Mass Spectrometry Advances: Lichen Metabolomics, CYP Assay Optimization, and Drug Quantification

Three recent studies highlight the expanding use of mass spectrometry-based analytical techniques in pharmaceutical and biomedical research. One study involved the comprehensive phytochemical profiling of 39 lichen species collected in eastern Poland using HPLC-QTOF-MS/MS, combined with chemometric analysis and biological activity testing. After data processing, a total of 91 compounds were identified, with depsides and depsidones constituting the dominant chemical classes. Multivariate analyses (PCA and PLS-DA) revealed distinct metabolomic patterns among the species and highlighted the relationship between metabolite composition and biological activity.

The anti-inflammatory potential of lichen extracts was evaluated in LPS-stimulated macrophages by measuring the levels of intracellular reactive oxygen species (ROS), nitric oxide (NO) and cyclooxygenase-2 (COX-2) activity. Most extracts significantly reduced ROS levels, indicating a consistent antioxidant effect. In contrast, modulation of NO and COX-2 was more variable and generally less pronounced. Chemometric analyses suggested that multiple metabolites contribute to the observed anti-inflammatory activity, with evidence of an inverse relationship between ROS reduction and COX-2 inhibition. Although some compounds were found to be associated with biological effects, the results indicate that these effects are likely to be driven by the complex interactions within the extracts rather than by the action of individual constituents. Overall, lichens emerge as promising sources of anti-inflammatory agents, primarily acting through antioxidant mechanisms.

In a second study, a validated LC-MS/MS method was developed following USFDA guidelines for the simultaneous estimation of Tramadol HCl and dexketoprofen trometamol in rat plasma using Tapentadol as an internal standard. Analytes were extracted from plasma by protein precipitation technique using acetonitrile. Chromatographic separation was achieved on a Waters Symmetry Shield RP-18 column (250 × 4.6 mm, 5 μm) with an isocratic mobile phase of acetonitrile and 0.1% formic acid in HPLC grade water (30:70 v/v) at a flow rate of 1.0 mL/min, yielding a 7-min runtime. Detection was performed using electrospray ionization with ion transitions of m/z 222.3398 → 70.0307 for Tapentadol, m/z 264.4351 → 96.4527 for Tramadol HCl, and m/z 376.4239 → 100.4520 for dexketoprofen trometamol. The method showed good accuracy, with mean recoveries of 92.79%–98.15% for Tramadol HCl and 91.78%–98.37% for dexketoprofen trometamol. Excellent linearity was obtained, with r2 values of 0.99983 (22.5–900 ng/mL) and 0.99963 (7.5–300 ng/mL), respectively. All validation parameters met acceptable criteria. The method is suitable for evaluating pharmacokinetic parameters that indicate drug efficacy and safety.

A third investigation examined the ionic strength of incubation buffers in CYP metabolism assays. CYP substrates were incubated with NADPH-fortified human liver microsomes in buffer solutions containing 20, 50, or 100 mM potassium phosphate. The goal was to find the appropriate potassium phosphate level for samples tested with the SCIEX 7500+ system, Echo® MS+ system, and ZenoTOF 7600 system while retaining CYP activity. By comparing half-life values across buffer conditions, the study discovered ideal assay parameters that maintain CYP activity while providing the highest analytical sensitivity for the assay. Half-life comparisons between the Echo® MS+ system and conventional LC-MS for analytes including bupropion, dextromethorphan, and diclofenac showed percent bias ranging from -16.82% to 15.19% across the evaluated buffer strengths.