Journal of pharmaceutical and biomedical analysis最新文献

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.