Sheehan D, Li Y, Meckler E, Upton R, Zhang M.

J Am Soc Mass Spectrom

PMID:41989479

Free PMC article

Kratom ( Mitragyna specios a) products have become widely available in diverse commercial formulations, raising increasing analytical and regulatory interest due to the presence of potent alkaloids such as mitragynine (MG), 7-hydroxymitragynine (7-OH), and mitragynine pseudoindoxyl (MGP). The accurate identification of these compounds is analytically challenging because kratom matrices contain numerous structurally related alkaloids and isomeric species that produce similar mass spectral features. In this study, an ultrahigh-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) workflow was developed to characterize kratom alkaloids in authentic leaf materials and 38 commercial kratom products representing multiple formulations. Chromatographic separation combined with accurate mass measurement and diagnostic MS/MS fragmentation enabled reliable differentiation of MG, 7-OH, MGP, and related isomers. Multiple overlapping chromatographic peaks were observed at m / z 415.2227, demonstrating that reliance solely on accurate mass or commonly monitored transitions such as m / z 415 → 190 can lead to false-positive identification of 7-OH in complex botanical matrices. Application of the multicriteria confirmation strategy revealed substantial variability in alkaloid composition across commercial products, including the presence of 7-OH and MGP in several tablet and capsule formulations. Untargeted metabolomic profiling further distinguished kratom plant extracts, including the authentic leaf materials, from processed products enriched in specific alkaloids. These results highlight the importance of integrating chromatographic resolution with orthogonal MS/MS criteria for reliable identification of kratom alkaloids and provide an analytical framework for the characterization of complex botanical products.

Gao J, Shuang L, Yang X, Dong R, Wang G, Wang R, Zhang T.

J Lipid Res

PMID:41999981

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Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder characterized by dysregulated bile acid metabolism, which can lead to severe adverse pregnancy outcomes. Glycine-conjugated deoxycholic acid (G-DCA) is a small-molecule metabolite that has been reported to be elevated in the serum of patients with ICP; however, its clinical application remains limited because conventional chromatographic methods for its quantification are time-consuming, costly, and not well suited for rapid clinical screening. To address this gap, we developed a time-resolved fluorescence immunochromatographic test strip (TRF-ICTS) for the quantitative detection of serum G-DCA. Based on a small-molecule competitive binding design, the assay requires only 10 μL of serum sample and enables rapid detection within 16 min. The TRF-ICTS demonstrated good analytical performance, with a linear range of 0.05-10.0 ng/mL and a detection limit of 0.074 ng/mL, and maintained stable performance over two weeks of storage at both 4 °C and 37 °C. The results showed good quantitative agreement with LC-MS/MS measurements. Evaluation of sensitivity, specificity, and precision demonstrated that all parameters were within acceptable ranges. Clinical validation using serum samples from ICP patients and healthy pregnant women showed that G-DCA levels were elevated across different trimesters and were more pronounced in patients with severe ICP and adverse pregnancy outcomes. Overall, the developed TRF-ICTS provides a rapid and sensitive approach for quantifying G-DCA and supports its use as a complementary biomarker for ICP diagnosis and disease stratification, with potential to provide complementary insights into bile acid changes following UDCA treatment.

Rayhan A, Limbach PA, Addepalli B.

RNA

PMID:41991242

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Understanding the location of modified nucleosides in RNA sequences is crucial to understanding their biochemical significance. Mapping the sequence location of modified nucleosides from low abundance RNAs is challenging. Here, we report the development of a liquid chromatography tandem mass spectrometry (LC-MS/MS) exclusion list strategy that enhances sequence information from modified oligonucleotides. This approach, compatible with standard RNA modification mapping methods that utilize LC-MS/MS, enables the exclusion of any unmodified oligonucleotide from fragmentation during MS/MS thereby enabling enhanced dissociation of modified oligonucleotides. This universal exclusion list is applicable to natural RNAs of any type from any organism. We find this approach generates at least 10% more mapped RNase T1 digestion products than using DDA alone. To demonstrate the broad utility of this approach for discovery-based analyses, RNA modification mapping of total tRNAs from four distinct organisms spanning both prokaryotic and eukaryotic domains was conducted.

Xie Y, Butler M.

PMID:PPR1178224

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Abstract Protein hydrolysates have attracted increasing interest as cost-effective media supplements for mammalian cell culture, including Chinese hamster ovary (CHO) cells used widely in biopharmaceutical production. However, the biological basis of their beneficial effects remains poorly understood because of their compositional complexity and batch-to-batch variability. In this study, time-resolved compositional profiles of culture media supplemented with different batches of cottonseed hydrolysates were analysed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) and related to CHO DG44 cell growth, antibody productivity, cellular metabolism, and antibody glycosylation. During 10-day batch cultures, hydrolysate supplementation prolonged high cell viability and significantly enhanced antibody productivity, despite lower peak viable cell densities than the control. Hydrolysate-supplemented cultures also showed reduced lactate and ammonia accumulation, consistent with altered nutrient utilization and metabolic activity. In addition, cottonseed hydrolysates significantly increased antibody galactosylation to varying degrees. Chemometric analysis further linked hydrolysate compositional variability to culture performance and identified 25 signature features associated with cell growth and antibody production. These findings provide complementary insight into how hydrolysate composition relates to functional performance in CHO cell culture and support the development of a method to identify hydrolysates that support high performance biopharmaceutical manufacturing.

Rajapaksha RD, Farmer JT.

Bioanalysis

PMID:41988666

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Bioanalytical method validation is a foundation of drug development, ensuring that pharmacokinetic (PK), toxicokinetic (TK), and biomarker assays produce accurate, precise, reliable, and decision-enabling data. Over the past several decades, advances in analytical technologies including ligand-binding assays (LBAs) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) platforms have transformed the field of bioanalysis. Regulatory guidance from the U.S. Food and Drug Administration (FDA), the European Medicines Agency (EMA), and the International Council for Harmonization (ICH) has evolved to reflect these advancements. This review provides a comprehensive comparison of PK and biomarker assay validation, highlighting critical parameters such as selectivity, sensitivity, calibration, accuracy, precision, dilution/parallelism, specificity, and stability. Best practice recommendations are provided for method development, qualification, and validation, with a focus on fit-for-purpose (FFP) approaches and context-of-use (CoU) considerations. Areas of ambiguity in current regulatory expectations for biomarker validations, common pitfalls encountered during regulatory interactions, and emerging trends in bioanalysis are also reviewed. This review paper aims to guide bioanalytical scientists in developing robust, compliant assays that support regulatory submissions and facilitate informed drug development decisions.