delta variants) obtained from five different manufacturers. Thus, we present a new robust, reliable, and general method for measuring the antigenic content of SARS-CoV-2 protein subunit vaccines. In addition to currently marketed and emergency vaccines, it is suitable for vaccines in development containing antigens derived from pre-Omicron mutant strains