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Although proteins in snake venoms have been extensively studied and characterized, low-mass molecules remain relatively unexplored, mainly due to their low abundance, secondary role in envenomation, and some analytical technique limitations. However, these small molecules can provide new important data related to venom toxins' molecular structure, functions, and evolutionary relationships. This research aimed to characterize molecules below 10 kDa in the venoms of snakes from the Viperidae families (Bothrops, Agkistrodon, and Bitis) and compare two chromatographic approaches: reverse-phase chromatography (RP), a classic technique, and hydrophilic interaction liquid chromatography (HILIC), an alternative technique, both coupled with high-resolution mass spectrometry (HRMS). The results showed that the separation of the HILIC column provided a more efficient evenly distributed ion profile than RP, contributing to a 25.6% increase in the sequences identified. Homologous sequences for Bradykinin-potentiating peptides (BPPs) and fragments of major venom proteins, possibly cryptids, were found. In addition, BPP 13a, peptides rich in histidine and glycine (pHpG), and spacer sequences were identified in all snakes analyzed, especially with HILIC separation, suggesting that these sequences may be conserved within Viperidae. These findings indicate that the use of the HILIC column, compared to RP, is a promising approach for characterizing peptides in snake venom obtained by the ultrafiltration process. It contributes to the study of these still poorly understood molecules and is also a good option for studying other complex protein/peptide mixtures., 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 © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

The exact moment method for the determination of the dispersion tensor in retentive porous media has been adopted to compute the dispersion coefficients, the plate height curves and the kinetic performance factors of eight different 3D printable stationary phases based on triply periodic minimal surfaces (TPMS). The two cases in which the stationary phase is impermeable (hydrodynamic dispersion) or superficially retentive have been analyzed in detail. The Carman-Kozeny relationship between permeability K v , hydraulic diameter d h and hydrodynamic tortuosity τ holds true for all the geometries investigated with a unique shape coefficient K 0 . The analysis of plate height curves indicates that best performing geometries are associated with lower values of the effective diameter d eff , and thus lower values of permeability. When compared in terms of kinetic performance factor, the best performing geometries are those characterized by lower tortuosity and higher coefficient of uniformity δ of the axial velocity field. Among all the geometries investigated, sheet-based Gyroid and Primitive are the best performing, both in terms of maximum kinetic performance factor e c,max ∈(1,1.4) and in terms of column void time t 0 ∈(0.4s,1.6s) for ΔP=500 bar., 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 © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Rheology modifiers (RMs) are polymeric molecules providing rheological control of formulations, which are important in product application, shelf-life, and aesthetic perception. Bio-derived polyethylene glycol (PEG)-based RMs thicken formulations through nonionic-associative thickening where at least two hydrophobic end groups from a RM molecule interact with other hydrophobic groups of other RM molecules or ingredients in the formulation to form an associative network. We report a comprehensive two-dimensional liquid chromatography (2D-LC) separation of partly bio-derived PEG-based RMs in size exclusion chromatography (SEC) × reversed-phase liquid chromatography (RPLC) mode for the separation of RM components based on both molecular weight distribution and end group hydrophobe distribution. Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is first performed to gain understanding of the molecular weight, number of distributions, and approximate sample complexity, which helps with the SEC column selection. A thorough solubility screening is then conducted to identify the compatible SEC mobile phases and the gradient range for the RPLC column. Then, both SEC and RPLC methods are developed for the successful comprehensive SEC × RPLC separation of polymers with 0, 1, 2, 3, and 4 terminal hydrophobes. Finally, high resolution mass spectrometry mass profiler is used to further explain the minor secondary interaction mechanism of the SEC separation and confirm the MALDI results., 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 © 2025 Elsevier B.V. All rights reserved.)

The analysis of mineral oil aromatic hydrocarbons (MOAH) in vegetable oils is currently associated with high uncertainty due to various factors ranging from sample preparation to data interpretation. One significant factor is the coelution of biogenic compounds of terpenic origin with the MOAH fraction during chromatographic analysis. The common purification method is epoxidation, a chemical reaction that changes the polarity of the interferences, allowing their separation from MOAH. However, this reaction is non-selective and can lead to losses of both MOAH and internal standards. This variation is especially noticeable when using epoxidation procedures with higher reaction kinetics, such as those employing performic acid. MOAH losses also vary depending on their composition, which is unpredictable. Furthermore, 2-methylnaphthalene (2MN) and 1,3,5-tri‑tert-butylbenzene (TBB), which are the common quantification standards, are lost at different rates. As a consequence, the final MOAH quantity will vary both depending on the standard used, and on its initial composition. This work presents a new purification approach based on liquid chromatography fractionation on silica using the same column and eluents as for the usual MOSH/MOAH fractionation. This approach efficiently removes squalene, carotenes, and derivatives, without inducing inconsistent losses between the internal standards and MOAH as epoxidation does. On average, MOAH recovery using this new method was 94% (± 8%) in coconut, palm, sunflower, and olive oil, using different MOAH sources and concentrations. Additionally, perspectives are presented regarding the separate analysis and quantification of mono-/diaromatic MOAH and other polyaromatic MOAH. This is of particular interest as these two sub-fractions are associated with different toxicological 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 © 2025 Elsevier B.V. All rights reserved.)

Two-dimensional liquid chromatography (2D-LC) separation systems, based on two independent columns with different separation mechanisms, have exhibited strong resolving power for complex samples. Therefore, in recent years, the exceptional resolution of 2D-LC has significantly advanced the chemical separation of natural products, such as complex herbs, greatly facilitating their qualitative and quantitative analysis. This paper aims to review the latest strategies of 2D-LC in the quantitative analysis of complex chemical compositions in natural products. To this end, the major advantages and disadvantages of various column couplings in 2D-LC are discussed based on specific studies, along with suggested solutions to address the identified drawbacks. Moreover, the applications of different detectors combined with the latest chemometrics in 2D-LC for accurate quantitative analysis of natural products are reviewed and discussed., 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 © 2025 Elsevier B.V. All rights reserved.)

24-h urinary free cortisol (UFC) measurements are fundamental in the diagnosis and follow-up of Cushinǵs syndrome (CS) and immunoassays (IA) are the most widely used tests for its quantification in clinical laboratory practice. However, their suitability has been questioned mainly due to their limitations concerning analytical specificity. The aim of this research project was to evaluate a novel algorithm for CS diagnosis and follow-up in the clinical laboratory, based on the combination of IA tests with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for UFC quantification. A quantitative LC-MS/MS approach based on liquid-liquid extraction for sample preparation was developed and fully characterized. A population-based reference range was established and the level of agreement for UFC values when compared to IA approach was assessed in patients under CS follow-up or clinical suspicion for hypercortisolism. Significantly higher UFC values were observed for IA when compared to LC-MS/MS approach, therefore population-based reference range was established for the latter (i.e. 4 - 41 µg/day). The clinical application of the herein presented LC-MS/MS approach to be used as a confirmation procedure for CS management was assessed and a high level of agreement with IA UFC values, except in the case of IA undetectable results, was observed. However, IA potential false negative (FN) and false positive (FP) findings were also noted. Higher clinical sensitivity for CS diagnosis / follow-up was observed for LC-MS/MS when compared to IA, supporting the advantage and necessity of implementing LC-MS/MS as a confirmation procedure in the clinical laboratory., 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 © 2025 Elsevier B.V. All rights reserved.)

Background: Zhenzhu Tongluo pills (ZZTL) have been utilized for the treatment of neurobrucellosis (NB) in the Inner Mongolia region of China. However, the specific mechanism underlying the neuroprotective effects of ZZTL in NB remains insufficiently explored. Therefore, this study aimed to analyze the metabolite profiles across different groups to identify potential biomarkers for the diagnosis and treatment of NB., Methods: LC-MS analysis was used to screen differential metabolites in cerebrospinal fluid (CSF) samples from control subjects, NB patients, and NB patients undergoing ZZTL therapy., Results: We identified 225 common metabolites across two comparison groups (NB vs. control group and NB + ZZTL vs. NB group). Among these metabolites, 138 were downregulated, and 87 were upregulated in the NB group compared to the control group, while the levels of these metabolites were reversed by ZZTL therapy. Creatinine exhibited the highest VIP score in both comparison groups, with elevated levels observed in CSF samples from NB patients compared to controls; however, its levels were reduced following ZZTL therapy. Additionally, hypoxanthine and uric acids levels were also increased in CSF samples from NB patients, indicating dysregulation of purine metabolism in NB; however, these changes were reversed by ZZTL. Furthermore, pantothenic acid (vitamin B5) and niacinamide (vitamin B3) levels were decreased in CSF samples from NB patients, suggesting a potential link between NB and vitamin B3 and B5 deficiency; however, these changes were reversed by ZZTL., Conclusion: Collectively, CSF metabolomics may effectively differentiate NB patients from control subjects, providing valuable insights for NB diagnosis and treatment., (© 2024 John Wiley & Sons Ltd.)

Processing techniques are critical factors influencing the quality of hawk tea, yet systematic studies on their effects are limited. This study investigates the impact of four key processing procedures-fixation, reddening, fermentation, and compressing-using sensory evaluation, LC-MS/MS, and GC × GC-TOF-MS. Analysis identified 6951 non-volatile metabolites, including 107 marker metabolites, primarily in flavonoid synthesis and degradation pathways. Fermentation increased sweetness and richness by enhancing sugars and amino acid content, while significantly reducing flavonoid levels. Reddening improved flavor, color, and retained more beneficial flavonoids. Volatile analysis detected over 1800 compounds, including 398 volatile marker metabolites. Fermentation increased alcohols and heterocyclic compounds, reducing hydrocarbons and ketones. Additionally, 26 camphoraceous and 12 pungent aroma compounds characteristic of hawk tea were identified, and an aroma-flavor correlation map was established. By elucidating the impact of processing methods on its chemical and sensory properties, this study lays the groundwork for targeted quality control of hawk tea., 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 Ltd.)

An online solid phase extraction-ultra performance liquid chromatography-tandem mass spectrometry detection method was developed for the simultaneous determination of sacubitril and seven sartan drugs in blood serum in this study. The compounds were separated through a C 18 column. Mass spectrometry of the samples was performed using a jet stream electrospray ion source (AJS, ESI+). The samples were detected via a multiple reaction monitoring (MRM) mode and quantified via a stable isotope internal standard method. 900 μL of prepared sample was injected and a run time of 12.5 minutes was obtained in this proposed method. The eight examined target compounds showed good linearity (r 2 ＞0.994) in the corresponding mass concentration range and a lower limit of quantitation (LLOQ) was in the range of 0.05-0.1 μg/L. The recovery of the spiked serum samples ranged from 90.90 % to 106.20 % with relative standard deviations (RSDs) of 4.57 %-9.27 %. Five target compounds were detected in the actual serum samples using this method. The proposed method is simple to use, sensitive, accurate, and suitable for the trace detection of sacubitril and seven sartan drugs present in serum samples. The method can meet the needs for clinical monitoring of blood concentrations of this type of drug, while providing detection technology support for the development of compound preparations of sacubitril and other sartan drugs., 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 prevalence of major depressive disorder (MDD) is higher in females than males, emphasizing the need to identify gender-specific biomarkers to improve diagnosis accuracy. In this study, a cross-sectional investigation with 258 samples was conducted to evaluate the discriminative power of potential gender-specific biomarkers for MDD. Eighteen MDD-related differential metabolites have been identified, involving pathways of phospholipids, glycerolipids, fatty acids, sphingolipids, cholesterol, vitamin E, and heme. A potential biomarker combination consisting of palmitelaidic acid, gamma carboxyethyl hydroxychroman (gamma-CEHC), and lysoPE(16:0) was confirmed for predicting depression in women using binary logistic regression analysis. To evaluate the panel's specificity, nine generalized anxiety disorder (GAD) samples, which share highly similar clinical symptoms with MDD, were included in the validation set. The discovery and validation sets yielded an area under the receiver operating characteristic curve of 0.86 and 0.83, respectively. All nine female GAD samples were correctly predicted as non-MDD, demonstrating the panel's specificity in diagnosing female MDD. Remarkably, this composite panel achieved a 75 % prediction accuracy in female samples in both the discovery and validation sets, but it did not reach 60 % prediction accuracy in male samples in either set. Our findings highlight the importance of gender-specific molecular diagnostics in developing practical and accurate diagnostic methods for MDD., Competing Interests: Declaration of Competing Interest The authors declare no 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.)

A flat membrane-based liquid-phase microextraction (FM-LPME) method was developed for the first time to simultaneously separate and extract basic and acidic pesticide analytes from the same sample. Using carbendazim and pirimicarb as representative basic pesticides, along with bromacil, diflubenzuron and 1,2-dibenzoyl-1‑tert-butylhydrazine (RH-5849) as representative acidic pesticides, the performance of FM-LPME for the extraction of these model pesticides has been optimised individually. Under optimal extraction conditions, the developed FM-LPME-LC-MS/MS method exhibited excellent linearity, with R 2 values exceeding 0.9903 in concentration ranges of 0.1-1400 ng mL -1 for carbendazim and pirimicarb, and 1-1400 ng mL -1 for bromacil, diflubenzuron and RH-5849. The limits of detection ranged from 0.01 to 0.09 ng mL -1 . The recoveries and relative standard deviations of all the tested pesticides at three spiked concentrations (100, 500 and 1200 ng mL -1 ) were in the ranges of 80.0 %-125.0 % and 0.3 %-9.3 %, respectively. The FM-LPME-LC-MS/MS method demonstrated advantages in terms of being environmentally friendly, possessing a wide linear range, high sensitivity, high precision and satisfactory recoveries, thus highlighting its notable potential for the separation and extraction of basic and acidic pesticide residues in water 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 © 2025 Elsevier B.V. All rights reserved.)

Optimization algorithms play an important role in method development workflows for gradient elution liquid chromatography. Their effectiveness has not been evaluated for chromatographic method development using standardized comparisons across factors such as sample complexity, chromatographic response functions (CRFs), gradient complexity, and application type. This study compares six optimization algorithms - Bayesian optimization (BO), differential evolution (DE), a genetic algorithm (GA), covariance-matrix adaptation evolution strategy (CMA-ES), random search, and grid search - for the development of gradient elution LC methods. Utilizing a multi-linear retention modeling framework, these algorithms were assessed across diverse samples, CRFs, and gradient segments, considering two observation modes: dry (in silico, deconvoluted), and wet (search-based, requiring peak detection). The optimization algorithms were evaluated based on their data (i.e. number of iterations) and time efficiency. Of the algorithms compared in this study, DE proved to be a highly competitive method for dry optimization purposes in terms of both data and time efficiency. BO outperformed all other algorithms in terms of data efficiency and was found to be most effective for search-based optimization, which requires a low number of iterations (<200). However, BO was found to be impractical for dry optimization requiring a large iteration budget due to its unfavorable computational scaling. It was observed that both the CRF and the sample have a strong influence on the efficiency of the algorithms, emphasizing the need for better benchmark samples and highlighting the importance of assessing CRF-induced complexity in the optimization landscape., 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 comprehensive identification of peaks in untargeted lipidomics using LC-MS/MS remains a significant challenge. Confidence in lipid annotation can be greatly improved by integrating a highly accurate machine learning-based retention time prediction model. Such an approach enables the identification of lipids for understanding pathogenic mechanisms, biomarker discovery, and drug screening. In this study, we developed a machine learning model to predict retention times and facilitate lipid peak annotations in LC-MS-based untargeted lipidomics. Our model achieved high correlation coefficients of 0.998 and 0.990, with mean absolute errors (MAE) of 0.107 min and 0.240 min for the training and test sets, respectively. External validation showed similarly strong performance, with correlations of 0.991 and 0.978, and MAE values of 0.241 min and 0.270 min. We also compared the impact of molecular descriptors and molecular fingerprints on the model's performance, finding that molecular descriptors outperformed molecular fingerprints across all datasets when using Random Forest (RF) for model construction. Notably, this retention time calibration model demonstrates robust performance across chromatographic systems with comparable gradients and flow rates. Overall, this machine learning model enhances lipid annotation accuracy and reduces errors in untargeted lipidomics, improving data analysis across multiple datasets., Competing Interests: Declaration of competing interest The authors confirm that they have no financial conflicts of interest or personal relationships that could have influenced the work presented in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Non-destructive chromatographic methods were used to determine the hold-up volumes of four self-packed columns containing embedded phosphate groups. The stationary phases are named Diol-P-C10, Diol-P-C18, Diol-P-Benzyl and Diol-P-Chol. The hydrophobicity of organic ligands bound to the phosphate group increases in the benzyl< decyl < octadecyl

Polysorbates (PS), as non-ionic surfactants, contribute significantly to the stability of proteins in formulations. However, their lack of chromophore groups makes them difficult to detect with high sensitivity and simplicity. The charged aerosol detector (CAD) is an emerging and universal detector that can provide highly sensitive response signals to non-volatile or semi-volatile substances, such as esters, acids, oxidized aldehydes, and contaminant ions in PS. This article provides a comprehensive review of the qualitative and quantitative analysis of PS, profiling its composition, investigating the reasons for its degradation, and discussing its reaction mechanisms. This review aims to promote the quality control of PS production for the development of stable and safe protein formulations., 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.)

Given the complexities of continuous bioprocessing, it is critical to thoroughly investigate the process parameters unique to multi-column chromatography (MCC) and their potential impacts. However, existing studies have focused on either loading densities or residence time at steady states only, and their combined impact on critical quality attributes (CQAs) especially during transient phases were less known. In this study, we investigated the impact of critical process parameters during both steady-state and transient phases (start-up, close-down, and intermediate perturbation) through full factorial design. Our findings revealed that MCC operation with static control results in a maximum delta UV variation (breakthrough percentage from 1 st loading column) of 30 % between the start-up phase and steady state. Additionally, eluate concentration showed a slight increase (with a maximum difference of 1.9 mg/mL, equivalent to 25 % deviation) when transitioning from the start-up phase to steady state but significantly decreased (with a maximum difference of 8.2 mg/mL, equivalent to 93 % deviation) when shifting from steady state to the close-down phase. The monomer purity during start-up and steady-state phases remained above 97 % while the close-down phase eluate purity ranged from 57 % to 98 %, depending on the operating conditions. Nevertheless, the variation in product quality can be potentially mitigated by product diversion or pooling with high-purity eluates from steady-state, highlighting the robustness of MCC operation in maintaining consistent product quality. Therefore, our study underscores the critical need to comprehensively understand the process dynamics of MCC operation at both steady state and transient phases., 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.)

Determination of quality attributes of antisense oligonucleotides (ASOs) such as purity, potency, and sequence is challenging due to their relatively large size, polyanionic nature, and large number of synthetic modifications. Chromatography technologies are evolving rapidly to meet these challenges, and one area of particularly rapid change at this time is the use of hydrophilic interaction liquid chromatography (HILIC) for oligonucleotide (ON) separations. Relatively little has been published on the factors that dictate the kinetics of these separations. This knowledge gap consequently makes it difficult to know what gains might be made during method development by changing flow rate or particle size, for example. In this work we have taken initial steps to address this gap by examining the dependence of plate height and resolution on flow rate for separations of 23-mer ASOs under HILIC conditions. Such work is complicated by the fact that the retention of these molecules decreases dramatically with increasing pressure. After adjusting mobile phase composition to hold retention factor nominally constant for each flow rate used, we find that plate height increases strongly with increasing flow rate such that the plate height increases about ten-fold over the range of flow rate of 0.1 to 4.0 mL/min. when using a 4.6 mm i.d. column. However, the minimum reduced plate height observed at the lowest flow rate is quite impressive at around 2. Finally, we find that this dependence of plate height on flow rate translates, as expected, to an improvement in resolution as flow rate is decreased, both in conventional one-dimensional separations, and in the second dimension of a two-dimensional separation. We expect to use this work as a foundation to build on as we deepen our understanding of the kinetics of ON separations., 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.)

Neuronanomedicine harnesses nanoparticle technology for the treatment of neurological disorders. An unavoidable consequence of nanoparticle delivery to biological systems is the formation of a protein corona on the nanoparticle surface. Despite the well-established influence of the protein corona on nanoparticle behavior and fate, as well as FDA approval of neuro-targeted nanotherapeutics, the effect of a physiologically relevant protein corona on nanoparticle-brain cell interactions is insufficiently explored. Indeed, less than 1% of protein corona studies have investigated protein coronas formed in cerebrospinal fluid (CSF), the fluid surrounding the brain. Herein, we utilize two clinically relevant polymeric nanoparticles (PLGA and PLGA-PEG) to evaluate the formation of serum and CSF protein coronas. LC-MS analysis revealed distinct protein compositions, with selective enrichment/depletion profiles. Enhanced association of CSF precoated particles with brain cells demonstrates the importance of selecting physiologically relevant biological fluids to more accurately study protein corona formation and subsequent nanoparticle-cell interactions, paving the way for improved nanoparticle engineering for in vivo applications.

Background: The increasing prevalence of xylazine in the illicit drug supply is a growing concern for major health consequences in individuals who use fentanyl mixed with xylazine, but limited data are available regarding the pharmacokinetics of xylazine in humans., Methods: Xylazine was quantified in serial remnant plasmas collected from 28 patients starting at the initial patient encounter and continuing for up to 52 h from presentation, using LC-MS/MS to calculate the terminal half-life for xylazine. Xylazine metabolites were identified by product ion scanning, and multiple reaction monitoring was used to estimate the relative abundance of xylazine metabolites in 74 collected plasma samples., Results: The median terminal half-life for xylazine was calculated to be 12.0 h (range: 5.9-20.8). Oxo-xylazine and sulfone-xylazine metabolites were detected in all plasma specimens that contained xylazine., Conclusions: The half-life of xylazine in humans is longer than previously observed in animal studies, which furthers the current understanding of the expected duration of effects in individuals who use fentanyl mixed with xylazine and the window of detection. Both oxo-xylazine and sulfone-xylazine appear to circulate in plasma for as long as xylazine., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Serum total 25-hydroxyvitamin D [25(OH)D] concentration is the most widely used clinical biomarker for vitamin D status. Under certain physiological and pathological conditions, however, total 25(OH)D may not always be the best index for vitamin D status. Instead, the nonprotein-bound (free) fraction of total 25(OH)D has been suggested as a more appropriate marker in certain clinical situations., Content: Free 25(OH)D levels can either be calculated or measured directly. Calculated free 25(OH)D depends on the concentrations of total serum 25(OH)D, vitamin D binding protein (VDBP), and albumin, as well as the affinity between analyte and binding proteins. Differences in VDBP concentrations are observed between populations as a result of health status, gene polymorphisms, and the assay used for determination. Direct measurement methods for free 25(OH)D are often complicated (dialysis, ultrafiltration) or susceptible to interferences, cross-reactivity, and type of antibody (immunoassays). Therefore, it is very important to develop tools that allow either accurate and precise measurement of VDBP or direct measurement of free 25(OH)D. For the latter, liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) has recently shown promise for analysis of free vitamin D. In the current review, we present the importance and challenges regarding free 25(OH)D determination and the role of LC-MS-based methods in future studies., Summary: More research is required to determine the role of free 25(OH)D in the assessment of vitamin D status in healthy subjects and in various clinical conditions. Recent advances in technology, including mass spectrometry, can provide the required assays for this purpose., (© Association for Diagnostics & Laboratory Medicine 2024. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Background: Carbohydrates exhibit diverse functions and extensive biological activities and are notable in the field of life sciences. However, their inherent diversity and complexity-steaming from variations in isomeric monomers, glycosidic bonds, configurations, etc.-present considerable challenges in structural analysis. Considering these challenges, the disaccharide building blocks with simpler structures could provide more structural information. Although various approaches have been explored, sufficient standards or specialized equipment are required to differentiate and characterize isomers. Therefore, a strategy that addresses these challenges is urgently needed., Results: A Composition-dependent MRM Transitions and Structure-indicative Elution Segments (CMTSES)-based liquid chromatography-triple quadrupole mass spectrometry (LC-QQQ-MS) strategy was developed to comprehensively profile disaccharide units and differentiate isomers. First, the composition-related precursor and structure-specific product ions of disaccharides were generated by QQQ-MS. Thereout, MRM transitions were proposed to enable the comprehensive profiling of disaccharides and rapid annotation of their compositions and saccharide types at both termini. Next, the linkage, composition, and configuration isomers of disaccharides were effectively differentiated and presented characteristic LC elution. Furthermore, low-cost and available "location references" (mannose, galactose, and isomaltose) were sought to define structure-indicative elution segments for the identification of isomeric hexose disaccharides. Building on this foundation, the novel CMTSES-based LC-MS strategy was designed, and its feasibility was further verified by successfully differentiating and identifying mixed homogenous and/or heterogenous disaccharide isomers in real samples. Sufficient structural information was obtained even for those consisting of diversified monomer types., Significance and Novelty: This strategy comprehensively profiles both major and minor disaccharides and effectively differentiates multiple types of isomers. The use of readily available "location references" facilitated the identification of isomeric hexose disaccharide with reduced dependence on standards, thereby broadening the applicability of this strategy. However, the characterization of disaccharides with other compositions is challenging. Further in-depth investigations into intramolecular hydrogen bond simulation should provide solutions. Additionally, CMTSES-based LC-MS strategy is promising to analyze complex structures and 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 Elsevier B.V. All rights reserved.)

Panax notoginseng (P. notoginseng) is one of the most famous natural medicines and widely used to promote blood circulation in health care. However, the active component group of P. notoginseng for activating blood is not clear. We aim to screen and validate the pharmacodynamic component group (PCG), which could exert the same blood-activating effect as P. notoginseng. To clarify the active components, the chemical components were determined by liquid chromatography-tandem mass spectrometry, and the fingerprint of P. notoginseng was established. Twenty candidate active monomers were selected through the spectrum-effect relationship analysis. Eleven active monomers, including Ginsenoside Rg1, Rb1, Rd, F1, Rh1, Rg2, Rb2, Rg3, and Rk1 and Notoginsenoside R1 and R2, were screened out as the PCG through validation by platelet aggregation test. Among them, the antiplatelet aggregation activity of Ginsenoside Rh1 was directly confirmed for the first time. The active component group could exert similar efficacy to the P. notoginseng extract in vitro and in vivo through the validation of in vitro platelet aggregation test and the rats with cerebral ischemia. This study laid the foundation for the quality evaluation of P. notoginseng and provided a reference for the research on the material basis of the pharmacodynamics of other Chinese herbs., (© 2025 John Wiley & Sons Ltd.)

The antioxidant, total phenolic, flavonoid, and anthocyanidin properties of extracts prepared from Cotoneaster frigidus Wall. ex Lindl. "Cornubia" fruit were examined. In addition, the phenolic profile was analyzed using LC-HRMS, and the surface morphological features were studied using ATR FT-IR. Additionally, the inhibitory potential of the extracts on tyrosinase and α-glucosidase/α-amylase was tested. The highest antioxidant activity values were found in ethyl acetate and methanol extracts. The methanol extract was found to be rich in TFC, TPC and TPA. In the research conducted to designate the phenolic profile, it was found that the extracts contained high levels of fumaric acid and chlorogenic acid. In studies conducted for enzyme inhibition, it was observed that methanol extract gave better results than the standard inhibitor used in studies conducted for tyrosinase. For α-glucosidase/α-amylase, it was determined that ethyl acetate extract gave results close to standard inhibitors. Considering the information obtained, it has been evaluated that the fruits of this plant, which are mostly grown for ornamental purposes plants, may have different application areas., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yakup KOLCUOGLU reports financial support was provided by The Scientific and Technological Research Council of Türkiye (TUBITAK). Yakup KOLCUOGLU reports financial support was provided by Karadeniz Technical University. Yakup KOLCUOGLU reports a relationship with The Scientific and Technological Research Council of Türkiye (TUBITAK) that includes: funding grants. Yakup KOLCUOGLU reports a relationship with Karadeniz Technical University that includes: funding grants. 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 Elsevier Ltd. All rights reserved.)

Background: Chemical derivatization is a common technique in liquid chromatography-mass spectrometry (LC-MS) metabolomics used to improve the ionizability and chromatographic properties of metabolites in complex biological samples. This process facilitates better detection and separation of a wide array of compounds. The reagent 2-(4-boronobenzyl) isoquinolin-2-ium bromide (BBII), developed as a glucose labeling reagent for matrix-assisted laser desorption/ionization MS, enhances ionization for glucose and other hydroxyl metabolites. Its quaternary ammonium group increases ionization efficiency, and its rapid reaction time simplifies pretreatment procedures., Results: We developed a novel post-column derivatization (PCD) method using BBII to boost the detection sensitivity of hydroxyl metabolites in LC-MS. By optimizing this BBII PCD approach with 14 hydroxyl-containing compounds, we were able to detect previously undetectable metabolites such as glucose, ribose, and long-chain alcohols. Sensitivity enhancements for these metabolites ranged from 1.1 to 42.9-fold. Applying this method to metabolic profiling of hydroxyl metabolites in the DBTRG-05MG glioblastoma cell line, with and without treatment with the new drug MFB [1-(4-chlorobenzyl)-2-(5-methyl-2-furfurylideneamino)benzimidazole], revealed several hydroxyl metabolites with significantly reduced levels post-treatment., Significance and Novelty: This study presents a new BBII PCD method that substantially improves the detection sensitivity of hydroxyl metabolites in LC-MS. This innovative approach is highly valuable for untargeted metabolomics studies in biological and clinical research, offering a robust tool for identifying metabolite changes and advancing our understanding of metabolic processes in disease and therapeutic contexts., 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: Tissue metabolomics analysis, alongside genomics and proteomics, offers crucial insights into the regulatory mechanisms of tumorigenesis. To enhance metabolite detection sensitivity, chemical isotope labeling (CIL) techniques, such as dansylation, have been developed to improve metabolite separation and ionization in mass spectrometry (MS). However, the dissolution of hydrophobic derivatized metabolites in solvents with high acetonitrile content limits the use of liquid chromatography (LC) systems with small-volume reversed-phase (RP) columns. In this study, we established a nano-LC-MS system with an online dilution design to address this issue, enabling sensitive analysis of oral cancer tissue metabolomes., Results: Our nano-LC system features a flow path design with online sample dilution before an RP trap column and backflushing of the trap column before entering the analytical column. Compared to other nano-LC systems, both with and without online dilution designs, our system demonstrates the superiority of the T-connector-based dilution method. Using only 1/20th of the sample required for popular micro-LC systems, our nano-LC detects a larger number of peak pairs with similar recovery rates for both hydrophilic and hydrophobic metabolites, ensuring unbiased results. Thirty-two matched pairs of oral squamous cell carcinoma (OSCC) tissue samples and adjacent noncancerous tissues (ANTs) underwent high-throughput CIL-metabolome analysis using our nano-LC system. Compared to our previous micro-LC methods, the nano-LC-MS system exhibits enhanced detection sensitivity, significantly reducing sample requirements., Significance: Our findings highlight the efficacy of our platform for metabolomic analysis with limited sample amounts. The nano-LC system's ability to analyze samples dissolved in strong eluents suggests potential applications for handling other hydrophobic compounds using RPLC or other separation methods facing similar solvent incompatibility issues. This approach holds promise for identifying novel metabolite biomarkers for oral cancers, advancing our understanding of tumorigenesis, and enhancing clinical applications., 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.)

Free fentanyl is responsible for its pharmacological effects, but its total concentration is typically determined for therapeutic drug monitoring purposes. Determination of fentanyl concentration can help reduce the prescribed doses, leading to fewer side effects and increased effectiveness. Therefore, predicting free drug concentration in pharmaceutical research is crucial. The aim of this study was to determine free fentanyl in exhaled breath condensate. These samples were extracted using a dispersive micro solid phase extraction method with a new adsorbent made of graphene oxide, magnetic iron oxide nanoparticles, and covalent organic framework. 10 mg of the adsorbent was added to the sample solution adjusted to pH 10. After sonication for 5 min, the sorbent was separated using an external magnet. The adsorbed analyte was then eluted from the sorbent surface using a mixture of acetonitrile, methanol, and deionized water in a ratio of 42.5:42.5:15 (v/v/v) and analyzed using liquid chromatography-tandem mass spectrometry system. The calibration curve showed high linearity in the range of 0.17-10000 μg L -1 with a coefficient of determination of 0.9998 and good repeatability with a relative standard deviation of 4.1 %. Additionally, this method provided a low detection limit of 0.05 μg L -1 and quantification limit of 0.17 μg L -1 ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Bexicaserin is a highly selective agonist at the 5-HT 2c receptor in clinical development for the treatment of seizures associated with developmental and epileptic encephalopathies (DEEs). We report an LC-MS/MS method for the quantitative estimation of bexicaserin in human plasma and urine. The sample preparation involves the extraction of bexicaserin and internal standard ( 13 CD 2 -bexicaserin; IS) from 150 μL plasma and 50 μL urine using a solid phase extraction method. The chromatographic separation of bexicaserin and IS was achieved on a Poroshell EC-C18 column using 5 min gradient program. The calibration curve ranged from 0.1 to 100 ng/mL in plasma and 1.0 to 1000 ng/mL in urine. Intraday and interday precision and accuracy, linearity, matrix effect, extraction recovery, carry-over, dilution integrity, a battery of stability studies, and incurred sample reanalysis were performed for bexicaserin in both plasma and urine. The intraday and interday accuracy and precision were well within the acceptable limits in both plasma and urine matrices. Stability studies in plasma and urine showed that bexicaserin was stable on bench for 24 h, in autosampler over 54 h, five freeze-thaw cycles, and long-term storage at -20°C and -80°C for > 368 days. The validated methods were successfully applied in clinical study., (© 2025 John Wiley & Sons Ltd.)

Authentication of gelatin sources are required for cultural beliefs and food integrity. This paper describes a sensitive and rapid detection of gelatin sources using liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The specific peptide markers were adopted to accurately identify bovine and porcine gelatin in pharmaceutical capsules and jellies. Multiple reaction monitoring (MRM) was employed to identify and quantify over five specific peptide markers, each characterized by its unique precursor and product ion transitions. The developed method was validated at three concentration levels in gelatin-containing products to assess its accuracy and precision. The recovery (accuracy) of the proposed method was between 80 % and 107 %, and relative standard deviation (precision) was in the range of 5.16-9.97 %. Linearity was obtained ≥ 0.99 (R 2 ). To ensure the accuracy of ingredient labeling, the LC-MS/MS results were compared with those obtained from PCR assays. The LC-MS/MS method demonstrated exceptional sensitivity, reliably detecting gelatin adulteration at concentrations as low as 0.01 %. The developed LC-MS/MS method provides a rapid and accurate results for authenticating gelatin sources in various food products within 4 hours., 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 © 2025 Elsevier Ltd. All rights reserved.)

To evade legal controls, new psychoactive substances (NPS), which have been designed as substitutes for traditional and synthetic drugs, are gradually dominating the drug market. Synthetic cannabinoids (SCs), which account for the majority of NPS, are rapidly being derivatized; consequently, controlling increasing abuse by merely listing individual compounds is difficult. Therefore, China has included the entire SC category of SCs listed as legal controlled substances since July 1, 2021. However, new SCs obtained through structural modification are still appearing and pose significant analytical challenges for forensic laboratories. Therefore, an efficient, green, and automated detection method is urgently required to provide technical support for the accurate screening actual samples. Meanwhile, the number of indazole-type SCs has increased sharply since 2013, which is ascribable to their stronger psychoactive effects. Indeed, forensic laboratories mainly analyze this key SC subclass. Therefore, in this study, we developed a new method for analyzing 51 indazole-type SCs in human urine and blood, which involves online solid-phase extraction (online SPE) as the preprocessing technology, with analysis performed using liquid chromatography-linear ion trap mass spectrometry. Deproteinization was achieved by adding acetonitrile, with dilution performed using 10 mmol/L ammonium acetate solution (pH 4.8) containing 0.1% formic acid. Samples were then analyzed directly using acetonitrile-10 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid) as the mobile phase. The mass-to-charge ratios of protonated molecular ions ([M+H] + ) in the mass spectra acquired in full-scan mode, and the retention times in the chromatograms of the analytes were selected with the aim of monitoring the MS 2 ions of the various compounds. Characteristic fragment ions of the various SC structures were summarized, with five groups of isomers, each containing ten compounds, successfully distinguished using multistage mass spectrometry and their retention times. Multistage MS was used to qualitatively screen 51 indazole-type SCs, which were then quantitatively analyzed using MS 2 ion pairs (as quantitative ion pairs). The analytes exhibited limits of detection (LODs) of 0.02-1 ng/mL, with limits of quantification (LOQs) of 0.04-4 and 0.1-4 ng/mL in urine and blood, respectively. Linear fitting (weighting factor 1/ x ) revealed good linearity for each analyte within its respective linear range, with correlation coefficients ( R 2 ) greater than 0.99 in both urine and blood. The validity of the analytical method was tested by determining precision and spiked recovery values ( n =6). Recoveries of 83.47%-116.95% were obtained at LOQ levels, with precisions of 2.29%-13.40%. In addition, recoveries of 86.63%-113.38% and precisions of 0.58%-13.79% were obtained at low, medium, and high levels. The method described herein is not only easy to operate but also can be automated. Indeed, high-throughput sample analysis was achieved when sample extraction, enrichment, and analysis were performed in dynamic mode through valve switching. Meanwhile, the method exhibited good sensitivity and is applicable to a wider range of compounds than those previously reported; it also provides a scientific basis and technical support for the rapid screening and quantitative analysis of SCs in actual relevant cases.

Praziquantel (PZQ) is the most effective treatment for schistosomiasis, commonly administered as a racemic mixture of the two enantiomers. Despite many reports on the pharmacokinetics of PZQ, the stereoselective pharmacokinetics of PZQ and its major metabolite 4-hydroxypraziquantel (4-OH-PZQ) remain poorly understood in goats. In this study, the chiral LC-MS/MS method was further optimized for separating and quantifying PZQ, trans-4-OH-PZQ, and cis-4-OH-PZQ and their enantiomers and then applied for the molecular pharmacokinetics of three analytes in black goat plasma. The findings showed that PZQ was rapidly absorbed and metabolized to 4-OH-PZQ. The C max of trans-4-OH-PZQ was about 3 times and 6 times higher than those of cis-4-OH-PZQ and PZQ, respectively. The stereoselectivity of the PZQ and cis-4-OH-PZQ enantiomers was insignificant in black goat plasma (p > 0.05), whereas the trans-4-OH-PZQ enantiomers exhibited obvious stereoselectivity (p < 0.05). The C max of S-trans-4-OH-PZQ were ~3.1 times higher than that of R-trans-4-OH-PZQ. Further computer simulations indicated that these differences in the stereoselectivity might mainly stem from the different binding energies of the corresponding R- and S-enantiomers of the target analytes to black goat plasma albumin. It has guiding significance for the research on the stereoselectivity of chiral veterinary drugs and their precision medication., (© 2025 John Wiley & Sons Ltd.)

Dried blood spot (DBS) assays to quantify novel and repurposed drugs for the treatment of rifampicin-resistant tuberculosis (RR-TB) would facilitate pharmacokinetic studies and therapeutic drug monitoring in low-middle income settings, considering their ease of application and simple sample storage requirements. We describe a DBS method for the simultaneous quantification of bedaquiline and metabolite N-desmethyl bedaquiline, linezolid, levofloxacin, and clofazimine. The analytes were extracted from the matrix and isolated by solid-phase extraction. Two LC-MS/MS systems were used, optimized for the separate analysis of the more polar compounds (linezolid and levofloxacin), and less polar compounds (bedaquiline, N-desmethyl bedaquiline, and clofazimine), employing gradient elution. Electrospray ionization and multiple reaction monitoring were used to quantify the analytes on a Sciex API3200 and an API5500 triple quadrupole mass spectrometer, for the more polar and less polar analytes, respectively. Isotopically labelled internal standards were used to compensate for variability in the quantification of each analyte. The method was validated according to international guidelines and applied to samples from a clinical trial. We performed correlation and agreement analysis of the DBS assay and in-house plasma methods using Deming regressions and Bland-Altman plots. Coefficients of correlation between measured plasma and DBS concentrations ranged from 0.866 (95% CI: 0.817-0.902) to 0.989 (95% CI: 0.985-0.992). More than 67% of the samples showed a difference between the observed and estimated plasma concentrations within 20% of their means, meeting EMA requirements for method reproducibility and demonstrating the interchangeability of our DBS and plasma LC-MS/MS methods., 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 © 2025 The Author(s). Published by Elsevier B.V. All rights reserved.)

An analytical quality by design-guided LC-ICP-MS method for simultaneous arsenic speciation analysis in HEK-293 cells was optimized and validated. Initially, critical method variables (CMVs) were identified to achieve the targeted critical quality attribute (CQA) of the analytical target profile (ATP). Based on knowledge and risk assessments, formic acid (X 1 ), citric acid (X 2 ), and pH (X 3 ) were studied for their effect on method responses such as resolutions (Y1, Y2) and retention time of As(V), As(III) and DMA (Y3, Y4, and Y5) using central composite design (CCD). ANOVA analysis indicated that variable interaction was significant in method responses with curvature effect on resolution (Y1 and Y2). The method operable design region (MODR) afforded a 0.1% formic acid, citric acid strength of 20-30 mM, and pH 5.6-6.8 as a robust region for the appropriate method performance. Hence, the final method was optimized on the ZORBAX RRHD SB-Aq column using a mobile phase consisting of 0.1% Formic acid: Citric acid (22.5 mM) (50:50 % v/v; pH 5.6). The optimized method eluted As(V), As(III), and DMA at 2.5 ± 0.1, 2.7 ± 0.1, and 3.1 ± 0.1 min, respectively, with an acceptable resolution. The LOD of the method was 4.78, 3.39, 5.35 ppb respectively for As(V), As(III), and DMA whilst the linearity was established at 30-1000 ppb for all species with respective r 2 -value of 0.9967, 0.9996, and 0.9972, respectively. The % recovery (77.11-99.64 %) and precision (0.25-1.95 %) were acceptable. The method has proven robust for method variables. Notably, we conducted the Green-white analytical chemistry assessment for the developed method by three different assessment tools viz., AGREES, GAPI, and RGB of 12 algorithms. The developed method demonstrated robustness, environmental friendliness, and user-friendliness., 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 © 2025 Elsevier B.V. All rights reserved.)

Background: Furfurals, key derivatives of Maillard reactions commonly found in everyday foods, have been identified as having significant toxic effects on human health. Excessive intake of furfurals can lead symptoms such as weight loss, poor nutrient metabolism which followed by disease occurrence. The severe carcinogenicity, mutagenicity and genotoxicity of furfurals were well recognized recently. In response, international organizations have established strict limits on the allowable levels of furfurals in food products. Therefore, it is of paramount importance to implement control which mediated by modern pretreatment and analytical techniques, towards the daily accessibility of furfurals., Scope and Approach: This review seeks to present a comprehensive overview of recent advancements in the pretreatment and analytical techniques for furfurals in food from 2017 to 2023. Various pretreatment methods, such as liquid phase microextraction, solid phase extraction, solid phase microextraction, and QuEChERS, as well as analytical technologies like liquid chromatography-based methods and gas chromatography-based methods, are thoroughly discussed in terms of their mechanisms, benefits, and limitations., Key Findings and Conclusions: Currently, various pretreatment and analytical techniques with advantages and limitations had been proposed. The development of novel materials does facilitate the optimization and application of microextraction based pretreatment platforms which share with enhanced extraction efficiency. In addition, the development of novel targeting/sensing materials along with the utilization of high-resolution mass spectrometry could promote the determination sensitivity. In future, development of novel absorbents which mediates more desirable pretreatment methods, and automated and miniaturized on-site analytical instruments for furfurals determination still deserve indepth invesigation., 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 Ltd. All rights reserved.)

A simple LC method has been developed and validated for estimating budesonide (epimer B + A) and formoterol fumarate dihydrate in dry powder inhalation. The development results of this study make it very significant. The degradation and process impurities in EP and ChP were identified in addition to budesonide and formoterol fumarate. As of yet, no one has reported all impurities using a single method. It is a unique research because it analyzes APSD (Aerodynamic Particle Size Distribution), DDU (Delivered Dose Uniformity), BU (Blend Uniformity), Assay, and cleaning test samples. It enhances the quality of medicine and separates all organic impurities and isomers through a suitable stationary phase (YMC-Pack Pro C18, 150 × 4.6 mm × 3 μm). We optimized the chromatographic conditions: Injection volume was 20 μL, and flow rate was 1.0 mL/min. The wavelength was optimized at 220 nm. After experimental and validation results. An example is A, which contains sodium dihydrogen orthophosphate monohydrate, sodium 1-decane sulfonate, adjusted pH 3.0, and acetonitrile at a ratio of 80:20 (v/v), and B, which contains pH 3.0 buffer and acetonitrile at a ratio of 20:80 (v/v) respectively. In addition to being optimized, the test method was validated according to ICH Q2(R2)., (© 2025 The Author(s). Biomedical Chromatography published by John Wiley & Sons Ltd.)

Introduction: High-dose systemic prednisolone is the cornerstone treatment of many autoimmune- and inflammatory diseases. Since prednisolone shows non-linear protein binding at higher serum concentrations, quantification of the unbound prednisolone concentration is important to understand prednisolone pharmacokinetics. We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify protein-unbound prednisolone in serum., Methods: Protein-unbound prednisolone was obtained using an equilibrium dialysis technique. Prednisolone was extracted from the dialysate using methyl tert-butyl ether. After evaporation to dryness, the organic phase residue was reconstituted and ready for injection onto the LC-MS/MS. Prednisolone was analysed by selected reaction monitoring with MS/MS operating in positive ion mode., Results and Discussion: The equilibrium between bound and unbound prednisolone was stable after 24 h. The calibration model for prednisolone in serum ranged from 0.25 to 811 µg/L and had an average linearity of 0.998. The coefficient of variation (CV) for precision at the lower limit of quantification was ≤ 4.3 % and for the other quality control samples ≤ 7.8 %. Prednisolone protein binding showed no significant degradation after 30 months of storage at -80 °C and was not influenced by multiple cycles of freezing and thawing. The recovery for the tested matrix effects in serum ranged from 85 % to 115 % (CV 10.3 %) and throughout the validation, no carry-over was observed., Conclusion: An LC-MS/MS assay for prednisolone in serum was developed and validated, with a successful equilibrium dialysis technique to obtain protein-unbound prednisolone prior to quantification. This assay is considered suitable for pharmacokinetic studies., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: University Hospital Centre Utrecht Location Wilhelmina Children’s Hospital reports financial support was provided by Harmannus Erhardt foundation. 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.)

Bruton's tyrosine kinase inhibitors (BTKis) exhibit significant interindividual pharmacokinetics, making therapeutic drug monitoring (TDM) a promising approach for personalized therapy. However, simultaneous quantification of multiple BTKis poses technical challenges. A unified protocol for BTKis detection would be clinically desirable. Herein, we developed and validated a novel LC-MS/MS method for the simultaneous analysis of four BTKis including ibrutinib (IBR), zanubrutinib (ZAN), orelabrutinib (ORE), and acalabrutinib (ACB) and active metabolite of IBR and ACB (DIH and ACBM, respectively) in human plasma. The samples were prepared by liquid-liquid extraction using tert-butyl methyl ether. Ibrutinb-d4 (IS) was used as an internal standard. Chromatographic separation was obtained on an XBridge C18 column and connected to an LC-30AD system coupled to an API 4000 + mass spectrometer. The mobile phase comprised 10 mM ammonium acetate containing 0.1% formic acid and acetonitrile containing 0.1% formic acid. The optimized multiple reaction monitoring transitions of m/z 441.4 → 138.3, 475.4 → 304.2, 472.5 → 455.5, 428.3 → 411.5, 466.1 → 372.2, 482.2 → 388.4, and 445.5 → 142.5 were selected to inspect IBR, DIH, ZAN, ORE, ACB, ACBM, and IS, respectively. The method exhibited linearity from 1 to 1000 ng/mL (r > 0.99) for all analytes, with intra-day and inter-day precision of 1.8 to 9.7% and accuracy below 15%. Recovery ranged from 90.4 to 113.6%, and matrix effect varied from 89.3 to 111.0%. All compounds demonstrated stability under relevant conditions. Application of the method to 57 blood samples from 18 patients demonstrated high interpatient variability, with ORE plasma concentrations ranging from 25.6 to 89.9%. The validated LC-MS/MS method provides a feasible, specific, and rapid approach for quantification of BTKis in clinical settings. Simultaneous determination of four BTKis and their metabolites in a single extraction process and chromatographic run reduces analysis time, cost, and resources. The observed variability among individuals highlights the value of TDM for personalized treatment., Competing Interests: Declarations. Ethics approval: This study was conducted according to the guidelines of the Declaration of Helsinki, and ethical approval was obtained from the Medical Ethics Committee of Peking University Third Hospital (No. IRB00006761-M2023451). Source of biological material: The biological material used in this study consisted of blood samples collected from patients diagnosed with B-cell lymphoma. All samples were obtained with informed consent from participants, following the ethical guidelines and under the approval of the (Medical Ethics Committee, Peking University Third Hospital, and No. IRB00006761-M2023451). Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Chinese hamster ovary (CHO)-K1 cells are widely used in biomedical research relevant to cancer, toxicity screening, and viruses, as well as in the production of recombinant proteins for biopharmaceuticals. In this study, liquid chromatography (LC)-electrospray ionization (ESI)-higher energy collisional dissociation (HCD)-tandem mass spectrometry (MS/MS) was used to characterize the surface and lysate N-glycans of CHO-K1 cells and analyze their structures. The relative quantity (%) of each N-glycan and absolute quantity (pmol) of total N-glycans were also obtained. In total, 37 surface and 30 lysate N-glycans were identified. Each of these two fractions contained eight high-mannose type (required for protection against proteolysis and N-glycosylation of recombinant proteins) at 28.8 % (the sum of the relative quantities of each N-glycan) and 66.5 %, respectively. Additionally, the surface and lysate N-glycans differed in their levels of sialyation (affect cell-cell interactions; 48.1 % and 13.5 %), fucosylation (affect cell signaling; 37.9 % and 25.5 %), and terminal-galactosylation (prerequisite for subsequent sialylation; 36.6 % and 20.9 %). These results indicate that the lysate of CHO-K1 cells contained more mannosylated (2.3-fold) N-glycans compared to the surface, which contained relatively more sialylated (3.6-fold), slightly more highly fucosylated (1.5-fold), and more terminal-galactosylated (1.8-fold) N-glycans. The sum of the absolute quantity of each N-glycan was obtained as a ratio of 1 (1,778.7 pmol; surface):2.2 (3,887.3 pmol; lysate) from approximately 5 × 10 6 CHO-K1 cells. This study is the first to compare the surface and lysate N-glycans of CHO-K1 cells using LC-ESI-HCD-MS/MS. The results can be used to control and optimize biotechnology and biomedical research using CHO-K1 cells., 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.)

Paxalisib is a dual PI3K/mTOR inhibitor, being used in advanced cancer treatment. In this research, we report a validated LC-MS/MS method for quantifying paxalisib from mouse dried blood spot (DBS). We validated the method in-line with the FDA guidelines. Liquid-liquid extraction technique was used to extract paxalisib from the DBS discs. We used a Chromolith RP-18 end cap (100 × 4.6 mm) column and isocratic mobile phase for the chromatographic separation of paxalisib and the internal standard (IS, dasatinib). The flow was 0.80 mL/min. In the optimized chromatographic conditions, the retention of paxalisib and the IS was ~2.13 and 2.06 min, respectively. Each injection total run time was 2.50 min. The MS/MS ion transitions monitored were m/z 383.2 → 309.1 and 488.1 → 410.1 for paxalisib and the IS, respectively. We have used a broad calibration range (1.24-3762 ng/mL) with a determination coefficient (r 2 ) of 0.995. All the validation parameters assessed met the acceptance criteria, and hematocrit had no effect on DBS Paxalisib concentrations. We have used the validated method to derive the intravenous and oral pharmacokinetic parameters by quantifying paxalisib in mouse blood and correlated with mice pharmacokinetic data., (© 2024 John Wiley & Sons Ltd.)

Mobocertinib is a potent selective tyrosine kinase inhibitor approved for the treatment of non-small cell lung cancer with activating EGFR exon 20 insertions. The aim of this study was to develop a procedure for liquid chromatography tandem mass spectrometry (LC-MS/MS) for the determination of mobocertinib and its metabolite desmethyl-mobocertinib in human plasma. The human plasma samples were precipitated with acetonitrile and analyzed using a Waters ACQUITY BEH C 18 column coupled to a triple quadrupole mass spectrometer. Separation was executed using the acetonitrile-0.1% formic acid solution with gradient elution, at a flow rate of 0.4 mL/min. Mobocertinib and desmethyl-mobocertinib were monitored by multiple reaction monitoring (MRM) with m/z 586.5  >  72.2 and 572.4  >  473.2, respectively. The procedure demonstrated excellent linearity (r > 0.997) within the concentration range of 0.1-200 ng/mL for both analytes. Precision in relative standard deviation was <  9.37% for mobocertinib and <  12.03% for desmethyl-mobocertinib. Accuracy in relative error was within -7.23% to 9.18% for mobocertinib and -2.78% to 9.87% for desmethyl-mobocertinib. Extraction recovery was >  80% for both analytes. The validated LC-MS/MS method was successfully applied to the pharmacokinetic study of mobocertinib and desmethyl-mobocertinib in healthy human volunteers with K 2 EDTA as anticoagulant after a single dose of mobocertinib (160 mg)., (© 2024 John Wiley & Sons Ltd.)

To date, there are more than 80 ergot alkaloids identified; their distribution depends on different factors (e.g. geographic regions, host plants). These toxins can cause acute and chronic toxic effects on human health and commonly infect cereal crops such as triticale and rye, wheat, barley and oats. Considering the growing consumption of plant-based foods, the European Food Safety Authority has highlighted the need to develop risk assessment strategies. This work focused on the optimization of extraction efficiency, to quantify the main ergot alkaloids and their epimers, that are available on the market without any legal restriction (ergosine, ergocristine, ergocriptyne, ergocornine, ergosinine, ergocristinine, ergocriptinine and ergocorninine). Considering the quantification of 8 out of 12 regulated compounds by EU (sum of -ine and -inine forms), this approach can be defined as a screening method for a reliable estimation of the risk, specifically devoted to industrial stakeholders that can then possibly outsource to authorized external labs only the samples suspected of significant positivity. The effectiveness of three different extraction conditions (acidic, alkaline and neutral) followed by a rapid clean-up using dispersive solid-phase extraction with C 18 sorbent was evaluated by ultra-performance liquid chromatography tandem quadrupole mass spectrometry (UPLC-MS/MS), resulting in a short chromatographic run (16 min). The method was developed and validated in five different cereal production chains (rye, oat, wheat, wheat gluten and baby food). The applicability of the method was examined by analyzing a set of 54 samples, including also other cereals like spelt, tritordeum and triticale, and evaluating also some reference materials., Competing Interests: Declarations. Competing interest: The authors declare no competing interests, (© 2024. The Author(s) under exclusive licence to Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature.)

Interest in bile acids (BAs) is growing due to their emerging role as signaling molecules and their association with various diseases such as colon cancer and metabolic syndrome. Analyzing BAs requires chromatographic separation of isomers, often with long run times, which hinders BA analysis in large studies. Here, we present a high-throughput method based on liquid chromatography-tandem mass spectrometry to quantify BAs in mouse samples. After acidic protein precipitation in the presence of a comprehensive mixture of stable isotope-labeled internal standards (SIL-ISs), BAs are separated on a biphenyl column by gradient elution at basic pH. Quantification is performed using a six-point calibration curve. Except for the separation of β- and ω-muricholic acid (MCA) species, a rapid separation of 27 BA species was achieved in a run time of 6.5 min. Plasma quality controls (QCs) were used to evaluate intra- and inter-day precision. The CV was less than 10% for most BA species and exceeded 20% only for glycohyodeoxycholic (GHDCA) and taurohyodeoxycholic acid (THDCA) due to the lack of a corresponding SIL-IS. The limit of quantification (LoQ) was tested using diluted QCs and was found to be compromised for some BA species as a result of insufficient isotopic purity of the SIL-IS, leading to significant interference with the respective analyte. Finally, we tested the mouse sample material requirements for plasma, bile, and liver samples and determined BA concentrations in C57/BL6N wild-type mice. In conclusion, the LC-MS/MS method presented here permits a rapid and reproducible quantification of the major murine BAs., Competing Interests: Declarations. Statement on animal welfare: Breeding, handling, and sampling of mice were performed in accordance to a breeding permit under §11 of the German Law for Animal Welfare, and no animal experiments were performed as defined under §7 Sect. (2). Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s).)

Dried urine spots have recently been proposed as an alternative matrix in the anti-doping field. Drying urine may open the opportunity to limit microbial and thermal degradation of the prohibited substances during transportation to the anti-doping laboratories without the need for refrigeration or freezing. In this study, a multi-targeted initial testing procedure was developed for the determination of 237 prohibited drugs/metabolites from 11 different classes in dried urine spots. The comparability between two different microsampling techniques (i.e., Whatman ® FTA DMPK-C cards and Mitra ® tips) was evaluated. The developed method was then used to evaluate the stability of the target compounds in urine for 7 days under different environmental conditions to simulate the transportation of the urine samples from the collection sites to anti-doping laboratories. Sample preparation consists of (i) extraction of the analytes from the collection device using a mixture of acetonitrile/methanol (1/1) for 30 min at 40 °C, (ii) enzymatic hydrolysis, and (iii) sample concentration by solid-phase extraction. Analysis was performed using liquid chromatography coupled to high-resolution mass spectrometry. The entire workflow was validated in terms of specificity (analytes were distinguishable from the matrix interferences), sensitivity (only with the Mitra ® tips the limits of detection comply with the World Anti-Doping Agency's requirements for the majority of the target compounds), carry-over (no signals in the negative urine injected after the positive urine), matrix effect (16-28% for Mitra ® tips and 22-35% for DMPK-C cards), and extraction yield (Mitra ® tips: 51-88%; DMPK-C cards: 40-76%). As proof of concept, authentic urine samples were analyzed: results obtained in dried urine were compared with those of fluid urine, providing good agreement. Stability studies showed that the target compounds were stable for the whole duration of the study (7 days) at -20 and 4 °C in both fluid and dried urine. At 50 °C or at 20-25 °C, several thiazide-based compounds were completely degraded to their degradation product in the first 24 h or after 3-4 days in fluid urine, whereas in dried urine the compounds were detectable for the entire duration of the study., Competing Interests: Declarations. Ethics approval: The use of negative urine from healthy volunteers was approved by the Medical Ethics Committee (CME) affiliated with Ghent University (UGent) and the Ghent University Hospital (UZ Gent) (B6702024000326). Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

The aim of this study was to investigate the pharmacokinetics, bioequivalence, and safety of generic trazodone hydrochloride sustained-release tablet and its reference listed product in healthy Chinese subjects. An open, randomized, single-dose, and 2-period crossover study was involved under fasting and fed conditions, with a 7-day washout period. A single oral dose of 150 mg of 2 trazodone hydrochloride sustained-release tablets was administered to 84 healthy volunteers, with 36 in the fasting group and 48 consuming a high-fat diet, respectively. The plasma concentrations of trazodone were analyzed using a liquid chromatography-tandem mass spectrometry method, and pharmacokinetic parameters were obtained from concentration-time profiles. The geometric mean ratio with 90% confidence intervals of the maximum trazodone concentration, area under the plasma concentration-time curve (AUC) from time 0 to the last measurable concentration, and AUC from time 0 to infinity were within the bioequivalence acceptance criteria (80%-125%) under fasting and fed conditions, which indicated that the test and reference formulations were bioequivalent. Compared with the fasting study, the concomitant administration of trazodone with a high-fat diet had a negligible influence on the drug pharmacokinetic behavior. Adverse events were recorded, and no serious adverse events were observed during either fasting or fed conditions. Trazodone has proven to have an acceptable safety profile in the Chinese population, with bioequivalence successfully established under both fasting and fed conditions., (© 2024, The American College of Clinical Pharmacology.)

Contaminants released into the atmosphere that undergo regional and long-range transport can deposit back to Earth through snowfall. When snow melts, these contaminants re-enter the environment, sometimes far from their original emission sources. Here we present the first comprehensive characterization of organic contaminants in snow from North America. Fresh snowfall samples were collected in the central United States over a three-year period and measured by liquid chromatography high-resolution mass spectrometry for suspect screening and non-targeted analysis. The resulting data set was screened against experimental MS/MS libraries and underwent supplemental in silico MS/MS analysis. In total, 91 possible compounds were tentatively identified in snow, and 17 were successfully confirmed and semi-quantified with reference standards. These contaminants were mostly anthropogenic in origin and included six herbicides, three insect repellants, one insecticide metabolite, and one fungicide. The most prominent compounds present in all samples were N-cyclohexylformamide (known contaminant in tire leachate), DEET (insect repellent), and dimethyl phthalate (plasticizer), with median deposition fluxes of 4032, 284, and 262 ng m -2 , respectively. Three additional compounds were detected in 100% of samples: coumarin (phytochemical and fragrance additive), 5-methylbenzotriazole (antifreeze component), and quinoline (heterocyclic aromatic). The Peto-Peto test revealed statistically significant differences in deposition fluxes for these six contaminants (p < 0.05), with weak but statistically significant positive associations between coumarin and DEET and between coumarin and quinoline according to a Kendall's tau correlation analysis. These findings demonstrate the utility of in silico analysis to complement MS/MS matching with experimental databases. Even so, thousands of unidentified features remained in the data set, highlighting the limitations of current strategies in non-targeted analysis of environmental 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 Inc.)

Montelukast sodium is a leukotriene type 1 receptor antagonist that can be used for the prophylaxis and treatment of asthma. However, the pharmacokinetics of montelukast sodium tablets (10 mg) remain unclear in healthy Chinese subjects. Here, a single-dose randomized, open-label, 2-sequence, and 2-period crossover (7-day washout period between treatments) study was performed to compare the pharmacokinetics and bioequivalence between the test products and the reference at a single dose of 10 mg among healthy Chinese subjects under fasting and fed conditions. Blood samples were collected at specified time points to analyze the plasma concentrations of montelukast by a validated liquid chromatography-tandem mass spectrometry method. The results showed that the 90% confidence interval values of the geometric mean ratio of test/reference for the maximum plasma drug concentration, area under the concentration-time curve from time 0 to the end, and area under the concentration-time curve from time 0 to infinity were within the range of 80%-125%. Moreover, both the test and reference formulations were safe and well tolerated, with no occurrence of severe adverse events. These results demonstrate that both the test montelukast sodium tablets and the reference showed similar bioequivalence, safety, and tolerability among healthy Chinese subjects under fasting and fed conditions., (© 2024, The American College of Clinical Pharmacology.)

Organic precipitation of proteins with acetonitrile demonstrated complete protein recovery and improved chromatography of human plasma proteins. The separation of 25 μL of human plasma into 22 fractions on a QA SAX resin facilitated more effective protein discovery despite the limited sample size. Micro chromatography of plasma proteins over quaternary amine (QA) strong anion exchange (SAX) resins performed best, followed by diethylaminoethyl (DEAE), heparin (HEP), carboxymethyl cellulose (CMC), and propyl sulfate (PS) resins. Two independent statistical methods, Monte Carlo comparison with random MS/MS spectra and the rigorous X!TANDEM goodness of fit algorithm protein p-values corrected to false discovery rate q-values (q ≤ 0.01) agreed on at least 12,000 plasma proteins, each represented by at least three fully tryptic corrected peptide observations. There was qualitative agreement on 9393 protein/gene symbols between the linear quadrupole versus orbital ion trap but also quantitative agreement with a highly significant linear regression relationship between log observation frequency (F value 4,173, p-value 2.2e-16). The use of a QA resin showed nearly perfect replication of all the proteins that were also found using DEAE-, HEP-, CMC-, and PS-based chromatographic methods combined and together estimated the size of the size of the plasma proteome as ≥12,000 gene symbols., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The site-specific antibody-drug conjugates (ADCs), particularly those utilizing the engineered cysteine in Fc fragments of mAbs (THIOMAB™ antibodies), have emerged as a novel class of biotherapeutics for cancer treatment. The engineered cysteine residues in these antibodies are capped by cysteine or glutathione through a disulfide bond. Prior to conjugation with linker-payloads, these caps need to be removed through a reduction process. However, monitoring the efficiency of the decapping process has been challenging due to the lack of effective analytical methods. Intact reversed-phase liquid chromatography-mass spectrometry and hydrophobic interaction chromatography methods failed to separate decapped and capped intact THIOMAB™ mAbs in our study. Instead the fragmentation of mAbs provided a novel strategy to analyze the decapping effiency. After cleavage using a hinge specific enzyme, the generated Fc fragments with and without cysteine and/or glutathione caps displayed different hydrophobicity and were well separated by RPLC, allowing quantitative determination of the decapping efficiency. Enzymes that cleave both above and below the hinge disulfide bonds were tested. The use of FabALATICA can determine percentages of molecules with 0, 1, and 2 cysteine and/or glutathione caps, respectively, regardless of whether the antibody contains the hinge LALA mutations. On the other hand, FabRICATOR enzyme can only be utilized for antibodies without LALA mutations for the overall decapping percentage and cannot be used to estimate intact antibody each with 0, 1, and 2 caps. Therefore, FabALACTICA cleavage followed by RPLC provides a wider application of monitoring the decapping efficiency of all antibodies with the engineered cysteine in Fc., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

A growing number of hemp-derived cannabidiol (CBD) products are available with negligible amounts (< 100 ppm) of delta-9-tetrahydrocannabinol (THC) due in part to consumer concerns regarding the risk of positive drug screens. There are, however, no published studies that report whether repeated use of these products may lead to positive urine drug tests for THC. There is also scant research on the effects of these products on physical and mental well-being. Twenty healthy adults consumed a hemp-derived broad-spectrum CBD product every day for 2 weeks. Participants attended study visits at the beginning and end of the 2-week period. At each visit, participants underwent urinalysis testing for CBD, THC, and metabolites (analyzed via liquid chromatography with tandem mass spectrometry) and completed a validated assessment of physical and mental well-being. Participants reported using an average of 1.09 ± 0.51 ml (34.20 ± 16.00 mg CBD) of study product per day. Neither tetrahydrocannabinol nor its metabolites were detectable in urine following the 2-week period of use. Ingestion of the broad-spectrum product was associated with a significant reduction in sleep disturbance and pain intensity symptoms ( p < .05), which remained significant after correcting for possible confounds (i.e., age, sex, dosage). No adverse events were reported. (PsycInfo Database Record (c) 2025 APA, all rights reserved).

The infant flours produced in Burkina Faso are essentially a mixture of cereals and legumes. These raw materials are frequently contaminated with mycotoxins which pose a huge food safety and public health threat. The objective of this study was to determine mycotoxin levels in raw materials and infant flours in Ouagadougou and to investigate the impact of decontamination on the raw materials used in infant flour production. A total of 22 cereals and 17 legumes as raw materials and 26 infant flour samples were analysed for aflatoxins, fumonisin B 1 (FB 1 ), and ochratoxin A (OTA) by liquid chromatography coupled to tandem mass spectrometry, while saline treatment and hand-sorting of grains in mycotoxin reduction were tested. All the samples of raw materials and infant flours were contaminated with aflatoxins, whereas 20.5% and 38.5% of raw materials and 57.7% and 61.5% of infant flours, respectively, were contaminated by FB 1 and OTA. These decontamination assays significantly reduced the levels of mycotoxins. AFB 1 was reduced by 48% after soaking of maize for 6 h in a 6% NaCl solution. Sorting resulted in a 92% reduction in AFB 1 content in peanut. However, soaking in saline solution did not reduce the FB 1 and OTA contents. Sorting did not also reduce FB 1 contents in peanut. Sorting and soaking in 6% saline solution for 6 h are production processes that lead to a reduction in the level of contamination by aflatoxins in maize and peanut used as raw materials for infant flour production., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s) under exclusive licence to Society for Mycotoxin (Research Gesellschaft für Mykotoxinforschung e.V.) and Springer-Verlag GmbH Germany, part of Springer Nature.)

The detection of mycotoxins in Sichuan peppers is crucial for ensuring food safety. Hence, an efficient detection approach based on the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method followed by LC-MS/MS (liquid chromatography-tandem mass spectrometry) was established for the analysis of various mycotoxins in dried red and green Sichuan peppers. Samples were extracted with a solution containing 99 % acetonitrile and 1 % formic acid. A new adsorbent, namely, ENVI-Carb, and primary secondary amines were used as purifying agents. The proposed method demonstrated good linearity (R 2  ≥ 0.999), satisfactory recovery (77.3 %-109.2 %), and good repeatability (intraday and interday relative standard deviations ≤9.7 %) in dried red and green Sichuan peppers. Its limits of detection in red and green Sichuan peppers were 0.2-0.6 and 0.1-0.7 μg/kg, respectively. The established method can effectively eliminate complex matrix interference and provides an effective approach for the high-throughput detection of mycotoxins in various complex food matrices., 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 Ltd. All rights reserved.)