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The effectiveness of lapatinib (LAP) and trastuzumab (TRZ), the first-line therapies for HER2 + breast cancer, has been limited owing to the development of acquired resistance in patients with HER2 + . This study aimed to investigate the alterations in metabolic signatures in LAP-resistant HCC1954 and TRZ-resistant HCC1954 and pathways in human HER2 + breast cancer cells using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) and enrichment analysis. The HCC1954 parental cells were sequentially treated 13 rounds with LAP or TRZ to develop resistant cells and then tested for their cytotoxicity using the MTT assay. Metabolites were prepared from HCC1954 parental (MBXWT), HCC1954-LAP (MBXLAP), and HCC1954-TRZ (MBXTRZ) cells prior to LC-HRMS, chemometric, enrichment, and joint pathway analyses. LAP- and TRZ-resistant cells were successfully developed from HCC1954, and 29 and 17 differentially expressed metabolites (DEMs) were identified between MBXWT-MBXLAP and MBXWT-MBXTRZ, respectively. The analysis of DEMs between MBXWT and MBXLAP revealed significant enrichment in D-amino acid metabolism, while MBXWT and MBXTRZ identified valine, leucine, isoleucine biosynthesis, ascorbate, and aldarate metabolism. Joint pathway enrichment analysis of LAP-resistant DEMs and differentially expressed genes (DEGs) showed enrichment in glutathione metabolism, while that of TRZ-resistance and DEGs showed enrichment in carbohydrate metabolism, namely pentose and glucuronate interconversions, starch and sucrose metabolism, and galactose metabolism. The findings from this study indicate considerable metabolic changes in LAP- and TRZ-resistant HCC1954 cells, which are crucial for understanding the resistance mechanisms and developing strategies to overcome these problems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Wheat germ is one of the richest natural sources of polyamines, especially spermidine. Cell proliferation property of polyamines has given them inductive effects in the reduction of a variety of chronic diseases and fertility enhancement. Preparing a polyamine-rich extract powder from wheat germ for use in supplements is the aim of the present study. For the first time, the effects of three independent variables of clean-up replicate (A), extraction time (B), and solid-to-liquid ratio (C) on the response of total spermidine content (Y) were investigated using a central composite design optimizing polyamine enrichment. The optimal extraction conditions were 7 h, 3 clean-up replicates, and 1:4 solid to liquid ratio. This is the first production report of spermidine-enriched powder for encapsulation purposes. To obtain an acceptable rheological property, the polyamine-enriched extract was spray dried together with a selected group of excipients, among which glucose was evidenced as the best choice based on encapsulation properties., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of manuscript and there is no financial interest to report. We certify that the submission is original work and is not under review at any other publication., (Copyright © 2024. Published by Elsevier Ltd.)

Chemical warfare nerve agents (CWNAs) are potent and irreversible inhibitors of acetylcholinesterase (AChE). Oxime reactivators are an important part of the standard treatment for CWNA exposure as they can reactivate inhibited AChE. Evaluating the oxime candidates of interest in biological samples requires analytical detection methods and oxime reactivators as a class of compounds have historically been notoriously difficult to isolate, detect and analyze in an analytical laboratory, and there are currently no sensitive or robust analytical detection methods in the literature. The goal of this study was to develop reliable and robust novel extraction and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to detect and quantitate 2-PAM, HI-6, HLö-7, and MMB-4 in a human AChE knock-in, mouse carboxylesterase knock-out (KIKO) mouse in vivo model. This study identified an LC column that achieved retention for all four oxime compounds which is a major advancement over past oxime methods. A unique extraction and chromatographic method was developed for each oxime. The developed methods were sensitive down to 0.5 ng/mL for 2-PAM, 50 ng/mL for HI-6, and 15 ng/mL for both HLö-7 and MMB-4. These methods were validated to meet the Food and Drug Administration (FDA) bioanalytical method validation requirements under Good Laboratory Practice (GLP) conditions. The 4 methods were validated for performance by assessing linearity, sensitivity, precision, accuracy, selectivity, specificity, carryover, extraction recovery, dilution analysis, and stability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

A novel vardenafil analogue was discovered during adulterant screening of a dietary supplement. After extraction and purification, this analogue was identified using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) analyses. The molecular formula determined using LC-HRMS was C 24 H 34 N 6 O 5 S. Fragmentation data suggested that this unknown compound may have two modifications to vardenafil. NMR analysis confirmed the presence of a hydroxyl group on the piperazine moiety and a propyl group attached to the phenoxy group. Consequently, this compound was named 19-O-propyl hydroxy vardenafil., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The number of prescriptions for new direct oral anticoagulants (DOACs) apixaban, edoxaban, rivaroxaban and dabigatran has increased exponentially in recent years, increasingly replacing the old gold standard, vitamin-K-antagonists. Due to their wide therapeutic range, therapeutic drug monitoring (TDM) is not required, although it has been proven that this could significantly reduce side effects. In order to develop a cost-efficient and simple method for the simultaneous detection of the DOACs and phenprocoumon, a new technology for sample preparation from capillary blood in the ambulant sector named VAMS® was integrated and an LC-MS detector with on-line solid phase extraction (SPE) applying a Turboflow HTLC Cyclone TM 1.0x50 mm column was used. The mobile phase consisted of methanol with water (3/97 v/v) and 0.1 % ammonia solution with a flow rate of 2.5 mL/min. For the chromatographic separation, a Phenomenex LTD Kinetex 2.6 µm C18 100 Å, 100x3.0 mm column with a flow rate of 0.3 mL/min in gradient mode was utilized. The mobile phase consisted of acetonitrile, water and formic acid (A: 10:90:0.1 v/v and B: 95:05:0.1 v/v). The method was fully validated in the therapeutic range of the substances according to current guidelines. The LLOQ ranged from 3.5 µg/L for rivaroxaban to 88 µg/L for phenprocoumon and the intra-day and inter-day precision was less than 13 % and 12 %, while the accuracy was within a range of 85.7-113 % and 88.7-106 %, respectively. Samples could be stored in the Mitra® devices for at least seven days at room temperature except of dabigatran. Because the Mitras® were used, exactly 10 µL of blood could be drawn and no significant haematocrit effect was observed. A reliable, simple and cost-effective extraction and analysis LC-MS method could be developed and validated. This method is therefore applicable in ambulatory care., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Withania somnifera L. (Solanaceae), for over 3000 years, has been considered an essential herb in Ayurvedic medicine. The roots of W. somnifera contain metabolites mainly belonging to steroidal lactones called withanolides, which possess various pharmacological activities such as neuroprotective, cardioprotective, anti-diabetic, antioxidant and anti-inflammatory. Since the demand on the market for W. somnifera extracts is increasing, with the aim to find an ecological and environmentally friendly strategy of extraction, the roots were submitted to different extraction techniques (macerations, ultrasound-assisted extraction and solid-liquid dynamic extraction) using EtOH:H 2 O 50:50, 75:25, 100:0. W. somnifera extracts were investigated by an integrated LC-ESI/QExactive/MS/MS and NMR approach to obtain comprehensive metabolite profiles. Principal Component Analysis of LC-MS and NMR data revealed how the extraction method and the solvent can affect the chemical profile of the extracts. Extracts obtained by maceration exhibited the highest amount of withanolides and withanosides, while the SLDE-Naviglio EtOH extract showed the highest amount of metabolites as benzoic acid, tropane alkaloids and sarcosine, reported for their CNS activity. Moreover, based on the use of this plant in the treatment of neurological disorders, the tyrosinase inhibitory activity of all the extracts was herein tested by spectrophotometric assay, showing IC 50 values in a range of 32.86-85.36 µg/ml., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The CT143 protein of Chlamydia trachomatis (Ct) is a key immunodominant antigen and candidate type-III secretion substrate. Although CT143 expression has not been detected in the cytosol of infected cells, it is known to interfere with the physiological behavior of HeLa cells. This study aims to investigate how the CT143 protein affects the protein expression profile of HeLa cells, providing a basis for further research into Ct's pathogenic mechanisms., Methods: We constructed a stably transfected HeLa cell line, pCD513B-1-CT143-HeLa, and a control cell line, pCD513B-1-HeLa. Protein expression profiles of these cell lines were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differentially expressed proteins were identified, constructed into a database, and verified using parallel reaction monitoring (PRM). Bioinformatics software facilitated the preliminary analysis of the biological functions of these differential proteins., Results: A total of 221 host proteins were differentially expressed, with 68 upregulated and 153 downregulated. These variations influence the regulation of peptidase activity and are crucial in biological processes such as cell secretion and protease activity. Significant changes were noted in protein processing, alcohol dehydrogenase activity, Aldo-Keto reductase activity, and peptidase regulator activity. Furthermore, alterations were observed in cellular components like the plasma membrane and cell periphery. Pathways involving the hematopoietic system, glycosaminoglycan degradation, retinol metabolism, and cytochrome P450-mediated exogenous drug metabolism were notably affected. Indirect interactions among differentially expressed proteins included three key nodal proteins: C3, IFIT3, and IFIT1., Conclusion: The successful construction of a host differential protein expression profile was achieved through stable transfection of HeLa cells with the CT143 gene. The differential proteins identified are implicated in regulating various biological processes such as intracellular signal transduction, cell secretion, protein processing, hydrolysis, and enzyme activity. These findings suggest that the CT143 protein may influence the host cell's biological behavior by altering host protein expression, potentially hindering Ct growth and development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Microcystins (MCs) and nodularins (NODs) produced by cyanobacteria occur in ambient freshwaters and across the freshwater-marine continuum, and pose health threats through drinking and recreational waters, as well as food resources. Approximately 300 MC and NOD toxins have been published, but less than 15 of them are commercially available as toxin standards. Our aim herein was to rapidly identify and quantify all toxin congeners, including those without standards, in water samples even at low abundance by reversed-phase solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) to provide insights into toxin levels and potential toxicity., Results: Trizma instead of acid was used as an ion pairing reagent to increase retention of MCs without arginine residues on Strata X SPE. Toxin elution from Oasis HLB SPE was complicated by their hydrophilic and lipophilic interactions with HLB sorbent. Three stable isotope-labeled (SIL) toxin standards representing MCs carrying 2, 1, and 0 arginine residues served as internal standards (IS) for toxin determination and evaluation of cyanobacterial cell lysis methods. Average recoveries of toxin standards in lake water and seawater using the HLB sorbent for validation ranged from 90 to 109 % except MC-WR & LW (71-87 %) with detection limits from 1.3 to 23.7 ng L -1 . Doubly charged protonated toxin molecular ions were employed as precursors for tandem MS to screen and identify toxin congeners using a hybrid triple quadrupole linear ion trap MS. More than 30 (4 new) MCs were detected in Microcystis aeruginosa strain LE-3 culture., Significance: This is the first report to explain the issues with and possible mechanisms for SPE extraction of MCs from water, to use SIL-IS to evaluate methods for lysing cyanobacterial cells for MC release, and to show that doubly rather than singly charged toxin molecular ions as MS/MS precursors enabled efficient screening, identification, and quantification of all toxins at low levels. A MC with homoalanine residue at position 1 was reported for the first time., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

An isotope-dilution bioanalytical assay for abemaciclib and its metabolites in combination with olaparib was developed and validated in human plasma K2 EDTA. For the quantitative assay, human plasma samples (or human plasma QC samples) were spiked with internal standard solution before a simple protein precipitation with methanol. The extract was injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument where it was chromatographically separated by a polar end-capped reversed phase column and guard using gradient elution with water and methanol both modified with 0.2 % formic acid (v/v) as the mobile phases. The analytes and internal standards were measured by heated electrospray ionization (HESI) in positive polarity using selected reaction monitoring (SRM) on a triple quadrupole mass spectrometer. The assay was validated for linear ranges as follows: 0.4 - 1000 nM abemaciclib, 0.35 - 1000 nM M2 and M18, 0.5 - 1000 nM M20, and 0.75 - 1000 nM olaparib. The inter-day or between day precision for the quality controls (n = 18) was < 13 % and the accuracy was ± 12 %, for all analytes, including the lower limit of quantification (LLOQ). The intra-day or within day precision for the quality controls (n = 6) was ≤ 11 % and the accuracy was ± 12 % for low, mid, and high and < 19 % at LLOQ. The recovery in human plasma was determined to be between 92 % and 102 % for all analytes spanning the linear range. The validated, bioanalytical quantitative assay was designed to measure abemaciclib, its metabolites, and olaparib for pharmacokinetic evaluation of patients in clinical trials for breast, brain, and ovarian cancers., Competing Interests: Declaration of Competing Interest Kathleen Moore: Consultation or advisory fees from Astra Zeneca, Aravive, Aadi, Blueprint, Clovis, Caris, Duality, Eisai, GSK, Genentech/Roche, Immunogen, Mersana, Merck, Myriad, Novartis, Janssen, Regeneron, VBL Theraeputics, Verastem, and Zentalis. Other activities that could influence the work include GOG Foundation BOD, and ASCO BOD., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A multiparametric liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous quantification of 11 antifungal drugs and their metabolites in human plasma. This method addresses the critical need for therapeutic drug monitoring in the treatment of invasive fungal infections, which are increasingly prevalent among immunocompromised patients and those in intensive care units. The method quantifies flucytosin, fluconazole, itraconazole, hydroxy-itraconazole, posaconazole, isavuconazole, voriconazole, voriconazole-N-oxide, anidulafungin, caspofungin, and micafungin. Key challenges in method development included optimising mass spectrometer settings, chromatographic conditions, and sample preparation techniques to ensure accurate, sensitive, and specific detection. Validation of this method was conducted in accordance with the guidelines set by the USA Food and drug administration and the European Medicines Agency covering linearity, precision, accuracy, selectivity, matrix effect, and stability. The method exhibited robust performance with intra- and inter-assay precision under 10 % and average accuracy for intra- and inter-assay comparison of -2.35 % and 0.80 %, respectively. Limits of detection (0.002 to 0.110 mg/L) and a quantification range between 0.005 and 200 mg/L make this method suitable for clinical TDM applications. The ability to simultaneously analyse eleven antifungals and their metabolites within a single 5-minute run enhances its utility in clinical settings, particularly for critically ill patients who may experience significant pharmacokinetic variations. The method requires only 100 µL of plasma, demonstrating good analytical performances rendering it a valuable tool for optimising antifungal therapy and improving patient outcomes in ICU management., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Gimeracil, a component in S-1 (an oral anticancer agent comprising tegafur, a prodrug of 5-fluorouracil (5-FU), potassium oxonate, and gimeracil), inhibits metabolic enzymes, thereby impeding 5-FU degradation. Therefore, the blood level of gimeracil is closely associated with the disposition of 5-FU, and quantification of gimeracil can provide important information if a case shows an inappropriate 5-FU blood concentration. Nevertheless, methods for quantifying gimeracil in human plasma are rarely reported. Herein, we aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying gimeracil, in addition to tegafur and 5-FU, levels in human plasma using a clinically applicable simplified pretreatment process and faster elution time. Hence, an acetamide-functionalized monolith silica disk-packed spin column was used to extract gimeracil and internal standard (IS; nicotinamide), whereas diatomaceous earth-based solid phase for liquid-liquid extraction was used to extract tegafur, 5-FU, and IS (5-chlorouracil) from plasma. Each extract was analyzed within 4 min of elution via LC-MS/MS using a shared LC column and mobile phase. Accuracy and precision analyses indicated lower limits of quantification of 5, 10, and 2 ng/mL for gimeracil, tegafur, and 5-FU, respectively. The calibration curves showed good linearity between 5 and 500 ng/mL for gimeracil, 10 and 5000 ng/mL for tegafur, and 2 and 1000 ng/mL for 5-FU. We confirmed that the levels of all analytes in the plasma of patients with cancer undergoing S-1-inclusive therapy were within the calibration range for each analyte. Thus, this newly developed quantification method is likely to be useful for optimization of S-1 therapy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Validation of Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method was performed for estimation of imidacloprid (IM) and its metabolites in maize leaves, immature kernels, mature kernels, stalk, and soil using liquid chromatograph tandem mass spectrometry, coupled with electrospray ionization. The extraction in different matrices of maize and soil was performed using acetonitrile +0.1% formic acid followed by clean-up with primary secondary amine sorbent and anhydrous magnesium sulfate. The method was validated in terms of selectivity, linearity, limit of detection, limit of quantification, matrix effect, ion ratios, quality control, robustness, accuracy, and precision. The validation of all parameters was done in accordance with European Commission's Directorate-General for Health and Food Safety (DG SANTE) guidelines. A linear relationship with high correlation coefficients R2 > 0.99 was obtained for solvents and different matrices viz., maize leaves, immature kernels, mature kernels, stalk, and soil. The recovery and relative standard deviations were ˃78% and ˂5.4%, respectively. This method permits a simple, sensitive, accurate, cost-effective, precise, and rapid extraction of IM and its metabolites from maize leaves, immature kernels, mature kernels, stalks, and soil. This can help the residue analysts to address effective residue estimation, regular monitoring of residues and can also aid in the regulatory and food safety concerns about the usage of IM in maize., (© The Author(s) 2025. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background/objectives: Although approximately 160 human milk oligosaccharides (HMOs) have been identified, current studies on HMO quantitation are limited to the 10-19 most abundant HMOs. We assessed the variations in the relative concentrations of 71 HMO structures over lactation in human milk samples by an advanced liquid chromatography-mass spectrometry approach., Methods: Samples were collected from 64 mothers at 6 weeks, 6 months, and 12 months of lactation in the Ulm SPATZ Health Study, a German birth cohort. In this longitudinal study, we fitted linear mixed-effect models to analyze changes in the log2-transformed and standardized HMO concentration over time. Based on the profile of 71 HMOs, we also fitted a group-based multi-trajectory (GBMT) model to cluster mothers secreting cluster type I milk, who account for the majority of lactating mothers., Results: We found that 52 HMOs had a decreasing trend (regression coefficients ranging from -1.41 to -0.17) and 9 had an increasing trend (regression coefficients ranging from 0.25 to 0.64) during lactation, and the findings were statistically significant after multiple testing corrections. Using human milk samples of 49 mothers with type I milk, we further identified two novel sub-clusters with distinct longitudinal trajectories of concentrations of 71 HMOs during lactation: Type I-a (N = 20) and I-b (N = 29). These sub-clusters were not associated with maternal non-genetic characteristics., Conclusions: Our findings extend existing knowledge about the structural diversity of HMOs and their variations over lactation. These may pave the way to investigate the potential nutritional benefits of various HMOs on infant health and early life development in the future.

Maximizing the extraction of true, high-quality, nonredundant features from biofluids analyzed via LC-MS systems is challenging. Here, the R packages IPO and AutoTuner were used to optimize XCMS parameter settings for the retrieval of metabolite or lipid features in both ionization modes from either faecal or urine samples from two cohorts ( n = 621). The feature lists obtained were compared with those where the parameter values were selected manually. Three categories were used to compare feature lists: 1) feature quality through removing false positives, 2) tentative metabolite identification using the Human Metabolome Database (HMDB) and 3) feature utility such as analyzing the proportion of features within intensity threshold bins. Furthermore, a PCA-based approach to feature filtering using QC samples and variable loadings was also explored under this category. Overall, more features were observed after automated selection of parameter values for all data sets (1.3- to 3.7-fold), which propagated through comparative exercises. For example, a greater number of features (on average 51 vs 45%) had a coefficient of variation (CV) < 30%. Additionally, there was a significant increase (7.6-10.4%) in the number of faecal metabolites that could be tentatively annotated, and more features were present in higher intensity threshold bins. Considering the overlap across all three categories, a greater number of features were also retained. Automated approaches that guide selection of optimal parameter values for preprocessing are important to decrease the time invested for this step, while taking advantage of the wealth of data that LC-MS systems provide.

Oligonucleotides are currently one of the most rapidly advancing classes of therapeutic modalities. Understanding critical quality attributes, such as the impurity profile, stability, potential metabolites, and sequence conformity, is the key to their ultimate success. To obtain the information presented above, liquid chromatography-mass spectrometry (LC-MS) is often employed. However, data interpretation can be challenging due to multiple charge states, unknown species, chemical noises, and overlapping isotope envelopes (OIEs) of distinct but unresolved species. To address these challenges, we have developed an MS-platform agnostic, multifunctional R package and a web-tool for automated and interactive MS and MS 2 data analysis, OligoDistiller . From the MS spectrum of a complex mixture of two synthetic strands, our tool OligoDistiller annotated and quantified 45 oligonucleotide-related features including 13 unknown impurities and 6 OIEs, which all together explained 90.8% of the detected peaks. Also, major product ions were assigned from the MS 2 spectrum of a 47-mer DNA strand, covering 80.3% of the oligonucleotide sequence. We provide not only diverse isotope quality metrics for each annotated feature but also an interactive data review module allowing for direct inspection of the part of raw spectrum linked to a selected feature. This tool OligoDistiller is freely available at https://github.com/daniellyz/OligoDistiller.

Quantification of intact proteins in serum by liquid chromatography high-resolution mass spectrometry (HRMS) may be a useful alternative to bottom-up LC-MS or conventional ligand binding assays, due to reduced assay complexity and by providing additional information, such as isoform differentiation or detection of post-translational modifications. The 47.2 kDa lung cancer tumor marker neuron-specific enolase γ (NSEγ) was quantified in a clinically relevant concentration range of 6.25 to 100 ng/mL in NSE-depleted human serum using magnetic bead immunoprecipitation coupled to LC-high-resolution quadrupole-time-of-flight MS. The novelty of the described approach is in the combined setup of immunoaffinity extraction and the use of a full-length NSEγ calibrator and labeled NSEγ internal standard (IS) to reliably quantify the post-translationally acetylated form of this protein tumor marker in a top-down proteomics workflow. Isolation parameters and quantification using deconvolution and reconstructed extracted ion chromatograms were evaluated, and the development of a suitable liquid chromatography method was demonstrated. Various validation parameters were determined using both quantification methods, both showing acceptable performance. Additionally, deconvolution-based quantification enabled an accurate mass determination. The developed method was compared to a commercially available ECLIA and showed good correlation in sera of patients suspected of lung cancer. This assay may form the starting point for the development of a reference method for the standardization of immunoassays.

A sensitive, specific, reliable and low-cost LC-UV method has been developed and validated for simultaneous quantification of brimonidine tartrate (BM) and brinzolamide (BZ) in rabbit aqueous humor (AH) in the presence of N-desethyl-brinzolamide (NDBZ); BZ is a major degradation product, and it is also considered to be its major metabolite. Dorzolamide hydrochloride (DZ) was used as an internal standard (IS). The analytes were extracted from rabbit AH samples by a simple pre-treatment utilizing ZnSO4.7H2O as a deproteinizing agent. The analytes were separated on a cyanopropyl Waters column (4.6 × 200 mm, 5 μm) with an isocratic mobile phase consisting of 25 mM ammonium acetate pH 4.5 (adjusted with 85% phosphoric acid):methanol:acetonitrile (94:4.5:1.5, v/v) at a flow rate of 1.0 mL min-1. The detection was done at 254 nm. The lower limit of quantification in rabbit AH was 100.0 ng mL-1. The method was validated according to EMA guidelines. The method was confirmed to be accurate, precise and linear over a range of 100.0-1000.0 ng mL-1 for BM and BZ. The method developed herein is simple, safe, eco-friendly, rapid and accurately applied for the quantification of BM and BZ, along with the successful separation of NDBZ, which is considered as a potential irritant degradation product of BZ., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Recently, the demand for respiratory disease-related products has surged due to the influence of coronavirus disease 2019, prompting warnings about illegal dietary supplements containing unauthorized substances. Additionally, adulterated dietary supplements are continuously detected in open markets, posing significant public health safety problem. In this study, we developed and validated an analytical method for 11 respiratory drug substances using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and proposed optimal conditions for LC-quadrupole time-of-flight MS (LC-QTOF-MS) to determine the fragmentation patterns of each substance. This method underwent thorough validation considering specificity, linearity, limits of detection and quantification, accuracy, precision, matrix effect, stability, etc. All results met international guidelines. These validated methods were applied to 52 dietary supplements advertised for treating respiratory diseases and enhancing respiratory function, among which one sample was found to contain 313.7 mg/g of theobromine. This determination was made by comparing the product ion ratios with the standards and subsequent quantification. To re-confirm the detected substances, their fragmentation patterns were compared with those of the standards using LC-QTOF-MS. In conclusion, the mass-based information, coupled with the LC-ESI-MS/MS method development, can be successfully applied to rapidly identify 11 respiratory drug substances in illegal dietary supplements used for respiratory disease treatment. The developed simultaneous detection method contributes to public health and safety improvements., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Many analytical methods have been developed for performing targeted metabolomics. By combining multiple analytical techniques, comprehensive coverage of the metabolome can be achieved. We combined multiple analytical techniques to comprehensively and quantitatively characterize the widely studied NIST human plasma reference material, SRM 1950. Our goal was to provide a large, well-validated list of confident metabolite concentration values (i.e., benchmarks) to assist the metabolomics community in its calibration and comparison efforts. We used four analytical platforms: high-resolution NMR spectroscopy, direct injection tandem MS (DI-MS/MS), liquid chromatography tandem MS (LC-MS/MS), and inductively coupled plasma MS (ICP-MS). Eight validated analytical assays were run, yielding accurate quantitative measurements for 728 unique metabolites or metabolite species. Through computer-aided literature mining, we identified another 330 unique metabolites previously quantified in SRM 1950. We compared NIST-certified values along with literature-derived concentrations/ranges to the metabolite concentrations measured by our four platforms and eight assays. From these assays/platforms, we generated a list of high-confidence concentration values of 1058 metabolites or metabolite species in SRM 1950 including data for 60 amino acids/related compounds, 48 bile acids, 72 amines/sugars/alcohols, 21 metals, 8 catecholamines, 11 vitamins, 92 organic acids, 40 fatty acids/steroids/nucleobases/indole derivatives, 5 polyfluorinated compounds, 7 carotenoids, 39 acylcarnitines, 76 oxylipins, 13 sterols, and 566 lipids/lipid species. This data set represents the most complete quantitative characterization of SRM 1950. An online database (SRM1950-DB) containing 1058 plasma metabolites/metabolite species in SRM 1950, their structures, HMDB IDs, mass, chemical class, concentrations, references, and reliability is freely available at https://srm1950-data.wishartlab.com.

Here we show that when using a mix of 274 light synthetic peptide standards (NAT) as surrogates for 270 human plasma proteins, as well as stable isotope-labelled standards (SIS) as normalizers (both from MRM Proteomics Inc.) for targeted quantitative analysis by liquid chromatography multiple reaction monitoring mass spectrometry (LC/MRM-MS), the Seer Proteograph™ platform allowed for the enrichment and absolute quantitation of up to an additional 62 targets (median) compared to two standard proteomic workflows without enrichment, representing an increase of 44%. The nanoparticle-based fractionation workflow resulted in improved reproducibility compared to a traditional proteomic workflow with no fractionation (median 8.3% vs. 13.1% CV). As expected, the protein concentrations in nanoparticle coronas were higher and had more compressed dynamic range in comparison to the concentrations determined either by a 3-hour Trypsin/LysC or overnight tryptic digestion methods. As the nanoparticle-based fractionation technology gains popularity, additional steps such as establishing technique-specific protein reference ranges across plasma samples and comparisons to well-established protein quantitation methods like enzyme-linked immunosorbent assay (ELISA) and LC/MRM-MS may be explored to enable absolute quantification of plasma proteins based on nanoparticle-based fractionation data.

Biological aminothiols (BATs) typically exist in both reduced and oxidized forms, each exhibiting diverse biological activities. Monitoring the levels and ratios of the two forms is crucial for clinical diagnosis and understanding their roles in biological systems. In this study, we developed a method for simultaneous analysis of both reduced and oxidized BATs using a double derivatization approach combined with liquid chromatography-mass spectrometry (LC-MS). The method employed a sequential derivatization strategy: initially, 2‑bromo-N,N-dimethylacetamide (Br-DMA) reacted with the thiol groups of reduced BATs, followed by derivatization of both reduced and oxidized BATs with stable isotope labeling reagents, [d 0 ]-/[d 3 ]-6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate ([d 0 ]-/[d 3 ]-DMMIC). The methodology validation showed excellent linearity (R 2 > 0.99), accuracy (85.07-119.94 %), precision (intraday: 5.26-18.78 %; interday: 6.52-19.01 %), recovery (70.09-119.27 %), and matrix effect (92.69-126.79 %). Finally, the method was successfully applied to nontargeted BAT screening in lung A549 cells, assessing changes in BAT levels in A549 cells upon treatment with the anticancer compounds triptolide and bufalin, and comparing differences in BAT levels between lung adenocarcinoma and paracarcinoma tissues. The results indicated that the developed method could be a comprehensive practical protocol and serve as a platform for profiling reduced and oxidized BATs in biological samples while meeting various analysis demands., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Asari Radix et Rhizoma (ARR) is a traditional Chinese herbal medicine derived from the genus Asarum in the Aristolochiaceae family, which has been widely used for years. Aristolochic acids in it significantly restrict the clinical application of ARR due to its nephrotoxic and carcinogenic properties, of which aristolochic acid I (AA I) is the most representative. The present paper describes two 2D-LC systems modulated by a sample loop and a trap column respectively for the detection of trace aristolochic acid I in ARR. An Inertsil ODS-3 C18 column and a Luna 3u Phenyl-Hexyl column were separately utilized in the first-dimension (1D) and second-dimension (2D) chromatography. A mixed-mode column with reversed-phase and ion-exchange was selected for trap column modulation of 2D-LC system. Method validation results showed that these two 2D-LC methods performed well in terms of repeatability, linearity, intra/interday precision, stability, and accuracy, which were demonstrated qualified for the simultaneous determination of trace AA I in ARR. The AA Ⅰ contents in three batches of ARR were 0.466∼0.812 μg/g by the two 2D-LC methods, which were below the limit requirement in the Chinese Pharmacopoeia. The comprehensive comparison between the two methods showed that a significant solute focusing effect was obtained by trap column modulation, which led to four-time lower LOD compared with loop modulation, while the loop-modulation-based method achieved higher analytical efficiency when analyzing a large batch of samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Metabolomics based on hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (MS) is a powerful tool for polar metabolite identification and quantification to further contribute to biomarker discovery and disease mechanism elucidation. However, matrix effect (ME), which may lead to altered ionization efficiency due to co-eluting compounds, is a significant challenge during biological analysis. Therefore, ME evaluation plays a crucial role during method development. Two approaches to evaluate ME are using stable isotope labelled-internal standards (SIL-IS) and post-column infusion (PCI) of standards. In this study, we developed an untargeted HILIC-MS method by applying four PCI standards for ME evaluation. We found PCI is a compelling approach for ME assessment compared to SIL-IS method due to its advantage in untargeted analysis. Through the ME evaluation and chromatographic performance comparison of 18 SIL standards across three columns and three different mobile phase pH conditions, our findings revealed that the BEH-Z-HILIC column operated at pH 4 with 10 mM ammonium formate exhibited minimal ME and superior performance. The method showed exceptional linearity (R² > 0.98), reliable repeatability (RSD < 15 %), good inter-day precision (RSD < 30 %), and acceptable recovery (>75 %) for all SIL standards. Absolute matrix effect (AME) and relative matrix effect (RME) assessment in three plasma donors revealed a high consistency between PCI and SIL-IS approaches. Finally, this method coupled with the PCI approach was applied to 40 plasma samples. Fifty endogenous compounds were detected and their AME and RME were evaluated. Results showed that many compounds experienced severe ion suppression, though their ME variation between 40 samples is low. In conclusion, PCI method is a robust alternative for monitoring ME and evaluating ME of endogenous compounds during untargeted method optimization and biological analysis., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Thomas Hankemeier, Anne Charlotte Dubbelman, Amy C Harms has patent #NL2022019B1, EP3881068A1, US20220003726A1, CN113196052A, WO2020101499A1 pending to Universiteit Leiden. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In this work, a DNPH doped PDMS based membrane was developed to facilitate carbonyl compound derivatization. This membrane delivers DNPH in presence of carbonyl compounds to form hydrazones. Subsequently, the resulting hydrazones are preconcentrated, separated and detected by in-tube solid phase microextraction (IT-SPME) coupled on-line with capillary liquid chromatography (CapLC) with Uv-Vis diode array detection (DAD). The method proposed allowed the determination of the analytes up to 200 µg/L. The limits of detection were between 0.1 and 3.3 µg/L and intra-day precision and inter-day precision were lower than 7 % and 21 %. The applicability of the strategy proposed was tested in environmental (waters and PM10) and biological samples (saliva). Samples were satisfactorily analysed with recoveries in the range of 68 %-80 %. The main advantages of the proposed strategy were the increment of DNPH stability, a reduction in the solutions and wastes required for the analysis and protection of the chromatographic column minimizing the excess of DNPH, thus providing a more sustainable analytical method to estimate carbonyl compounds., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

The retention time of a polymer in liquid chromatography depends on the details of its microstructure and topology. Despite the number of separation modes and methods available for polymers, gaining quantitative information from chromatograms remains a challenge. A model able to predict the LC retention time of a polymer accounting for all possible variations in its microstructure could provide some valuable insight during method development and produce the information necessary to establish unambiguous structure/property relationships. In a previous article, we reported on the separation of end-functionalized oligomers with the Hypercarb™ column using interaction polymer chromatography. In this article, the chromatograms for the oligomer were simulated using the general model for the partition coefficient of linear polymers in adsorbing pores developed by Gorbunov and Skvortsov [1]. The chromatograms of the oligomer were simulated under a variety of conditions mimicking the experimental ones. The results confirmed the predictive strength of the model. To explain some unexpected results for high molecular weight polymers under size exclusion conditions, hydrodynamic effects were considered as well as a sorbent consisting of two pore networks. This provided new insight into the Hypercarb™ column properties., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Biochars (BCs) derived from waste-branches of apple tree, grape tree, and oak were developed for direct solid-phase extraction (SPE) of five benzodiazepines (BZDs) in crude urine samples prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination. Scanning electron microscopy, elemental analyzer, X-ray diffractometry, N 2 adsorption/desorption experiments, and Fourier transform infrared spectrometry characterizations revealed the existence of their mesoporous structure and numerous oxygen-containing functional groups. The obtained BCs not only possessed high affinity towards BZDs via π-π and hydrogen bond interactions, but also afforded the great biocompatibility of excluding interfering components from undiluted urine samples when using SPE adsorbents. Variables affecting SPE of target analytes were systematically optimized including pH, ionic strength, dilution ratio, washing solution, desorption solvent, and its volume. The method of BC-based SPE combined with LC-MS/MS exhibited a wide linear range of 0.03-100 ng/mL, a low detection limit of 0.01-0.08 ng/mL, and satisfactory recovery of 77.6-106% for the studied BZDs. Notably, this method allowed the possibility of direct loading of undiluted urine samples and avoided tedious filtration and dilution steps, which significantly simplified the pretreatment process. Additionally, these BC sorbents derived from waste-branches were ecofriendly and cost-effective, providing a sustainable alternative for the traditional SPE sorbents. Thus, the proposed method has promising application for ecofriendly, simple, efficient, and reliable monitoring of BZDs in urine samples., Competing Interests: Declarations. Ethics approval: The study was approved by the Ethics Committee of North China University of Science and Technology (Approval number: 20240313065). The procedures used in this study adhere to the tenets of the Declaration of Helsinki. Consent to participate: All participating volunteers were informed that the samples provided will be used for scientific research. Competing interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Nowadays, synthetic cathinones (SCs) is the second more representative subclass of New Psychoactive Substances, accounting for 104 analogues in the illegal market. Since its first report in 2011, α-pyrrolidinovalerophenone (α-PVP) gained popularity among drug users, provoking an increased number of intoxications. Nonetheless, pharmacokinetics data is still limited in the literature. An observational non-controlled naturalistic study on 8 healthy volunteers was conducted to assess the α-PVP and β-OH-α-PVP concentrations in OF and urine, after snorting 10 mg or 20 mg of α-PVP. A multi-analytical approach based on GC-EI-MS/MS and LC-HESI-HRMS/MS was developed and fully validated for the analytes quantification, while four untargeted LC-HESI-HRMS/MS methods in full-MS and ddMS 2 were set up for unknown metabolites characterization in urine samples assisted by a dedicated data mining software. In OF, α-PVP reached a mean C max of 762 ± 323 ng/mL at 1 h after 10 mg administration, while a C max of 2,900 ± 1,373 ng/mL at 47 min after 20 mg dose. In urine, a total α-PVP mean amount of 179.2 ± 94.9 µg was accumulated after 10 mg dose, (27.2 ± 9.8 µg between 0-2 h and 152.0 ± 98.2 µg between 2-5 h), while a total amount of 122.9 ± 44.0 µg, of (36.2 ± 16.5 and 86.7 ± 28.3 µg between 0-2 and 2-5 h, respectively) was detected after 20 mg dose. Among the 10 identified metabolites, β-OH-α-PVP was a minor metabolite (total amount: 56.4 ± 27.1 and 69.1 ± 38.1 µg after 10 mg and 20 mg). The N-butanoic acid metabolite was the most abundant, detected also as glucuronide. In conclusion, α-PVP showed a later time peak than non-pyrrolidine SCs, with comparable C max. The pyrrolidine ring oxidative opening produced the most abundant urinary metabolite, independently from the dose., Competing Interests: Declarations. Competing Interest: All the authors declare no competing interest for this research., (© 2025. The Author(s).)

Ibrutinib is an orally administered compound that functions as an irreversible covalent inhibitor of the Bruton tyrosine kinase, an essential element in multiple cellular processes including B-cell differentiation, proliferation, migration, survival and apoptosis. The compound has been found to demonstrate efficacy against a range of B-cell malignancies. The drug product is available in oral tablet and capsule formulations. The drug degradation profiles of tablets dosage form were assessed in accordance with regulatory guidelines. The results indicate that the drug substance is susceptible to alkaline and oxidative stress. The oxidation degradation led to the identification of three significant unknown degradation impurities. The three compounds were isolated through the application of preparative liquid chromatography, and their structures were determined using analytical techniques such as liquid chromatography-mass spectrometry, high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Utilizing structural elucidation data, predictions were made regarding the composition of impurities, revealing them to be novel degradation impurities that bear structural resemblance to ibrutinib. Additionally, potential pathways for the formation of the impurities were proposed., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

N-nitrosamine impurities have been detected in a vast variety of drug substances and drug products, showing concern for regulatory aspects. To meet the regulatory requirement for the concerned impurity, a sensitive analytical method capable of quantifying these impurities at a lower level with accuracy and precision is required. This article focuses on the development and validation of an analytical method for the simultaneous detection of nine nitrosamine impurities in a single method for nebivolol drug product using liquid chromatography-mass spectrometry/mass spectrometry-atmospheric pressure chemical ionization (LC-MS/MS-APCI). The chromatographic separation was performed using the LC-MS column Allure BiPh C18 (250 × 4.6 mm), 5 μm employed a gradient mode elution program using 0.002 M Ammonium acetate buffer pH 4.5 as mobile phase A and methanol as mobile phase B. The method was challenged for accuracy, precision and linearity in accordance with International Council for Harmonization guidelines to ensure its suitability for the intended usage. The developed method was specific, accurate and linear with square of correlation coefficient (r2) found to be greater than 0.99 (0.9970-0.9992). The LOQ obtained in the range of 9.85-19.62 ppb for nine nitrosamines showed good sensitivity. The results demonstrated that method can be applied to quantify the nitrosamines in nebivolol drug products., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In 2019, the U.S. Department of Agriculture's (USDA) Food Safety and Inspection Service (FSIS) provided revised guidance for labeling claims of "raised without antibiotics" (RWA) and similar terms for meat and poultry produced in the US. In 2022, Price et al. published a study reporting that 15% of RWA-labeled cattle contained antibiotic residues in urine samples. In 2023, the USDA embarked on the project reported herein to independently determine the extent of antibiotic drug residues present in kidney and liver tissues from slaughtered cattle purported to be RWA. In this study, FSIS inspectors randomly collected kidney and liver tissues from 189 RWA animals in 79 slaughter establishments across the US. The samples were monitored for 185 veterinary drugs multiple times by different means, including liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quadrupole high-resolution MS with an orbital ion trap instrument (LC-Q/orbitrap). Samples from 37 animals (20%) met the analytical identification criteria for at least one antibiotic confirmed in at least two analyses. Multiple antibiotics were determined in 11 RWA animals. Macrolides (tulathromycin, tildipirosin, tilmicosin, and gamithromycin) were confirmed in 20 (11%) of the RWA animals. The ionophore, monensin, was also confirmed in tissues from 11 samples, with unconfirmed/partial identifications in 8 others. Tetracyclines were confirmed in samples from 12 animals, several of which were found to contain multiple antibiotic residues. One animal each was also found to be positive for sulfamethazine (a sulfonamide antibiotic) and metabolites of penicillin G (a β-lactam antibiotic). Although they are not antibiotics, two anthelmintics (fenbendazole and eprinomectin) were confirmed in tissues from 3 RWA animals. FSIS has informed the slaughter establishments with positive results and advised them to conduct a root cause analysis and implement corrective actions.

In a new respiratory virus pandemic, optimizing allocation of scarce medical resources becomes an urgent challenge. Infection prognosis takes on particular importance when allocating scarce antiviral antibodies and drugs, which are most effective when administered before the onset of severe disease. During arrival of the COVID-19 pandemic to the United States in 2020, we conducted a prognostic biomarker discovery and validation effort based upon metabolomic profiling with a liquid-chromatography-mass spectrometer (LC-MS) type used clinically for rapid toxicology. We obtained urine specimens from 163 patients presenting for evaluation. We obtained LC-MS profiles in the initial cohort and used machine learning methods to define a simplified urine metabolomic signature associated with respiratory failure or death by 90 days. This signature was composed of three metabotypes linked to intestinal microbiome metabolism and anticonvulsant use, with a receiver-operator characteristic area under the curve (ROC AUC) of 89.4%. Blinded application of this signature to the subsequent validation cohort yielded a ROC AUC of 81.2%. A model trained on the two baseline metabotypes present before intubation exhibited similar performance in the validation cohort. This study demonstrates the plausibility and promise of rapid metabolome-based prognostic discovery and validation in the opening wave of a pandemic. The approach used here could be used to inform therapeutic and resource allocation decisions early in a future epidemic.IMPORTANCEIn a new respiratory virus pandemic, the ability to identify patients at greatest risk for severe disease is essential to direct scarce medical resources to those most likely to benefit from them. Tools to predict disease severity are best developed early in a pandemic, but laboratory-based resources to develop these may be limited by available technology and by infection precautions. Here, we show that an accessible metabolic profiling approach could identify a prognostic signature of severe disease in the initial wave of COVID-19, when patients presenting for care often exceeded the available doses of convalescent plasma and remdesivir. In a future pandemic, this approach, alongside efforts to identify clinical disease severity predictors, could improve patient outcomes and facilitate therapeutic trials by identifying individuals at high risk for severe disease., Competing Interests: The authors declare no conflict of interest.

Antibody-drug conjugates (ADCs) are intricate compounds that pose significant challenges in bioanalytical characterization. Therefore, multiple bioanalytical methods are required to comprehensively elucidate their pharmacokinetic (PK) profiles. In this study, we investigated DS001, an ADC consisting of a humanized monoclonal antibody (hRS7), a cleavable chemical linker, and the microtubule inhibitor monomethyl auristatin E (MMAE), with a drug-to-antibody ratio (DAR) of 8. This study established a rapid and sensitive hybrid immunoaffinity liquid-chromatography-tandem-mass-spectrometry (LC-MS/MS) approach for the simultaneous quantification of the total antibody and the enzymatically cleavable conjugated payload of DS001. This method is capable of monitoring fluctuations in average DAR values during PK assessments. The sample preparation procedure involved immunocapture, denaturation, trypsin digestion, papain digestion, and termination, all completed within a total processing time of less than 4 h. The method demonstrated linearity for the total antibody in the range of 100 ng/mL (lower-limit-of-quantification, LLOQ) to 100,000 ng/mL, and for the conjugated payload from 3.495 ng/mL (LLOQ) to 3495 ng/mL in rat serum. Both analytes exhibited standard curve correlation coefficients (r) greater than 0.990 within their respective linear ranges. The precision and accuracy of the method were within ± 15% (± 20% for LLOQ). The verified LC-MS/MS approach was successfully employed in the PK analysis following intravenous administration of 0.2 mg/kg DS001 in rats via tail vein injection., Competing Interests: Declarations. Conflict of Interest: The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this article., (© 2024. The Author(s), under exclusive licence to American Association of Pharmaceutical Scientists.)

Yersinia pestis, a Gram-negative bacterium is the causative agent of the fatal communicable disease plague. The disease had a profound impact on human history. Plague bacteria are usually transmitted to humans through the bite of an infected rat flea. Earlier studies have indicated that Y. pestis can survive in environmental matrices e.g. water and soil. This study aimed to generate a peptide-based screen for identification of Y. pestis particularly from environmental matrices. We employed a shotgun proteomic approach using nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) to discover Y. pestis-specific peptides. The pure cultures of Y. pestis and related species were grown, their proteome were delineated and analyzed by in silico tools to discover 61 Y. pestis specific peptides. Additionally, 148 peptides were discovered from proteins of Y. pestis-specific plasmids and chromosomal-associated virulence markers. To validate this screen of 209 peptides, various concentrations of Y. pestis (ranging from 1.3 × 10 8 to 1.3 × 10 5 cfu) were spiked into garden soil. Y. pestis could be identified in all samples except un-spiked negative control soil sample. This study offers a valuable method for the identification of Y. pestis, by tandem mass spectrometry which may be used in environmental and clinical matrices., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Alzheimer's disease (AD) poses a global health challenge, demanding innovative approaches for effective treatments. Clerodendrum infortunatum Linn. (Lamiaceae) is a shrub traditionally used as a medicinal plant to treat inflammation, skin diseases, and bronchitis. This study aims to identify the main bioactive metabolites in C. infortunatum using LC-QTOF-MS/MS and investigate its potential in protecting against cognitive decline in rats with scopolamine-induced AD disease. Metabolite profiling was performed on the methanol extract of the plant's aerial parts using LC-QTOF-MS/MS. The inhibitory activity of the acetylcholinesterase enzyme was measured in vitro. To evaluate the cognitive effects, the methanol extract was orally administered at three doses (100, 200, and 400 mg/kg) to scopolamine-induced AD rats, and their cognitive functions were assessed using the novel object recognition test. Additionally, acetylcholinesterase enzyme activity, as well as the levels of acetylcholine, dopamine, noradrenaline, glutathione, malondialdehyde, tumor necrosis factor-α, interleukin-1β, and amyloid-β in the rat hippocampus, were measured using ELISA, followed by histopathological evaluation. A total of 79 metabolites, spanning various chemical classes, such as organic acids, phenolic acids, phenylpropanoids and phenylethanoids, flavonoids, coumarins, other phenolics, and fatty acids and their derivatives, were identified. The results showed that the extract promoted enhanced cognitive functions in the novel object recognition test. Scopolamine administration significantly altered the acetylcholinesterase enzyme activity and biomarker levels in the rat's hippocampus. However, treatment with C. infortunatum at 200 and 400 mg/kg almost restored these neurotransmitter levels to normal, which was further confirmed by histopathological analysis. This study demonstrates the therapeutic potential of C. infortunatum in mitigating cognitive decline in AD, with its first metabolite profiling revealing a range of bioactive compounds. The extract improved cognitive function in scopolamine-induced AD rats, restored acetylcholinesterase activity, normalized neurotransmitter levels, and reduced oxidative stress and inflammation. These findings suggest that C. infortunatum is a promising candidate for the development of natural therapies targeting AD., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: The experiments adhered to the “Guide for the Care and Use of Laboratory Animals” published by the US National Institutes of Health (NIH Publication No 85–23, 2011). Approval was granted by the Research Ethics Committee of the Faculty of Pharmacy of Cairo University (approval code: MP (3118)). ARRIVE guidelines: The study is reported in accordance with ARRIVE guidelines. A statement naming the person who identified the plant: The aerial parts of C. infortunatum were authenticated by Agriculture Engineer Therese Labib, a plant taxonomy consultant of the Ministry of Agriculture (Giza, Egypt). Voucher specimen statement: The plant material was collected with permission in compliance with national guidelines from the Agriculture Research Center, Giza, Egypt at “9 Cairo University Road, Giza District, Giza Governorate”. Samples of the plant material were deposited at the herbarium of the Faculty of Pharmacy, Cairo University, Cairo, Egypt (sample No.10.3.2021)., (© 2024. The Author(s).)

Polylactic acid (PLA) straws hold eco-friendly potential; however, residual diisocyanates used to enhance the mechanical strength can generate carcinogenic primary aromatic amines (PAAs), posing health risks. Herein, we present a rapid, comprehensive strategy to detecting PAAs in 18 brands of food-grade PLA straws and assessing their migration into diverse food simulants. Surface-enhanced Raman spectroscopy was conducted to rapidly screen straws for PAAs. Subsequently, qualitative determination of migrating PAAs into various food simulants (4 % acetic acid, 10 % ethanol, 50 % ethanol) occurred at 70 °C for 2 h using liquid chromatography-mass spectrometry. Three PAAs including 4,4'-methylenedianiline, 2,4'-methylenedianiline, and 2,4-diaminotoluene were detected in all straws. Specifically, 2,4-diaminotoluene in 50 % ethanol exceeded specific migration limit of 2 μg/kg, raising safety concerns. Notably, PAAs migration to 10 % and 50 % ethanol surpassed that to 4 % acetic acid within a short 2-hour period. Moreover, PLA straws underwent varying degrees of shape changes before and after migration. Straws with poly(butylene succinate) resisted deformation compared to those without, indicating enhanced heat resistance, while poly(butyleneadipate-co-terephthalate) improved hydrolysis resistance. Importantly, swelling study unveiled swelling effect wasn't the primary factor contributing to the increased PAAs migration in ethanol food simulant, as there was no significant disparity in swelling degrees across different food simulants. FT-IR and DSC analysis revealed higher PAAs content in 50 % ethanol were due to highly concentrated polar ethanol disrupting hydrogen bonds and van der Waal forces holding PLA molecules together. Overall, minimizing contact between PLA straws and alcoholic foods is crucial to avoid potential safety risks posed by PAAs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Dai-Bai-Jie, the root of the plant Marsdenia tenacissima from the Asclepiadaceae family, is well-known for its therapeutic effects in clearing heat, detoxifying, reducing swelling, and relieving pain as one of the most commonly used Dai medicine. Due to numerous structurally similar C21 steroidal compounds in Dai-Bai-Jie, chemical composition profiling has been substantially challenged. In this study, an offline two-dimensional chromatographic method (LC × SFC separation system) was developed to address these issues. Using the Hypersil Gold ( 1st D LC column) and 2-PIC ( 2nd D SFC column) based on 40 reference standards, the orthogonality was as high as 83.83 %. Most profiled ion peaks were tentatively identified through quadrupole time-of-flight mass spectrometry and a self-built compound virtual library. Consequently, the integrated method effectively addressed and resolved the issues associated with co-elution, thus significantly expanding the peak capacity. This advancement identified 362 C21 steroidal components, 319 of which were speculated to be potentially novel compounds. Furthermore, 86 groups of isomeric compounds were distinguished. This method provides a comprehensive understanding of chemical composition of Dai-Bai-Jie and an integrated qualitative analysis method for the C21 steroids., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hydrophilic Interaction Liquid Chromatography (HILIC) and Mixed-Mode Chromatography (MMC) excel in separating polar, hydrophilic, and charged analytes due to unique hydrophilic or mixed-mode retention mechanisms. They represent a complementary approach to the widely used Reversed Phase Liquid Chromatography (RPLC). Often, where RPLC struggles, HILIC and MMC thrive. The applications of HILIC and MMC in pharmaceutical analysis are expanding rapidly across a variety of drug modalities. This article reviews advances in the applications of HILIC and MMC in seven major areas of pharmaceutical analysis: synthetic small molecules, counterions and salts, lipids and surfactants, carbohydrates, amino acids and peptides, proteins, and nucleic acids in the past two decades. We aim to provide comprehensive information and strategic guidance to facilitate future research, development and applications in these areas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abnormal glycosylation and phosphorylation are strongly associated with brain aging. N-glycosylation and phosphorylation are closely involved in pathological processes in a crosstalk dependent manner. However, simultaneous characterization of glycosylation and phosphorylation together in aging brain was uncommon. Herein we developed a novel tandem HILIC-IMAC strategy for simultaneous analysis of N-glycoproteomics and phosphoproteomics. This tandem method showed higher enrichment repeatability, more identifications of glycopeptides and phosphopeptides, and lower overlap. Application of the established method to mouse brain at two ages (8 weeks and 65 weeks) to explore changes in glycosylation and phosphorylation during aging. Up to 10,990 N-glycopeptides and 11,409 phosphopeptides were identified from mouse brain. Among these, differentially expressed phosphoproteins were involved in regulation of microtubule depolymerization, synapse, and transmission of nerve impulse. And glycoproteins differentially expressed with age were mostly related to cell adhesion processes and extracellular matrix. Furthermore, we found the opposite expression trends in glycosylation and phosphorylation during aging on Grin2b. Together, the HILIC-IMAC strategy has potential to discover aging biomarkers and analyze complex biosamples, paving the way for in-depth investigations for the changes of protein glycosylation and phosphorylation in aging brain., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Prostaglandin analogs (bimatoprost, travoprost, tafluprost, etc.) have similar effects to prostaglandins and are effective drugs for treating glaucoma. These compounds exhibit abnormal reactions such as causing eyelash growth, with several cases being reported of people purchasing them to increase eyelash growth; however, some cases have reported side effects such as pigmentation and dry eyes. In the Republic of Korea, cosmetics are not medicines for treating diseases; therefore, cosmetics cannot contain drugs or have labels that could mislead people. However, there are cases in which products claim to elongate and enrich eyelashes. Concerns about the abnormal reactions of these products are constantly growing, and the absence of analytical methods for illicit compounds (prostaglandins and their analogs) in cosmetics (eyelash growth serums) renders monitoring challenging. Accordingly, in this study, we sought to develop an LC-MS/MS method for facile and fast analysis of compounds illegally mixed into eyelash growth serums. Green analytical chemistry has recently emerged because of environmental concerns. In line with this trend, we developed an optimal method by comparing the methods mainly used in cosmetic pretreatment (solvent extraction, QuEChERS, and solid phase extraction) with a method using deep eutectic solvents (DESs), which is an eco-friendly solvent. As a result of validation according to the International Conference on Harmonization guidelines, the limit of detection was 0.20-9.34 ng/mL, and the limit of quantification was 0.60-28.31 ng/mL. Recovery, linearity, precision, and accuracy were within acceptable ranges. Additionally, using the Analytical GREEnness calculator and complex green analytical procedure index tools, we confirmed that the method using the DES was greener than the other methods. In this study, we developed an analytical method for illicit compounds contained in eyelash growth serums, offering an eco-friendly approach for the prevention of the distribution of illegal cosmetics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The routine monitoring of per- and polyfluoroalkyl substances (PFASs) in drinking water has become an important task in the field of public health. In this study, a fluorinated covalent organic framework (COF) was synthesized at room temperature using tetra-(4-aminophenyl) methane (TAM) and 2,3,5, 6-tetrafluoro-terephthalal (TFTA) as building blocks and named as TAM-TFTA-COF. The adsorption characteristics of PFASs on the TAM-TFTA-COF were investigated through adsorption model-fitting and molecular calculation. The TAM-TFTA-COF was served as the solid phase extraction (SPE) cartridge packing for the enrichment of PFASs. Combined with liquid chromatography-tandem mass spectrometry, the proposed method showed good linearity in the range of 1.25-375 ng·L -1 , low limits of detection (0.03-0.24 ng·L -1 ), and excellent intraday and interday precisions with RSD <10.3 %. Furthermore, this analytical method can be utilized for the determination of PFASs in tap water, spring water, and lake water with satisfactory accuracy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

In recent years, mushroom poisoning has been one of the most important factors of food poisoning in China, timely identification of the toxins contained in mushrooms is crucial for the treatment of patients. In this study, boric acid carbon dots (BA-CDs) can undergo specific boron affinity reactions with amatoxins toxins containing o-dihydroxyl groups by means of boric acid groups. Functional covalent organic framework (COF) and BA-CDs were combined to design a adsorbent with boric acid group (COF@VBC@BA-CDs) was designed to meet the requirements of both fluorescent and pretreated materials for amatoxins. The mushrooms and urine samples were rapid screening using fluorescence detection, and then, for positive samples, the target analytes on the COF@VBC@BA-CDs are collected and eluted for next liquid chromatography tandem mass spectrometry (LC-MS/MS) detection. According to the fluorescence characteristics of COF@VBC@BA-CDs, the fluorescence quenching intensity was linearly correlated with the concentration (2-200 μg/L) and the detection limit was 1.2 μg/L. Meanwhile, the detection limit of LC-MS/MS was 0.5 μg/kg for musroom and 0.2 μg/L for urine, as well as the recovery rate was 72.7-110.1%. This noval method meets the methodological requirements and can be used for actual sample analysis., Competing Interests: Declaration of competing interest Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Diagnosis of non-small cell lung cancer (NSCLC) currently relies on imaging; however, these methods are not effective for detecting early stage disease. Investigating blood-based protein biomarkers aims to simplify the diagnostic process and identify disease-associated changes before they can be seen by using imaging techniques. In this study, plasma and frozen whole blood cell pellets from NSCLC patients and healthy controls were processed using both classical and novel techniques to produce a unique set of four sample types from a single blood draw. These samples were analyzed using 12 immunoassays and liquid chromatography-mass spectrometry to collectively screen 3974 proteins. Analysis of all fractions produced a set of 522 differentially expressed proteins, with conventional blood analysis (proteomic analysis of plasma) accounting for only 7 of the total. Boosted regression tree analysis of the differentially expressed proteins produced a panel of 13 proteins that were able to discriminate between controls and NSCLC patients, with an area under the ROC curve (AUC) of 0.864 for the set. Our rapid and reproducible (<10% CV for technical replicates) blood preparation and analysis methods enabled the production of high-quality data from only 30 μL of complex samples that typically require significant fractionation prior to proteomic analysis. With our methods, almost 4000 proteins were identified from a single fraction over a 62.5 min gradient by LC-MS/MS.

Advanced liquid chromatogram-mass spectrometry (LC-MS) and automated large-scale data processing have made MS-based quantitative analysis increasingly useful for research in fields such as biology, medicine, food safety, and beyond. This is because MS-based quantitative analysis can accurately and sensitively analyze thousands of proteins and peptides in a single experiment. However, the precision, coverage, complexity, and resilience of conventional quantification methods vary as a result of the modifications to the analytic environment and the physicochemical characteristics of analytes. Therefore, specially designed approaches are necessary for sample preparation. Dozens of methods have been developed and adapted for these needs based on stable isotopic labeling or isobaric tagging, each with distinct characteristics. In this review, we will summarize the leading strategies and techniques used thus far for MS-based protein quantification as well as analyze the advantages and shortcomings of different approaches. Additionally, we provide an overview of protein metrology development.

CKD is frequently diagnosed only after a significant progression. GFR is the most common indicator of kidney function but is limited in detecting early CKD cases and distinguishing glomerular, tubular, and global CKD. Aiming to provide a glomeruli specific biomarker assay, we developed a peptide immunoaffinity targeted mass spectrometry method for the quantitation of three podocyte specific proteins in human urine: nephrin, podocalyxin, and podocin. Proteins in urine were precipitated, stable isotope labeled peptide standards incorporated, and digested with trypsin. Target peptides were enriched using an online antibody column prior to LC-MS/MS. The performance metrics for nephrin, podocalyxin, and podocin were evaluated: The lower limits of quantitation were 3.8, 22.0, and 5.4 pM, respectively. The intraplate relative error (RE) was within ±10.6%, ± 10.4%, and ±16.1%, and coefficient of variation (CV) was ≤27.2%, ≤ 14.1%, and ≤20.7% accordingly. The interplate RE was within ±7.0%, ± 3.8%, and ±3.0%, and CV was ≤17.2%, ≤ 12.1%, and ≤20.0% for the three analytes. The urinary nephrin, podocalyxin, and podocin concentrations in 60 healthy volunteers and 20 disease samples was measured, thereby establishing the basal levels of these protein and enabling future evaluation of their roles as noninvasive biomarkers of glomerular injury in the clinic.

Host cell proteins (HCPs) coexpressed during the production of biotherapeutics can affect the safety, efficacy, and stability of the final product. As such, monitoring HCP populations and amounts throughout the production and purification process is an essential part of the overall quality control framework. Mass spectrometry (MS) is used as an orthogonal method to enzyme-linked immunosorbent assays (ELISA) for the simultaneous identification and quantification of HCPs, particularly for the analysis of downstream processes. In this study, we present an MS-based analytical protocol with improvements in both speed and identification performance that can be implemented for routine analysis to support upstream process development. The protocol adopts a streamlined sample preparation strategy, combined with a high-throughput MS analysis pipeline. The developed method identifies and quantifies over 1000 HCPs, including 20 proteins listed as high risk in the literature, in a clarified cell culture sample with repeatability and precision shown for digest replicates. In addition, we explore the effects of varying standard spike-ins and changes to the data processing pipeline on absolute quantification estimates of the HCPs, which highlight the importance of standardization for wider use in the industry. Data are available via ProteomeXchange with the identifier PXD053035.

Psoriasis, an immune-mediated chronic inflammatory skin disease, is primarily diagnosed through clinical assessment. Currently, specific markers for the accurate diagnosis and prediction of psoriatic disease are lacking. Here, we employed a three-step designed study to perform untargeted metabolomics, with the aim of identifying candidate biomarkers for psoriasis. Through comprehensive multivariate and univariate statistical analyses, we screened eight potential biomarkers specific to psoriasis, with five structurally identified. Two dipeptide biomarkers, γ-GluSer and ThrGly, along with a lysine glycation metabolite, N α-fructosyl-lysine (Fruc-Lys), were found to be psoriasis biomarkers for the first time. Receiver operating characteristic curve analysis revealed that the area under the curve (AUC) values of these eight metabolites/features ranged from 0.68 to 0.94. A biomarker panel comprising ThrGly and feature m / z 120.0656 (C 4 H 9 NO 3 ) demonstrated high diagnostic accuracy (AUC = 0.97) in distinguishing psoriasis patients from healthy controls. Overall, our study identified and validated a panel of plasma metabolites/features that could serve as potential biomarkers for the diagnosis of psoriasis, providing new insights into the diagnosis and pathogenesis of this disease.

Steroid hormones are essential for the regulation of various vital body functions. Therefore, the determination of steroid hormones in biological fluids is a significant issue to find out the mechanism of steroid hormones related to disorders. In this study, Fe 3 O 4 /reduced graphene oxide (Fe 3 O 4 /rGO) nanocomposite based dispersive solid phase extraction (DSPE) was coupled with liquid chromatography - quadruple isotope dilution - triple quadrupole mass spectrometry (LC-ID 4 -MS/MS) for the accurate and precise determination of aldosterone, testosterone, 17-hydroxyprogesterone and estrone in serum samples. There is no study in literature about the combination of DSPE and LC-ID 4 -MS/MS methods for the extraction and determination of selected steroid hormones. Significant improvements in accuracy and precision for the proposed method was achieved by the combination of DSPE and LC-ID 4 -MS/MS. The percent recovery results were calculated between 100.0 and 103.4% with low percent relative standard deviation values (≤2.0%). The obtained recovery results proved the application of the developed method with high accuracy and precision for the determination of steroid hormones in serum samples. Hence, the developed method is superior to the current analytical methods for the determination of selected steroid hormones in terms of sensitivity, accuracy and precision.

Data-independent acquisition has become a widely used strategy for peptide and protein quantification in liquid chromatography-tandem mass spectrometry-based proteomics studies. The integration of ion mobility separation into data-independent acquisition analysis, such as the diaPASEF technology available on Bruker's timsTOF platform, further improves the quantification accuracy and protein depth achievable using data-independent acquisition. We introduce diaTracer, a spectrum-centric computational tool optimized for diaPASEF data. diaTracer performs three-dimensional (mass to charge ratio, retention time, ion mobility) peak tracing and feature detection to generate precursor-resolved "pseudo-tandem mass spectra", facilitating direct ("spectral-library free") peptide identification and quantification from diaPASEF data. diaTracer is available as a stand-alone tool and is fully integrated into the widely used FragPipe computational platform. We demonstrate the performance of diaTracer and FragPipe using diaPASEF data from triple-negative breast cancer, cerebrospinal fluid, and plasma samples, data from phosphoproteomics and human leukocyte antigens immunopeptidomics experiments, and low-input data from a spatial proteomics study. We also show that diaTracer enables unrestricted identification of post-translational modifications from diaPASEF data using open/mass-offset searches., Competing Interests: Competing interests: A.I.N. and F.Y. receive royalties from the University of Michigan for the sale of MSFragger, IonQuant, and diaTracer software licenses to commercial entities. K.L. receives royalties from the University of Michigan for the sale of diaTracer software licenses to commercial entities. All license transactions are managed by the University of Michigan Innovation Partnerships office, and all proceeds are subject to university technology transfer policy. Other authors declare no other competing interests., (© 2024. The Author(s).)

The development of targeted therapies that correct the effect of mutations in patients with cystic fibrosis (CF) and the relevant heterogeneity of the clinical expression of the disease require biomarkers correlated to the severity of the disease useful for monitoring the therapeutic effects. We applied a targeted metabolomic approach by LC-MS/MS on saliva samples from 70 adult CF patients and 63 age/sex-matched controls to investigate alterations in metabolic pathways related to pancreatic insufficiency (PI), Pseudomonas aeruginosa (PA) colonization, CF liver disease (CFLD), and CF related diabetes (CFRD). Sixty salivary metabolites were differentially expressed, with 11 being less abundant and 49 more abundant in CF patients. Among these, the most relevant alterations involved salivary ADMA, N-acetylornithine, methionine and methionine sulfoxide levels. Furthermore, methionine was significantly lower in CF patients with PI and salivary histamine levels were significantly lower in patients colonized by PA. Moreover, ADMA as well as N-acetylornithine and methionine were significantly lower in CF patients with CFRD than in patients without CFRD. Finally, the levels of DOPA resulted significantly lower in saliva from patients with liver disease. Our study revealed an imbalance in arginine methylation and tryptophan pathway related to CFRD and PI as well as alterations in dopaminergic pathway and Krebs cycle related to CFLD. This study also highlights different salivary metabolites as new potential biomarkers in a non-invasive sample that could represent a useful tool for the stratification and management of CF patients., Competing Interests: Declarations. Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: The study was conducted in accordance with the Declaration of Helsinki and approved by CF regional Centre of Campania (Ethics Committee number 77/2021). The participants provided their written informed consent to participate in this study., (© 2024. The Author(s).)

Tibetan donkeys inhabit the harsh environment of the Qinghai-Tibet Plateau. Research on serum metabolites related to their high-altitude adaptation is limited compared to other livestock. We used liquid chromatography-mass spectrometry (LC-MS) to analyze serum samples from healthy adult donkeys in Shigatse, Changdu, and Dezhou to evaluate the effects of high altitudes on serum metabolites. Metabolomics analysis identified 443 differential metabolites (DMs) across the three groups, meeting criteria of VIP ≥ 1, p-value < 0.05, and Fold-Change ≥ 1.2 or ≤ 0.5. Significant upregulation was observed in deoxycholic acid, vitamin A, vitamin C, flavin mononucleotide, n-acetylserotonin, N'-formyl-kynurenine, calcidiol, and adenosine monophosphate in the high-altitude group compared to the low-altitude control group. The DMs were involved in processes such as bile secretion, vitamin digestion and absorption, tryptophan metabolism, and parathyroid hormone synthesis, secretion, and action. All these are crucial for nutrient metabolism, immune function, and antioxidant stress response. This study constructed a metabolomics dataset of Tibetan donkey serum and revealed differential metabolites among donkeys from different geographic regions and environments. The results offer crucial insights into the adaptive regulatory mechanisms., Competing Interests: Competing interests: The authors declare no competing interests. Ethics approval and consent to participate: This study was approved by the Animal Care and Use Committee of Qingdao Agricultural University (Qingdao, Shandong Province, China). This study was reported in accordance with ARRIVE guidelines. Consent for publication: Not Applicable., (© 2024. The Author(s).)