bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–05–18
fifty-four papers selected by
Sofia Costa, Matterworks



  1. J Sep Sci. 2025 May;48(5): e70162
      A sensitive and efficient method for simultaneous quantifying molnupiravir and its active metabolite β-d-N4-hydroxycytidine in human plasma was developed by combining chemical derivatization with liquid chromatography-tandem mass spectrometry. Through benzoyl chloride chemical derivatization, the analytes exhibited improved mass spectral responses and enhanced chromatographic retention. Besides, the fragmentation patterns were optimized to identify analyte-specific fragments, enhancing detection specificity beyond the common benzoyl fragments. These advancements enabled the method to achieve the lower limits of quantification of 0.4 ng/mL for molnupiravir and 1.0 ng/mL for β-d-N4-hydroxycytidine, which represents the lowest reported quantification limits values to date while demonstrating a 30-fold sensitivity enhancement compared to the non-derivatized method under identical instrument conditions. In addition, the sample preparation protocol was streamlined, combining derivatization and sample extraction in a single step, completed within 5 min, eliminating the need for additional handling or reaction time. After undergoing comprehensive validations, the method was successfully applied to clinical samples from patients receiving molnupiravir therapy, demonstrating its practicality for pharmacokinetic monitoring. By combining operational simplicity with sensitivity, this assay provides a reliable tool for advancing research on molnupiravir metabolism and therapeutic drug monitoring.
    Keywords:  COVID‐19; LC‐MS/MS; chemical derivatization; molnupiravir
    DOI:  https://doi.org/10.1002/jssc.70162
  2. Bioanalysis. 2025 May 14. 1-8
      A highly sensitive and selective LC-MS/MS assay was developed and validated for the simultaneous quantification of Chlordiazepoxide and Clidinium in human plasma for the first time, employing solid-phase extraction. Chromatographic separation of the analytes and their deuterated internal standards was performed on a reversed-phase Kinetex XB-C18 (150 × 4.6 mm, 5 μm) column with a gradient mobile phase. Mass spectrometric detection was achieved using electrospray ionization in positive ion mode, employing the ion transitions: m/z 300.0 → 227.1 for Chlordiazepoxide, m/z 352.1 → 142.1 for Clidinium, m/z 305.1 → 232.1 for Chlordiazepoxide D5, and m/z 357.2 → 142.2 for Clidinium D5. The assay demonstrated a linear calibration range of 504.0-500,198.3 pg/mL for Chlordiazepoxide and 5.0-3,004.7 pg/mL for Clidinium, ensuring precise pharmacokinetic evaluation. The method was validated with a lower limit of quantification (LLOQ) of 504.0 pg/mL for Chlordiazepoxide and 5.0 pg/mL for Clidinium, precision within 15% RSD, and accuracy within 85-115% of the nominal values. No matrix interference from haemolysed or lipemic plasma was observed, and recovery exceeded 90%. This study presents a novel LC-MS/MS method with significant improvements in sensitivity and specificity, facilitating its direct application in pharmacokinetic and bioequivalence studies.
    Keywords:  Chlordiazepoxide; LC-MS/MS; bioequivalence; clidinium; human plasma
    DOI:  https://doi.org/10.1080/17576180.2025.2501921
  3. Sci Rep. 2025 May 13. 15(1): 16640
      20(S)-Protopanaxadiol (PPD) is a saponin derivative of ginsenoside, with more potent biological and pharmacological activities than Rg3 and Rh2. The lack of ionizable centers leads to low mass spectrometry reactions and internal cleavage of three hydroxyl groups, making it challenging to establish highly sensitive PPD mass spectrometry methods. The aim of this study is to establish and validate a quantitative detection method for PPD in multiple matrices using mass spectrometry. The methods used Rh2 as the internal standard and organic solvent liquid-liquid extraction under alkaline conditions for biological sample pretreatment. Isometric separation was achieved through methanol, acetonitrile, and a 10 mmol/L solution of acetic acid (45:45:10, v/v/v) at a flow rate of 0.4 mL/min. Finally, perform mass spectrometry quantification. Comprehensive method validation was conducted on rat plasma samples, and partial method validations were performed on three types of rat tissues (adipose tissue, smooth muscle, and skeletal muscle), bile, urine, fecal samples, and dog plasma samples. The results were in accordance with the requirements of NMPA for bioanalytical method validation, ensuring the accuracy and reliability of our analytical measurements. This study employed a conventional liquid-liquid extraction sample pretreatment scheme, utilizing multiple biological matrices commonly found in a single treatment protocol and liquid chromatography-tandem mass spectrometry detection parameters. The consistency of processing and detection across diverse samples eliminated the need for methodological changes, providing exceptional convenience. Up to 90% of the organic phase and a 50 mm short chromatographic column achieved rapid and effective separation of PPD. A key aspect of our work is the use of a "programmed injection" technique, which significantly reduces the analysis time from 4.2 min during method exploration to 2.4 min. These methods have achieved a relatively low quantification limit of 2.5 ng/mL. The methods established were successfully applied to the kinetic process of PPD in rats, and the pharmacokinetic characteristics of PPD in dogs were studied for the first time.
    Keywords:  20 (S)-protopanaxadiol; Biological matrices; LC–MS/MS; Pharmacokinetic; Programmed injection
    DOI:  https://doi.org/10.1038/s41598-025-01432-1
  4. Anal Chem. 2025 May 15.
      The development of biocatalysis depends heavily on high-throughput screening (HTS) approaches to uncover engineered enzymes with superior biocatalytic activity. Recent advances in mass spectrometry (MS) enable label-free, high-speed analysis of biocatalytic samples in standard microplates by omitting the chromatographic separation. However, most MS methods need a complicated sampling interface system and face challenges due to biological matrix interferences, which can diminish the sensitivity and reliability. Herein, we established a direct microplate sampling (DMS)-MS technique for HTS of the enzymatic activity of 17β-hydroxysteroid dehydrogenase (17β-HSD) in steroid biocatalysis. The implementation of an open port interface (OPI) probe with a customized ion source for microwell direct insertion sampling and ionization has streamlined the setup, reduced operational complexity, and shortened the analysis time to 0.9-6.5 s/sample. A sample preparation strategy involving derivatization, followed by high-ratio dilution (e.g., 10,000-fold), was further integrated prior to DMS-MS for mitigating ion suppression effects across diverse biological matrices. Ultimately, this workflow, when applied for monitoring multiple steroids in 17β-HSD biocatalytic processes, demonstrated high stability (peak area RSDs < 7%), minimal carryover (<1%), and quantitative accuracy comparable to conventional liquid chromatography (LC) experiments while providing a >150-fold throughput enhancement. After screening a total of 5760 17β-HSD mutants across three different steroid conversion reactions, we identified and verified two new variants with enhanced biocatalytic activities. This study successfully established a DMS-MS-based HTS workflow for microplate screening, providing a simplified, robust, and highly reliable analytical platform for microbial or enzyme engineering projects involving complex biological matrices.
    DOI:  https://doi.org/10.1021/acs.analchem.5c00267
  5. Clin Lab Med. 2025 Jun;pii: S0272-2712(25)00007-1. [Epub ahead of print]45(2): 221-232
      Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has seen an enormous increase in applications, such as toxicology, biochemical genetics, endocrinology, therapeutic drug monitoring, and dietary monitoring. Although LC-MS/MS has the potential to offer significant clinical benefit to patients and clinicians, these assays often require significant effort and complex quality-assurance data review processes to ensure accurate results. Manual LC-MS/MS data review is time consuming and susceptible to human error. Automation of data review can reduce time spent on manually reviewing each chromatogram, reduce human error, and improve practice and ensure regulatory compliance with the rules and guidelines established by the College of American Pathologists (CAP), Clinical Laboratory Improvement Amendments (CLIA), and Clinical and Laboratory Standards Institute (CLSI) C62-A.
    Keywords:  CLSI 62-A; Chromatography; Data review; Data validation; LC-MS; LDT; Quality assurance
    DOI:  https://doi.org/10.1016/j.cll.2025.01.006
  6. Molecules. 2025 Apr 22. pii: 1866. [Epub ahead of print]30(9):
      Cystic fibrosis (CF) is a life-threatening disorder caused by mutations in the CFTR gene, leading to defective chloride ion transport and thickened mucus in the respiratory and gastrointestinal systems. CFTR modulators, including ivacaftor, lumacaftor, tezacaftor, and elexacaftor, have improved patient outcomes, but interindividual pharmacokinetic variability and potential drug-drug interactions require therapeutic drug monitoring (TDM) for optimal efficacy and safety. In this context, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the simultaneous quantification of CFTR modulators and their major active metabolites in human plasma to support pharmacokinetic studies and routine TDM. The multiplex LC-MS/MS assay was established using plasma protein precipitation, followed by chromatographic separation on an Xselect HSS T3 (Waters®) column and positive electrospray ionization mode detection. The method was validated based on FDA and EMA guidelines for specificity, linearity, accuracy (89.8-107.8%), repeatability (1.1-8.1%), intermediate fidelity (1.3-10.9%), matrix effects, and stability, demonstrating a robust performance with excellent precision and accuracy. International interlaboratory comparisons confirmed the reliability of the assay. The developed method can be applied for the clinical monitoring of caftors' plasma concentrations and preliminary data suggest that it can also be applied to alternative matrices, such as breast milk. This method will serve to characterize caftors' pharmacokinetic variability and monitor drug-drug interactions to further refine personalized dosing strategies and enhance precision medicine treatments for patients with CF.
    Keywords:  CFTR modulators; LC-MS/MS; cystic fibrosis; method development; pharmacokinetics; precision medicine; quantification; therapeutic drug monitoring; validation
    DOI:  https://doi.org/10.3390/molecules30091866
  7. Bioanalysis. 2025 Apr;17(8): 525-535
       AIMS: Omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) are indispensable for human health. Notably, imbalanced levels of maternal PUFAs during pregnancy can exert lasting impacts on the developing fetus. There is an urgent need for a robust and rapid method that can simultaneously quantify multiple n-3 and n-6 PUFAs in plasma and adapt to large-scale clinical screening.
    METHODS: Sample preparative process consisted of non-derivatized alkali hydrolyzing and liquid-liquid extraction followed by reconstituting after being blown dry with nitrogen, and the total chromatographic run time was only 5.6 min.
    RESULTS: The LC-MS/MS method was validated according to current national and international guidelines. Acceptable reproducibility and accuracy were shown based on the intra-day precision was 1.79%-7.02%, the inter-day precision was 0.01%-5.63%, and the accuracy was 87.42%-102.78%. The correlation coefficients of linearity were >0.9930. All measured plasma individual PUFAs levels in pregnant women significantly increased, apart from eicosapentaenoic acid (EPA, C20:5n3) and n-6/n-3 PUFA ratio levels decreased were observed. The analysis performance is satisfactory and suitable for clinical routine detection.
    CONCLUSION: This method could be a valuable tool for 10 kinds of n-3 and n-6 PUFAs nutritional assessment, supplementary guidance, and intervention during pregnancy.
    Keywords:  Liquid chromatography-tandem mass spectrometry; nutritional assessment; omega-3 polyunsaturated fatty acids; omega-6 polyunsaturated fatty acids; pregnant women
    DOI:  https://doi.org/10.1080/17576180.2025.2501935
  8. Molecules. 2025 Apr 30. pii: 2008. [Epub ahead of print]30(9):
      An HPLC-MS/MS method for the simultaneous determination of 32 polyphenolic compounds in berries was established. For method validation, the berry samples were extracted with 80% ethanol, purified on an HLB column, and separated on a C18 column via gradient elution with an acetonitrile-water mobile phase system before mass spectrometry detection with electrospray ionization in negative mode and multiple reaction monitoring. The results revealed that the 32 polyphenolic compounds had a good linear relationship in the concentration range of 1-500 μg/L, with R2 > 0.99, limits of detection, limits of quantitation, and recoveries of 0.2-0.6 μg/kg, 0.3-1.0 μg/kg, and 82.8-104.8%, respectively, and RSDs < 5.8%. The contents of polyphenolic compounds in the berries were determined, with 23 polyphenolic compounds in sea buckthorn, 18 in mulberry, 17 in black wolfberry, and 12 in red wolfberry. Eight polyphenolic compounds were found in all 4 kinds of berries, including 4-hydroxybenzoic acid, p-coumaric acid, ferulic acid, erucic acid, rutin, hypericin, kaempferol-3-O-rutinoside, and daffinoside. Additionally, six polyphenolic compounds, catechin, syringic acid, isorhamnetin-3-O-galactoside, isorhamnetin-3-O-glucoside, cinnamic acid, and isorhamnetin, were detected only in sea buckthorn.
    Keywords:  HPLC-MS/MS; berry; polyphenolic compounds; simultaneous determination
    DOI:  https://doi.org/10.3390/molecules30092008
  9. Rapid Commun Mass Spectrom. 2025 Aug 30. 39(16): e10064
       RATIONALE: Dried blood spot (DBS) sampling for the therapeutic drug monitoring of antiepileptic drugs offers practical advantages, including minimal invasiveness and ease of collection. However, for precise therapeutic management, its accuracy and reliability in quantification need to be validated.
    METHODS: This study validates DBS sampling for the analysis of 11 antiepileptic drugs using liquid chromatography-tandem mass spectrometry (LC-MS/MS), overcoming the physicochemical challenges associated with DBS samples.
    RESULTS: The acetonitrile-based DBS extraction method demonstrated high efficiency for the 11 antiepileptic drugs. Accuracy and precision within 6% were achieved in both intra- and inter-day assays, with good selectivity, minimal matrix effects, and negligible carryover. All antiepileptic drugs exhibited stability in DBS samples for at least 30 days at room temperature, confirming proper handling and storage of the DBS samples. A 3 mm diameter disc punched from a DBS produced accurate results for all target drugs.
    CONCLUSIONS: The optimized method provided a time- and cost-effective solution, showing a strong correlation between drug concentrations in whole blood, thereby supporting the suitability of DBS sampling as a promising and advanced method for antiepileptic drug monitoring.
    Keywords:  LC–MS/MS; antiepileptic drugs; dried blood spots; method validation; punch techniques; therapeutic drug monitoring
    DOI:  https://doi.org/10.1002/rcm.10064
  10. Anal Chim Acta. 2025 Jul 08. pii: S0003-2670(25)00493-3. [Epub ahead of print]1358 344099
      Ceramides (Cers) play a crucial role in sphingolipid metabolism with multiple biological activities and functions. Due to the high regularity and variability of their structures, there exist thousands of possible Cers. The structural diversity endows them with various biological functions but also poses significant challenges for qualitative and quantitative analysis. The lack of in-depth characterization methods for such lipids resulted in only a small fraction of Cers being reported, severely hindering the exploration of their biological functions and activities. This work presented a lipid analysis method based on a liquid chromatography-mass spectrometry platform, enabling the accurate quantification of 337 Cers simultaneously. Supported by a mathematical model, this work succeeded in generating a quadratic equation relationship between retention time and Cers carbon number. Subsequently, this method was applied to the large-scale quantitative detection of Cers in serum samples from Alzheimer's disease (AD) patients, identifying and characterizing 62 differential Cers. These could potentially serve as serum biomarkers for AD diagnosis. This study demonstrates a strategy for the large-scale in-depth characterization of complex endogenous lipid molecules with highly variable and regular structures in the absence of sufficient commercial standard materials. This work provides a novel analysis method and reference for exploring and developing the functions of such endogenous bioactive molecules.
    Keywords:  Alzheimer's disease; Ceramide; High performance liquid chromatography-mass spectrometry; Pseudo-targeted lipid analysis; Sphingolipid metabolism
    DOI:  https://doi.org/10.1016/j.aca.2025.344099
  11. Biomed Chromatogr. 2025 Jun;39(6): e70106
      To evaluate the pharmacokinetics, absolute bioavailability, and plasma concentrations of schaftoside and isoschaftoside in rats, an UPLC-MS/MS method was employed. For sample preparation, plasma proteins were precipitated using chilled methanol. The separation was achieved on a UPLC HSS T3 column with a mobile phase consisting of methanol and water (with 0.1% formic acid in water), at a flow rate of 0.4 mL/min. Detection was performed using electrospray ionization (ESI) in positive ion mode, coupled with multiple reaction monitoring (MRM) for quantitative analysis. Rats received oral doses of schaftoside (1 mg/kg) and isoschaftoside (5 mg/kg), and the pharmacokinetic profiles of both compounds were compared. The calibration curve for the method demonstrated excellent linearity within the concentration range of 1-2000 ng/mL, with correlation coefficients (r values) exceeding 0.99. Following intravenous and oral administration, significant differences were observed in the AUC(0-t) between schaftoside and isoschaftoside, whereas their half-lives (t1/2) remained comparable. The absolute bioavailability of schaftoside and isoschaftoside in rat plasma was determined to be 0.95% and 0.22%, respectively.
    Keywords:  UPLC‐MS/MS; isoschaftoside; pharmacokinetics; schaftoside
    DOI:  https://doi.org/10.1002/bmc.70106
  12. J Chromatogr A. 2025 May 10. pii: S0021-9673(25)00391-7. [Epub ahead of print]1754 466043
      Drug metabolite characterization from complex endogenous matrices such as plasma is a challenging task, especially for herbal product compounds. The analyses are generally carried out by flow-injection mass spectrometry (FI-MS) or combined with liquid chromatography (LC-MS), but the first one suffers from significant matrix effects, while the second is time-consuming and unsuitable for high-throughput analysis. In this study, a rapid method combining 2D-carbon microfiber fractionation (2DµCFs) with quadrupole time-of-flight high-resolution mass spectrometry (QTOF-HRMS) is proposed for the comprehensive characterization of herbal metabolites in plasma. Samples are separated into three fractions depending on their polarity (strong, medium, weak) and sequentially injected into MS, reducing matrix effects and enhancing metabolite coverage. Each 2DµCFs-QTOF-HRMS run takes only 5 min, enabling the detection of approximately 10,000 m/z features per injection-representing a 4-fold increase in the number of detected features compared to FI-MS. The proposed method maintains the high sensitivity offered by LC-MS, greatly simplifying the analytical procedure. By this approach, 14 potential effective compounds of Huangkui capsule (HKC), from dried flowers of Abelmoschus manihot (A. manihot), were characterized in blood plasma. Furthermore, the comparison of chemical components among different parts of A. manihot was performed, providing valuable insights into its active ingredients and creating a bridge between phytochemistry and clinical applications.
    Keywords:  Chromatographic separation; Matrix effect reduction; Metabolite analysis; Polarity fractionation
    DOI:  https://doi.org/10.1016/j.chroma.2025.466043
  13. Clin Lab Med. 2025 Jun;pii: S0272-2712(25)00003-4. [Epub ahead of print]45(2): 165-176
      The use of tandem mass spectrometry (MS/MS) for urine drug testing has expanded in recent decades due to an increasing demand for compliance testing and confirmation of positive immunoassay results. While it has been demonstrated to be a robust methodology, MS/MS cannot control all variables associated with sample collection and testing. This review will touch on many factors of targeted MS/MS assays pertaining to drugs, metabolism, and proper method development. Various preanalytical and analytical considerations will be discussed, and best practice guidelines are suggested for controlling sample-to-sample variability.
    Keywords:  Compliance; Drugs of abuse; Internal standard; LC-MS/MS; Liquid chromatography; Sample cleanup; Toxicology
    DOI:  https://doi.org/10.1016/j.cll.2025.01.002
  14. Commun Chem. 2025 May 14. 8(1): 147
      The development of analytical technology that allows investigation of the diversity of cells that form biological tissues based on molecular information is important to elucidate the heterogeneity of cells and pathological mechanisms. Here, we present a proof-of-concept demonstration of single-cell mass spectrometry imaging (SC-MSI) via tapping-mode scanning probe electrospray ionization (t-SPESI), which is an atmospheric-pressure sampling ionization technique. We developed a novel t-SPESI unit that can be used in combination with an inverted fluorescence microscope and basic technologies to extract components from microregions of cells and measure ions with high sensitivity. We performed multimodal (fluorescence, lipid ion, and topographic) imaging of two types of HeLa cells labeled with fluorescent dyes and chemically fixed and showed the potential for subcellular-scale analysis of both cell structure and chemical composition. Furthermore, we evaluated the lipid species by comparing the SC-MSI results with those of supercritical fluid chromatography tandem mass spectrometry. The technical advancement presented here is effective for distinguishing cell types based on the signal intensity of lipid ions in single cells and investigating differences in the subcellular localization of lipids in different types of cells.
    DOI:  https://doi.org/10.1038/s42004-025-01521-2
  15. Talanta. 2025 May 06. pii: S0039-9140(25)00758-1. [Epub ahead of print]295 128268
      Angiotensin I and II (Ang I, Ang II), aldosterone (ALD), 18-hydroxycorticosterone (18-OHB), 18-hydroxycortisol (18-OHF), and 18-oxocortisol (18-OXOF) are critical biomarkers for the screening, confirming, and subtyping of primary aldosteronism (PA). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables simultaneous quantification of multiple analytes; however, obstacles such as labor-intensive manual procedures, complex biological matrices, structural analog interference, and the physicochemical diversity of target analytes limit its application and promotion in clinical laboratories. This study established and validated a robust LC-MS/MS method based on automated magnetic-bead-assisted sequential extraction (MBASE) for the simultaneous detection and quantitation of Ang I, Ang II, ALD, 18-OHB, 18-OHF, and 18-OXOF. The method achieved complete chromatographic resolution with no interference from endogenous hormones or exogenous medications. The analytical recoveries ranged from 86.14 % to 112.89 %, with total imprecisions between 2.93 % and 12.89 %. The MBASE demonstrated a strong correlation with traditional SPE for all six analytes (R: 0.967-0.997), with biases ranging from -4.0 % to 5.1 %. Compared to traditional SPE process, the MBASE workflow reduced total processing time by 46 % and manual handling time by 77 %. The clinical application of this LC-MS/MS method indicated that Ang I and Ang II are effective screening biomarkers, and ALD, 18-OHB, 18-OHF, and 18-OXOF could be used to differentiate PA subtypes. Overall, the newly developed MBASE-LC-MS/MS method exhibits excellent performance and significant advantages for PA diagnosis. It can be a promising tool in the early screening and subtyping for PA and enhance automated mass spectrometry processes in clinical application.
    Keywords:  Liquid chromatography-tandem mass; Magnetic-bead-assisted sequential extraction; Polypeptide; Primary aldosteronism; Screening; Steroid hormone; Subtyping
    DOI:  https://doi.org/10.1016/j.talanta.2025.128268
  16. Methods Mol Biol. 2025 ;2916 109-119
      Extracting and analyzing polar lipids during conifer needle abscission is crucial for comprehending the associated lipid dynamics. A combination of polar and nonpolar solvents for lipid extraction is actively used in lipid analysis during conifer needle abscission. Conifer needle tissue is particularly difficult to work on for several reasons, which must be considered during sample preparation and lipid extraction. Further, conditions like in situ tissue harvest, postharvest tissue harvest, and extraction protocol influence the analysis of target lipids in the plant. The use of electrospray ionization tandem mass spectrometry (ESI-MS/MS) facilitates the simultaneous and thorough analysis of many polar lipids in conifer tissue samples.
    Keywords:  Chloroform:methanol; Conifer; Fatty acid; Galactolipid; Mass spectrometry; Phospholipid; Plant lipids
    DOI:  https://doi.org/10.1007/978-1-0716-4470-6_11
  17. Anal Chem. 2025 May 13.
      Imaging small polar metabolites and analyzing their in vivo dynamics with stable isotope-labeled (SIL) tracing through various biochemical pathways, including the citric acid (TCA) cycle, glycolysis, and amino acid metabolism, have gained substantial interest over the years. However, imaging these small polar metabolites across different tissue types is limited due to their lower ionization efficiencies and ion suppression from larger abundant biomolecules. These challenges can be further exacerbated with SIL studies, which require improvements in sample preparation and method sensitivity. Solvent pretreatments before matrix application on a tissue section have the potential to improve the sensitivity of metabolite imaging; however, they are not yet widely optimized across tissue types. Furthermore, there is a recurring concern about metabolite delocalization from such wash treatments that require "spatial validation". Here, we optimized a simple "basic hexane" wash method that improved sensitivity up to several folds for a broad range of polar and 2H-labeled metabolites across five different mouse organ tissues (kidney, heart, brain, liver, and brown adipose tissue). Notably, we provided region-specific quantification of 51 metabolites using laser microdissection (LMD)-LC-MS/MS to validate their localization observed in MALDI-MSI analysis after the basic hexane wash. Overall, we reported an improved MALDI-MSI sample pretreatment method with a "spatial validation" workflow for sensitive and robust imaging of polar metabolite distributions in mouse organs.
    DOI:  https://doi.org/10.1021/acs.analchem.5c00620
  18. J Pharm Biomed Anal. 2025 May 12. pii: S0731-7085(25)00301-2. [Epub ahead of print]264 116960
      In the present study, endogenous progesterone in plasma was quantified using a validated LC-MS/MS method. This bioanalytical technique was successfully applied to a bioequivalence study in postmenopausal female volunteers. The assay achieved a lower limit of quantification (LLOQ) of 20 pg/mL for endogenous progesterone. Electrospray ionization in positive mode was coupled to a triple-quadrupole mass spectrometer, with deuterated progesterone (progesterone-D9) as the internal standard. Chromatographic separation was performed on a Kinetex Biphenyl column (100 × 4.6 mm, 5 μm) using a time- and flow-gradient program to ensure symmetrical peak shapes and complete resolution from potential interferences. Progesterone was extracted from plasma using methyl tert-butyl ether, affording high recovery and negligible matrix effects. Quantitation was carried out in multiple-reaction monitoring (MRM) mode, monitoring the transitions m/z 315.5 → 97.2 for progesterone and m/z 324.3 → 113.1 for progesterone-D9. The calibration curve was linear over the range of 20.0-40 000.0 pg/mL and was fitted by weighted (1/x²) linear regression. The method fully complies with current regulatory guidelines for bioanalytical assay validation.
    Keywords:  Endogenous Molecule; LC-MS/MS; Progesterone; Stripped plasma; Surrogate Matrix
    DOI:  https://doi.org/10.1016/j.jpba.2025.116960
  19. Int J Mol Sci. 2025 May 02. pii: 4337. [Epub ahead of print]26(9):
      Strigolactones (SLs) are key hormones regulating branching and tillering in rice, impacting plant architecture and yield. A rapid, sensitive, and environmentally friendly method using dispersive liquid-liquid microextraction based on the solidification of floating organic droplets (DLLME-SFO), coupled with ultra-high-performance liquid chromatography and tandem mass spectrometry (UHPLC-MS/MS), has been developed for the determination of three SLs (strigol, orobanchol, and 5-deoxystrigol). The DLLME-SFO method integrates one-step low-temperature extraction and enrichment. The DLLME-SFO conditions were optimized through a single-factor experimental design. Under the best-tested conditions, the developed method exhibited excellent linearity, with the coefficient of determination (R2) values greater than 0.9993. The recoveries ranged from 83% to 96%, with precision values ranging from 4.5% to 12.4%. The limits of detection (LODs) varied from 0.6 to 1.2 pg/g fresh weight, indicating the high sensitivity of the method. Additionally, a novel assay protocol for the quantification of SLs in rice in response to nitrogen and phosphorus stress conditions was applied.
    Keywords:  UHPLC-MS/MS; dispersive liquid–liquid microextraction based on solidification of floating organic droplets (DLLME-SFO); rice; strigolactones
    DOI:  https://doi.org/10.3390/ijms26094337
  20. J Am Soc Mass Spectrom. 2025 May 13.
      Coffee is characterized by a complex chemical matrix that significantly influences its organoleptic properties and market value. This complexity is driven by factors such as botanical species, geographical origin, cultivation conditions, and post-harvest processing methods. Metabolomic studies aim to elucidate how these factors impact the biosynthesis of metabolites that contribute to the sensory qualities of high-quality coffee. Among various analytical techniques, liquid chromatography-mass spectrometry (LC-MS) is particularly effective for separating, identifying, and quantifying these compounds. Most metabolomic studies employ high-resolution mass spectrometry (HRMS) for its superior mass accuracy (<1 ppm), whereas the interpretation of low-resolution data requires additional effort, often relying on literature references and proposed fragmentation mechanisms. In this study, we applied LC-ESI(±)LTQ MSn to comprehensively profile coffee metabolites, identifying 60 compounds, including polar compounds and their isomers such as chlorogenic acids, carbohydrates, amino acids, alkaloids, glycosylated diterpenes, and flavonoids. Fragmentation mechanisms were proposed and discussed. The results demonstrate the effectiveness of LC-ESI(±)LTQ MSn in a detailed metabolomic analysis, providing a robust platform for future research in coffee metabolomics.
    Keywords:  Coffee; Linear ion trap; Liquid chromatography; Mass spectrometry; Tandem MS
    DOI:  https://doi.org/10.1021/jasms.4c00418
  21. Anal Chim Acta. 2025 Jul 08. pii: S0003-2670(25)00492-1. [Epub ahead of print]1358 344098
       BACKGROUND: Mass Spectrometry Imaging (MSI) is a label-free imaging technique used in spatial metabolomics to explore the distribution of various metabolites within biological tissues. Spatial segmentation plays a crucial role in the biochemical interpretation of MSI data, yet the inherent complexity of the data-characterized by large size, high dimensionality, and spectral nonlinearity-poses significant analytical challenges in MSI segmentation. Although deep learning approaches based on convolutional neural networks (CNNs) have shown considerable success in spatial segmentation for biomedical imaging, they often struggle to capture the comprehensive structural information of MSI data.
    RESULTS: We propose SagMSI, an unsupervised graph convolution network (GCN)-based segmentation strategy that combines spatial-aware graph construction of MSI data with a GCN module within a deep neural network. This approach enables flexible, effective, and precise spatial segmentation. We applied SagMSI to both simulated data and various MSI experimental datasets and compared its performance against three commonly used segmentation methods, including t-SNE + k-means, a pipeline implemented by the R package Cardinal, and a CNN-based segmentation method. Visual comparisons with histological images and quantitative evaluations using the silhouette coefficient and adjusted rand index demonstrate that SagMSI excels in segmenting complex tissues, revealing detailed sub-structures, and delineating distinct boundaries of sub-organs with minimal noise interference. The integration of graph-based neural networks with spatially structural information offers deeper insights into spatial omics.
    SIGNIFICANCE: The MSI data is modelled by graph structure so as to incorporate the biomolecular profiling and spatial adjacency within neighboring pixels. The GCN framework generates meaningful pixel representations by learning local and global contextual information through the graph-based structure, thus enabling precise segmentation of MSI. The approach demonstrated high flexibility, robustness to noise, and applicability in exploring complex tissue structures and identifying marker ions associated with microregions.
    Keywords:  Deep neural network; Dimensionality reduction; Graph convolutional network learning; Mass spectrometry imaging; Spatial segmentation
    DOI:  https://doi.org/10.1016/j.aca.2025.344098
  22. Anal Bioanal Chem. 2025 May 12.
      Accurate measurement of human insulin is critical for proper diagnosis, monitoring, and treatment of diabetes. But the insulin results of clinical immunoassay are inconsistent mainly due to antibody cross-reactivity. To standardize and ensure the accuracy of clinical assays, reference measurement procedures (RMPs) with higher metrological order are required. An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) for quantification of human insulin in serum as a candidate reference measurement procedure (cRMP) was developed and validated. Insulin was enriched from human serum by insulin antibodies immobilized on magnetic beads. The eluent was analyzed by ID-LC-MS/MS. The cRMP separated human insulin from potentially interfering compounds and enabled measurement over a range of 0.05-40 ng/g, with no matrix effects and carryover. The limit of detection (LOD) and the limit of quantitation (LOQ) in serum were 24.6 pg/g and 48.8 pg/g, respectively. Imprecision (intra-assay and inter-assay) was <2.77% at 0.436, 2.003, and 11.449 ng/g. Recoveries ranged from 99.5% to 101.7% at three spiked levels. Extraction recovery was measured at 85% or higher. Insulin analogues caused no interference for the determination of endogenous insulin. Expanded measurement uncertainty of target value-assigned samples was ≤3.5%. The cRMP was applied to measure human insulin in serum and was compared with two immunoassays using 46 serum samples. Also, a discrepancy of three candidate reference materials for the calibration of cRMP was discussed.
    Keywords:  Candidate reference measurement procedure (cRMP); Diabetes; Insulin; Isotope dilution-liquid chromatography-tandem mass spectrometry
    DOI:  https://doi.org/10.1007/s00216-025-05900-5
  23. Int J Mol Sci. 2025 May 01. pii: 4304. [Epub ahead of print]26(9):
      Dronedarone (DRO) is an antiarrhythmic drug that should be used under close supervision, and therapeutic drug monitoring (TDM) may be one of the tools supporting pharmacotherapy. The aim of our study was to develop an economical HPLC method for determining DRO and its active metabolite debutyldronedarone (DBD) in human plasma. An HPLC isocratic system with a manual injector was applied. The separation was performed on a Supelcosil LC-CN column (150 × 4.6 mm, 5 µm) at an ambient temperature. The mobile phase was a mixture of CH3OH:CH3CN:H2O:0.5 M KH2PO4 (170:85:237.2:7.8 (v/v)) + 0.1 mL 85% H3PO4 pumped at a flow rate of 1.8 mL/min. The UV detection was set at λ = 290 nm. A methyl tert-butyl ether was used for the extraction from a 0.4 mL alkalized plasma sample. The analytes were eluted at retention times of 4.0 min, 5.2 min and 6.0 min for DBD, internal standard bepridil and DRO, respectively. The method was calibrated in the range of 10-1000 ng/mL for both DRO and DBD. The adequate specificity, accuracy and precision were demonstrated in accordance with EMA guidelines, i.e., ≤15% (≤20% for the LLOQ), which ensures the reliability of the measurements. This method can be recommended for laboratories with basic HPLC equipment for TDM, adherence assessments and even in PK studies during chronic DRO therapy.
    Keywords:  HPLC-UV; bioanalysis; debutyldronedarone; dronedarone
    DOI:  https://doi.org/10.3390/ijms26094304
  24. Molecules. 2025 Apr 25. pii: 1906. [Epub ahead of print]30(9):
      Chromatography-mass spectrometry typically requires a time-consuming and costly pretreatment to detect illegal additives in cosmetics. Retinoic acid is classified as a prohibited additive in cosmetics by the European Union and China. Therefore, a rapid and convenient method is needed for its detection. In this study, a method for detecting retinoic acid using Reactive Paper Spray Ionization Mass Spectrometry was developed. N'-dimethylpiperazine was used as the derivatization reagent due to its ability to react with carboxyl functional groups at room temperature. Our results indicate that the derivatized retinoic acid compounds obtained using this method exhibited good linearity within the range of 0.0005~0.1 μg·mL-1, achieving a limit of detection of 0.107 ng·mL-1.
    Keywords:  chemical derivatization; cosmetics; mass spectrometry; retinoic acid
    DOI:  https://doi.org/10.3390/molecules30091906
  25. Anal Chem. 2025 May 13.
      Trapped Ion Mobility Spectrometry (TIMS) has demonstrated promising potential as a powerful discriminating method when coupled with mass spectrometry, enhancing the precision of feature annotation. Such a technique is particularly valuable for lipids, where a large number of isobaric but structurally distinct molecular species often coexist within the same sample matrix. In this study, we explored the potential of ion mobility for ether lipid isomer differentiation. Mammalian ether phospholipids are characterized by a fatty alcohol residue at the sn-1 position of their glycerol backbone. They can make up to 20% of the total phospholipid mass and are present in a broad range of tissues. There they are, for example, crucial for nervous system function, membrane homeostasis, and inter- as well as intracellular signaling. Molecular ether lipid species are difficult to distinguish analytically, as they occur as 1-O-alkyl and 1-O-alkenyl subclasses, with the latter being also known as plasmalogens. Isomeric ether lipid pairs can be separated with reversed-phase chromatography. However, their precise identification remains challenging due to the lack of clear internal reference points, inherent to the nature of lipid profiles and the lack of sufficient commercially available standard substances. Here, we demonstrate─with focus on phosphatidylethanolamines─that ion mobility measurements allow to discriminate between the ether lipid subclasses through distinct differences in their gas phase geometries. This approach offers significant advantages as it does not depend on potential retention time differences between different chromatographic systems. However, the current resolution in the ion mobility dimension is not sufficient to baseline separate 1-O-alkyl and 1-O-alkenyl isobars, and the observed differences are not yet accurately represented in existing collision cross section databases. Despite these challenges, the predictable properties of the ion mobility behavior of ether lipid species can significantly support their accurate annotation and hold promise for future advancements in lipid research.
    DOI:  https://doi.org/10.1021/acs.analchem.4c06617
  26. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 02. pii: S1570-0232(25)00187-4. [Epub ahead of print]1260 124633
      This study established two methods for detecting flunixin meglumine (FM) in pork. The MSPE-LC-MS/MS method utilizes the accuracy and quantifiability of liquid chromatography-tandem mass spectrometry. FM in pork can be accurately analyzed after optimizing the pretreatment, liquid phase conditions, and mass spectrometry parameters. It shows a good linear relationship (correlation coefficient > 0.999), with a detection limit of 0.2 μg/kg, limit of quantification of 0.6 μg/kg, spiked recovery rates of 90.6 % - 97.4 %, matrix effects of 96.7 % - 107.2 %, intra-day precision of 1.4 % - 5.6 %, and inter-day precision of 4.3 % - 11.5 %. The RP-MSPE-CGIA method combines the separation performance of reversed magnetic solid-phase extraction and the rapidness of colloidal gold immunochromatography. By optimizing conditions, it successfully extends the dairy product detection method to pork. It is easy to operate, has a detection limit of 5 μg/kg, and detection time less than half of MSPE - LC-MS/MS. Experiments show that the two methods complement each other in advantages and can meet the detection requirements of FM in pork in different scenarios.
    Keywords:  Colloidal gold immunochromatography; Flunixin meglumine; Magnetic solid phase extraction (MSPE); Pork; Ultra performance liquid chromatography-quadrupole-exactive orbitrap high-resolution mass spectrometry
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124633
  27. Gigascience. 2025 Jan 06. pii: giaf035. [Epub ahead of print]14
       BACKGROUND: Truly understanding the cancer biology of heterogeneous tumors in precision medicine requires capturing the complexities of multiple omics levels and the spatial heterogeneity of cancer tissue. Techniques like mass spectrometry imaging (MSI) and spatial transcriptomics (ST) achieve this by spatially detecting metabolites and RNA but are often applied to serial sections. To fully leverage the advantage of such multi-omics data, the individual measurements need to be integrated into 1 dataset.
    RESULTS: We present the Multi-Omics Imaging Integration Toolset (MIIT), a Python framework for integrating spatially resolved multi-omics data. A key component of MIIT's integration is the registration of serial sections for which we developed a nonrigid registration algorithm, GreedyFHist. We validated GreedyFHist on 244 images from fresh-frozen serial sections, achieving state-of-the-art performance. As a proof of concept, we used MIIT to integrate ST and MSI data from prostate tissue samples and assessed the correlation of a gene signature for citrate-spermine secretion derived from ST with metabolic measurements from MSI.
    CONCLUSION: MIIT is a highly accurate, customizable, open-source framework for integrating spatial omics technologies performed on different serial sections.
    Keywords:  image registration; mass spectrometry imaging; spatial transcriptomics
    DOI:  https://doi.org/10.1093/gigascience/giaf035
  28. J Sep Sci. 2025 May;48(5): e70155
      The liquid chromatography / mass spectrometry (LC/MS) quantification of per- and polyfluoroalkyl substances (PFAS) in complex matrices presents challenges. Despite advanced sample preparation techniques to isolate analytes, residual matrix components can still interfere with MS detection. Additionally, extracts often contain high levels of organic solvents limiting the volume that can be injected when using reversed-phase HPLC. Yet, increasing the injection volume could be a simple path to lower detection limits. Feeding PFAS samples, dissolved in pure acetonitrile, at controlled speed into the stream of mobile phase resulted in an analyte focusing effect on the column. By using this approach, the injection volume could be increased 10 times compared to the conventional injection mode, as did the detection sensitivity. Feed injection was coupled to two-dimensional liquid chromatography (2D-LC) in a configuration in which the same triple quadrupole MS could be used for detection in both separation dimensions. The improved separation markedly reduced ion suppression effects and allowed to distinguish the critical PFAS compound perfluorobutanoic acid (PFBA) from isomeric matrix components within tomato extracts interfering with its quantification when only LC/MS was used. This 2D-LC/MS approach therefore fulfilled recommendations that PFBA should be analyzed using a secondary LC approach, without adding a marked amount of time to the analysis.
    Keywords:  feed injection; perfluorobutanoic acid (PFBA); solvent incompatibility; two‐dimensional liquid chromatography (2D‐LC)
    DOI:  https://doi.org/10.1002/jssc.70155
  29. Anal Chim Acta. 2025 Jul 08. pii: S0003-2670(25)00490-8. [Epub ahead of print]1358 344096
       BACKGROUND: Bolting severely affects the medicinal value and yield of Saposhnikovia divaricata. Currently, spatial distribution of secondary metabolites in bolted and unbolted Saposhnikovia divaricata remains unknown. The commercial Mass spectrometry imaging (MSI) platforms has high sensitivity to high-polar compounds, but suppresses signals of less polar compounds, generating a gap of understanding how spatial distributed of such metabolites that is less prone to ionize.
    RESULTS: This study developed a highly sensitive Laser Ablation Carbon Fiber Ionization (LACFI) MSI method under atmospheric pressure to achieve the spatial distribution analysis of secondary metabolites in Saposhnikovia divaricata, including low-polar compounds which is difficult to be analyzed in other MSI methods. The laser efficiently desorbs compounds from Saposhnikovia divaricata tissue, and the desorbed compounds are rapidly ionized by a carbon fiber ion source under high pressure. The desorption and ionization are performed in two separate steps, reducing the matrix effect and enhancing the ionization efficiency of compounds, which increase sensitivity. The carbon fiber has good compatibility with polar and low-polar compounds, which increases the number of detectable metabolites. The spatial distribution of secondary metabolites undergoes a shift from the unbolted to the bolted Saposhnikovia divaricata. The result of imaging is consistent with the conclusion of pharmacopoeia that the bolted Saposhnikovia divaricata is not used medicinally.
    SIGNIFICANCE: Compared to commercial imaging methods, the LACFI-MSI method developed is ultra-sensitive, has a greater sensitivity in the analysis of less polar compounds. Spatial distribution analysis of secondary metabolites that previously difficult/unable to detect in bolted and unbolted Saposhnikovia divaricata is realized.
    Keywords:  Bolting; High sensitivity; LACFI-MSI; Saposhnikovia divaricata; Secondary metabolites; Spatial distribution
    DOI:  https://doi.org/10.1016/j.aca.2025.344096
  30. Clin Biochem. 2025 May 13. pii: S0009-9120(25)00078-5. [Epub ahead of print] 110949
       OBJECTIVE: Cancer cachexia is characterized by weight loss, muscle mass loss, and reduced food intake. Anamorelin is a ghrelin receptor agonist approved for the treatment of cancer cachexia. In this study, we established and validated an assay for quantification of anamorelin in human plasma.
    METHODS: For quantification of anamorelin, samples were pretreated with solid-phase extraction and analyzed by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). This analytical method was validated in accordance with the Food and Drug Administration (FDA) bioanalytical method validation guidance. We used the established assay to quantify plasma anamorelin concentrations in five patients with cancer cachexia treated with anamorelin.
    RESULTS: The validation results of this assay method met the acceptance criteria recommended by the FDA guidance. Within-batch and batch-to-batch precision at the lower limit of quantification and three quality control levels were within 6.20 % and 6.55 % coefficient of variation, respectively. Within-batch and batch-to-batch accuracies ranged from -2.58 to -1.33 % and -3.78 to -1.69 %, respectively. Recovery rates and matrix effects corrected by internal standard were 82.7-84.2 % and 102.7-104.6 %, respectively. Using the established assay with a calibration range of 0.1-2500 ng/mL, plasma anamorelin concentrations were successfully quantified in all 15 plasma samples from 5 patients with cancer cachexia.
    CONCLUSIONS: We established and validated a method to measure plasma anamorelin concentrations using UHPLC/MS-MS combined with SPE, and successfully applied the novel method to measure plasma anamorelin concentrations in patients with cancer cachexia. By measuring plasma anamorelin concentrations in large scale studies, the established quantitative method is expected to contribute to the pharmacokinetic study of anamorelin.
    Keywords:  Anamorelin; Cachexia; Solid-phase extraction; UHPLC-MS/MS
    DOI:  https://doi.org/10.1016/j.clinbiochem.2025.110949
  31. Analyst. 2025 May 15.
      The use of rapid mass spectrometry techniques, such as atmospheric-solids-analysis-probe mass spectrometry (ASAP-MS), in the analysis of metabolite patterns in clinical samples holds significant promise for developing new diagnostic tools and enabling rapid disease screening. The rapid measurement times, ease of use, and relatively low cost of ASAP-MS makes it an appealing option for use in clinical settings. However, despite the potential of such approaches, a number of important experimental considerations are often overlooked. As well as instrument-specific choices and settings, these include the treatment of background noise and/or contaminant peaks in the mass spectra, and the influence of consumables, different users, and batch effects more generally. The present study assesses the impact of these various factors on measurement accuracy and reproducibility, using human brain and cerebrospinal fluid samples as examples. Based on our results, we make a series of recommendations relating to optimisation of measurement and cleaning protocols, consumable selection, and batch effect detection and correction, in order to optimise the reliability and reproducibility of ASAP-MS measurements in clinical settings.
    DOI:  https://doi.org/10.1039/d5an00166h
  32. Anal Chim Acta. 2025 Jul 08. pii: S0003-2670(25)00494-5. [Epub ahead of print]1358 344100
       BACKGROUND: A reliable analysis of pesticide residues in fruits and vegetables is crucial for food safety and regulatory compliance. Traditionally, analytical-flow liquid chromatography coupled to mass spectrometry (LC-MS/MS) has been the gold standard but, shifting to low-flow LC-MS/MS presents an improved alternative that enhances sensitivity, reduces the injected amount of sample and aligns with the principles of green analytical chemistry. Operating at micro-flow rates significantly reduces solvent consumption and organic waste, which is particularly important for routine control laboratories. This approach maintains high sensitivity, robustness, and performance, addressing the critical need for greener, efficient, and reliable methodologies for multiresidue pesticide analysis.
    RESULTS: This study assessed the performance of a micro-flow LC system operating at a flow rate of 50 μL min-1, coupled to a triple quadrupole mass spectrometer, for the multiresidue analysis of 257 pesticides in tomato and orange matrices, in compliance with the ISO 17025. The method showed excellent retention time stability, with deviations under 2.1 s across 50 injections. Peak area repeatability was robust, with relative standard deviations of 3.4 % for tomato and 2.9 % for orange. The instrumental limits of quantification demonstrated high sensitivity, with 89 % of pesticides identified at very low concentrations (0.001-0.002 mg kg-1), outperforming the traditional analytical-flow LC-MS/MS method, which identified 75-81 % of compounds. Matrix effects were minimal, with less than 20 % signal suppression or enhancement for most compounds. The method was successfully tested through the analysis of proficiency tests samples, and the analysis of 39 real samples proved its applicability for routine food monitoring.
    SIGNIFICANCE: This work establishes micro-flow LC-MS/MS as an innovative method for multiresidue pesticide analysis in fruits and vegetables, combining high analytical performance with significant sustainability advantages, reducing solvent consumption by over fivefold compared to the conventional analytical-flow method. These benefits underscore micro-flow LC-MS/MS as a promising, efficient, and environmentally friendly option for multiresidue pesticide analysis, positioning it as a valuable tool for broader adoption in food safety analysis.
    Keywords:  Mass spectrometry; Micro-flow liquid chromatography; Pesticide residues
    DOI:  https://doi.org/10.1016/j.aca.2025.344100
  33. Molecules. 2025 May 03. pii: 2039. [Epub ahead of print]30(9):
      Albendazole is an anthelmintic drug commonly used in animals and humans against nematodes. A sensitive, accurate, precise, and time-saving high-performance liquid chromatography (HPLC) method for the simultaneous determination of albendazole and metabolites (albendazole sulfoxide and albendazole sulfone) in cattle plasma has been developed and validated. A solid-phase extraction (SPE) was carried out. Separation was performed with an XBridge® C18 column (4.6 mm × 250 mm, 5 µm) with gradient elution of acetonitrile:ammonium acetate buffer 0.025 M with pH adjusted to 6.6. The flow rate was 1.2 mL/min, and the PDA detector was set at 292 nm. Calibration curves were linear in the range from 0.025 to 2.0 µg/mL for the three compounds evaluated, with correlation coefficients ≥ 0.99. For the lower limit of quantification (LLOQ), within- and between-run precision and accuracy were satisfactory, with coefficients of variation (CV) ≤ 15.1% and deviations ≤ 117.7%, respectively. The method fulfilled all validation criteria established by the European Medicines Agency guideline (EMA/CHMP/ICH/172948/2019).
    Keywords:  EMA guideline; HPLC; albendazole; albendazole sulfone; albendazole sulfoxide; validation
    DOI:  https://doi.org/10.3390/molecules30092039
  34. Anal Chem. 2025 May 12.
      Mass spectrometry imaging is a developing technique that maps the molecular composition of samples in a label-free manner. However, highly heterogeneous samples, including bones, usually cannot be easily analyzed due to challenging sample preparation, particularly in minimizing cracks and maintaining flatness. To comprehensively address these issues, we developed a sample preparation method for the fresh frozen heterogeneous samples such as knee joint and skull of murine, which includes complex structures and tissue types, such as neuronal tissue, peripheral nerve, muscle, tracks, connective tissue, cartilage, mineralized bone, and bone marrow. By controlling the sample thickness and employing an optimized drying method, we achieved minimal cracking. We found that a combination of lyophilization and 5 μm section thickness, when attached to a cryofilm, was readily achievable and significantly reduced cracking in bone tissue. Additionally, we implemented a contactless spin-flattening technique to ensure surface uniformity. Centrifuging the section at 7000g improved surface flatness, bringing the height variation within the range typically observed in soft tissues while also removing excess mounting medium and bubbles. This approach enhances the sample quality and reliability without requiring complex manual skills, making it more practical and reproducible for routine analysis. High molecular coverage was achieved, including small metabolites, metals, and lipids, by using the N-(1-naphthyl) ethylenediamine dihydrochloride (NEDC) matrix. To further explore the potential of our workflow, high-resolution MSI was performed on rat tibial growth plates at different growth stages. Numbers of N-acetyl disaccharide sulfate and PE (34:1) are found to be complementary expressed in growth plate cartilage and have different intensities at different growth stages. Our findings suggested the potential involvement of those metabolites in bone development. By addressing the challenges of sample preparation, including surface flatness, bubbles, and severe cracking, our approach significantly improves the quality of the MS imaging. Additionally, this method offers a broad detection range that encompasses both metal ions and metabolites. This advancement enables detailed and accurate molecular characterization of rigid biological samples, enhancing the potential for applications in biomedical research.
    DOI:  https://doi.org/10.1021/acs.analchem.4c05410
  35. J Sep Sci. 2025 May;48(5): e70173
      Risdiplam is a new therapeutic agent developed to treat spinal muscular atrophy (SMA), a genetic neurodegenerative disease caused by mutations in the SMN1 gene. Unlike previous invasive therapies, risdiplam offers the advantage of oral administration, significantly improving patient comfort and accessibility. The review provides information on an SMA historical overview, breakthrough therapies of the development, and design of the methods used to treat SMA. We then focus on its structure and physicochemical properties. The analysis of risdiplam concentrates on developing improved analytical methods for the precise quantification of risdiplam and its metabolites by high-performance liquid chromatography with mass spectrometry in biological samples using octadecyl stationary phases. For sample preparation, only the protein precipitation method was used. Challenges associated with the risdiplam analytics include developing a highly sensitive and selective method in biological matrices and dealing with potential interferences from the biological matrix. Future research should focus on improving analytical methods, investigating metabolite activity, and expanding our knowledge of its long-term effects.
    Keywords:  liquid chromatography; mass spectrometry; protein precipitation; risdiplam; spinal muscular atrophy
    DOI:  https://doi.org/10.1002/jssc.70173
  36. Analyst. 2025 May 13.
      Encoding abstract information in chemical mixtures uses the selective presence or absence of specific analytes, creating a binary-based framework for data storage. Data storage capacity (C in bits) can be maximized by encoding with large analyte libraries (M) at distinguishable concentration levels (L), where C = M·log2L. However, roboust decoding of such complex libraries remains challenging for practical applications. This study introduces hyphenated mass spectrometry (MS) methods, liquid chromatography (LC) and flow injection analysis (FIA) that meet the dual requirements of high analyte coverage and precise quantitation to maximize data storage capacity. Encoding and decoding use plant metabolite libraries to create specific mixtures. Using LC-MS, it is feasible to encode and decode up to 200 bits per mixture, with scalability reaching 103-104 bits at the cost of low decoding rates (ca. 0.5 bits per sec). FIA-MS offers a high-throughput alternative, handling 100 bits at faster rates (ca. 3 bits per sec). The data storage capacity can be three-fold expanded by incorporating up to eight quantitation levels, supporting binary, quaternary, or octal encoding schemes. To demonstrate the practical application of these methods, we encode and decode various digital file formats such as texts and multicolor images.
    DOI:  https://doi.org/10.1039/d5an00353a
  37. Anal Chem. 2025 May 12.
      Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) allows spatial molecular profiling. Despite many successful applications, an appropriate control of technical variations is still lacking for result reproducibility assessment and for maximizing the MSI data quality. To address this, we introduce a novel quality control standard (QCS) design and data analysis pipeline accounting for variability due to sample preparation and instrument performance. Firstly, we created a tissue mimicking QCS consisting of propranolol in a gelatin matrix. We showed that this QCS mimics ion suppression of propranolol in the tissue. Next, a three-day batch experiment demonstrated the QCS's performance to longitudinal technical variations, establishing it as an effective indicator of batch effects. Then three computational approaches for batch effect correction were applied for the first time to MALDI-MSI data, leading to a significant reduction of QCS variation and to improved sample clustering by using multivariate principal component analysis. Altogether, we offer the designed QCS in combination with a data correction pipeline for MALDI-MSI users for batch effect evaluation and correction.
    DOI:  https://doi.org/10.1021/acs.analchem.5c02020
  38. Proteomics. 2025 May 12. e202400378
      Mass spectrometry has long been utilized to characterize a variety of biomolecules such as proteins, metabolites, and lipids. Most MS-based omics studies rely on bulk analysis; however, bulk approaches often overlook low-abundance molecules that may exert critical biological effects. Recently, multi-omics analyses have been driving an explosion of knowledge about how biomolecules interact within biological systems. In particular, spatial multi-omics has emerged as a groundbreaking approach for implementing multi-omic and multi-modal analyses. Broadly defined, spatial omics has the ability to analyze biomolecules within their native spatial contexts, offering transformative insights. This review focuses on mass spectrometry-based spatial omics, specifically matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We will explore how MALDI-MSI, in combination with laser capture microdissection (LCM) and traditional liquid chromatography-mass spectrometry (LC-MS) workflow, is advancing spatially resolved multi-omics research.
    Keywords:  LCM‐LC‐MS/MS; MALDI‐MSI; spatial multi‐omics
    DOI:  https://doi.org/10.1002/pmic.202400378
  39. J Sep Sci. 2025 May;48(5): e70170
      A modified QuEChERS method coupled with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to detect nine aromatic amines in mainstream cigarette smoke simultaneously. In this study, a Cambridge filter pad, impregnated with a hydrochloric acid-ethanol solution, was used to trap aromatic amines in vapor and particulate phases in mainstream cigarette smoke. The collection efficiency of aromatic amines for this method was significantly improved compared with conventional methods of trapping aromatic amines using a combination of Cambridge filter pad followed by impinger traps containing dilute acid solution. Utilizing primary secondary amine and C18 for purification effectively removed impurities of different polarities from smoke samples with reduced matrix effect. The limits of detection for the nine aromatic amines were within the range of 0.02-0.10 ng/cig, recoveries were within the range of 86.1%-113.7% and the intraday and interday precisions were both lower than 14%. A collaborative study was conducted involving 10 laboratories to validate developed method for the determination of nine aromatic amines in mainstream cigarette smoke. Statistical evaluation of the results gave good precision results, with an average repeatability relative standard deviation of 5.90%-35.38% and an average reproducibility relative standard deviation of 9.55%-75.72%. As a part of the validation procedure, the developed method was applied to evaluate aromatic amines yields for 17 commercially available cigarettes in China. Compared to the existing methods, this analytical approach affords high sensitivity and efficiency, improved accuracy, and lower cost.
    Keywords:  QuEChERS; aromatic amine; collaborative study; high performance liquid chromatography–tandem mass spectrometry; mainstream cigarette smoke
    DOI:  https://doi.org/10.1002/jssc.70170
  40. Nat Methods. 2025 May 12.
      Despite being information rich, the vast majority of untargeted mass spectrometry data are underutilized; most analytes are not used for downstream interpretation or reanalysis after publication. The inability to dive into these rich raw mass spectrometry datasets is due to the limited flexibility and scalability of existing software tools. Here we introduce a new language, the Mass Spectrometry Query Language (MassQL), and an accompanying software ecosystem that addresses these issues by enabling the community to directly query mass spectrometry data with an expressive set of user-defined mass spectrometry patterns. Illustrated by real-world examples, MassQL provides a data-driven definition of chemical diversity by enabling the reanalysis of all public untargeted metabolomics data, empowering scientists across many disciplines to make new discoveries. MassQL has been widely implemented in multiple open-source and commercial mass spectrometry analysis tools, which enhances the ability, interoperability and reproducibility of mining of mass spectrometry data for the research community.
    DOI:  https://doi.org/10.1038/s41592-025-02660-z
  41. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 May 09. pii: S1570-0232(25)00199-0. [Epub ahead of print]1261 124645
      Tacrolimus is a potent macrolide immunosuppressant widely used in pediatric liver transplant patients. However, its narrow therapeutic window and significant inter-individual pharmacokinetic differences result in a poor correlation between blood concentrations and administered doses. To support its individualized dose, a sensitive, fast and robust ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to measure tacrolimus concentrations in whole blood of pediatric liver transplant patients. The method employed acetonitrile for protein precipitation, and sample separation was achieved using an Acquity UPLC CSH C18 column (2.1 × 50 mm, 1.7 μm) with gradient elution. The mobile phase consisted of ammonium solution-water (0.5:1000, v/v) and methanol. The method demonstrated good linearity within a concentration range of 0.20-50.00 ng/mL. The intra- and inter-day precisions of tacrolimus in whole blood were less than 13.5 %, with the accuracy ranged from -7.2 % to 13.0 %. Selectivity, carryover, matrix effects, recovery, dilution reliability, and stability all met the standards set by relevant guidelines. The established UPLC-MS/MS method was successfully applied to measure the concentration of tacrolimus in the whole blood of pediatric liver transplant patients and support its individualized dose. Under the rapid UPLC-MS/MS method, 25 (65.8 %) patients required a dose increase, 3 (7.9 %) patients needed a dose reduction, and 10 (26.3 %) patients maintained their original dosage without adjustment.
    Keywords:  Pediatric liver transplantation; Tacrolimus; UPLC–MS/MS
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124645
  42. Biomed Chromatogr. 2025 Jun;39(6): e70110
      This study analyses the nutritional and medicinal properties of Moringa oleifera leaves from sub-Saharan Africa using HPLC-PDA-ESI-MS. A method for simultaneous polyphenol quantification was developed to understand how different habitats influence the quality and polyphenolic profile of M. oleifera. The study specifically aimed to analyze the polyphenolic profile of phenolic compounds extracted from M. oleifera leaves from the Tabelbala region in Bechar, Algeria. The extract's complete polyphenolic profile was determined using liquid chromatography, photodiode array, and mass spectrometry detection via an electrospray ionization interface. A total of 16 compounds were identified, with variations observed between different extracts. The most abundant among these were quercetin-3-O-glucoside (964.43 μg/g dry matter), kaempferol (839.71 μg/g dry matter), and rutin (835.51 μg/g dry matter). The acetonic extract was the only source of gallic acid, which was measured at 496.14 μg/g dry matter. It provides a database for qualitative assessments and clinical applications of M. oleifera, laying the groundwork for future germplasm selection and development research. Quantitative analysis methodology can be applied to quality assessment protocols. Findings show compounds with low gastrointestinal absorption and skin permeability prevent CYP-related medication interactions, but poor bioavailability and efflux transport capabilities limit their therapeutic potential, necessitating formulation strategies.
    Keywords:   Moringa oleifera ; ADMET; HPLC‐PDA‐ESI‐MS; Tabelbala; polyphenols
    DOI:  https://doi.org/10.1002/bmc.70110
  43. Clin Lab Med. 2025 Jun;pii: S0272-2712(25)00004-6. [Epub ahead of print]45(2): 177-186
      The current overdose crisis in the United States is a polydrug epidemic. Old drug testing strategies no longer keep up with the rapidly changing drug supply and patterns of use. Untargeted mass spectrometry is the testing approach most likely to detect novel psychoactive substances, active cuts in the drug supply, and any polypharmacy that may be complicating patient presentations. High-resolution mass spectrometry platforms offer untargeted data acquisition and different modes of data analysis, depending on the clinical applications. While they are currently costly and require skilled/trained personnel, more resources are becoming available to increase their accessibility to more laboratories.
    Keywords:  Broad spectrum drug screening; Clinical toxicology testing; High-resolution mass spectrometry; Untargeted mass spectrometry
    DOI:  https://doi.org/10.1016/j.cll.2025.01.003
  44. Biomed Chromatogr. 2025 Jun;39(6): e70105
      Viscum coloratum (Kom.) Nakai has been demonstrated to be an effective treatment for rheumatoid arthritis (RA), but the pharmacodynamic substances are still unclear. In this study, a four-step strategy integrated nontargeted metabolomics, multivariate statistical analysis, and UNIFI software. An ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) method was employed to characterize 56 flavonoids from V. coloratum, 25 prototypes and 133 metabolites in biological samples of rats following oral administration of V. coloratum. The endogenous interference peaks in plasma, urine, and feces were reduced by 83.79%, 91.60%, and 86.02%, respectively, through the application of nontargeted metabolomics approaches. The distinctions and commonalities in the flavonoid metabolic pathways of V. coloratum under normal and RA conditions were summarized. Phase II metabolism was significantly affected in the RA rats, especially the prototype exposure and its metabolites in plasma and excreted by urine and feces. Utilizing the aforementioned methods, we identified 109 differential metabolites, including 17 RA-specific metabolites. Twenty-two flavonoid prototypes and their metabolites were identified as potential pharmacodynamic substances in plasma. All the information gained from this study will significantly contribute to elucidating the potential biological and pharmacological mechanisms of flavonoids in V. coloratum, thereby opening new avenues for drug development.
    Keywords:   Viscum coloratum ; UHPLC‐Q‐TOF‐MS; metabolite identification; nontargeted metabolomics; rheumatoid arthritis
    DOI:  https://doi.org/10.1002/bmc.70105
  45. Anal Methods. 2025 May 16.
      Residues of pharmaceuticals and personal care products (PPCPs) in drinking water pose significant public health risks. To assess these residues in water, this study developed a solid-phase extraction combined with ultra-performance liquid chromatography-tandem mass spectrometry method (SPE-UPLC-MS/MS) for the simultaneous quantitative determination of 98 PPCP residues in drinking water. By optimizing the pretreatment method and analysis conditions, the method exhibits excellent linearity, with all PPCPs showing correlation coefficients (R2) greater than 0.99 within the concentration range of 0-100 μg L-1. Recovery rates ranged from 60.7% to 119.0%, and the relative standard deviations (RSDs) were below 20%. The limits of detection (LODs) were between 0.034 and 4.001 ng L-1. Residues such as amisulpride and 1,7-dimethylxanthine were detected in water samples. This method is simple, sensitive, and suitable for high-throughput detection of trace PPCPs in drinking water, providing reliable technical support for water quality monitoring.
    DOI:  https://doi.org/10.1039/d5ay00307e
  46. Anal Chem. 2025 May 12.
      The development of gene therapy techniques introduces a potential risk of gene doping, which threatens the integrity of sport. In response to this challenge, we have developed a novel analytical method that employs a multiplex polymerase chain reaction (PCR) in conjunction with liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) for the simultaneous identification of multiple transgenes in equine plasma within a single reaction. The method targets three potential doping transgenes: equine growth hormone 1 (eGH1), equine growth hormone-releasing hormone (eGHRH), and equine interleukin 10 (eIL10), along with an internal control. The artificial internal control (AIC) has been developed to be coextracted, coamplified, and codetected with the transgenes, thereby reducing the risk of false negatives. We proposed design and optimization strategies for the multiplex PCR-LC-MS method to attain high sensitivity, with estimated limits of detection at 25 copies/mL and estimated limits of confirmation at 50 copies/mL for all targets. The method has been validated and successfully applied for the confirmation of the eIL10 transgene in plasma samples from horses administered with the transgene. The utilization of LC-HRMS/MS enabled unequivocal identification of transgenes and provided the flexibility to include multiple doping transgenes. This simple setup offers an alternative approach that enables sensitive and reliable multiplex detection of transgenes in the majority of doping control laboratories.
    DOI:  https://doi.org/10.1021/acs.analchem.5c00770
  47. Molecules. 2025 Apr 24. pii: 1900. [Epub ahead of print]30(9):
      Anxiety and stress-related disorders affect all ages in all geographical areas. As high anxiety and chronic stress result in the modulation of mitochondrial pathways, intensive research is being carried out on pharmaceutical interventions that alleviate pertinent symptomatology. Therefore, innovative approaches being currently pursued include substances that target mitochondria bearing an antioxidant moiety. In this study, a newly synthesized antioxidant consisting of triphenylphosphine (TPP), a six-carbon alkyl spacer, and hydroxytyrosol (HT) was administered orally to mice via drinking water. Cerebellum and liver samples were collected and analyzed using ultra-high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-MS/MS) to assess the levels of TPP-HT in the respective tissues to evaluate in vivo administration efficacy. Sample preparation included extraction with appropriate solvents and a preconcentration step to achieve the required sensitivity. Both methods were validated in terms of selectivity, linearity, accuracy, and limits of detection and quantification. Additionally, a workflow for evaluating and statistically summarizing multiple fortified calibration curves was devised. TPP-HT penetrates the blood-brain barrier (BBB), with a level of 11.5 ng g-1 quantified in the cerebellum, whereas a level of 4.8 ng g-1 was detected in the liver, highlighting the plausibility of orally administering TPP-HT to achieve mitochondrial targeting.
    Keywords:  LC-MS/MS; anxiety; cerebellum; chemometrics; liver; mitochondria; triphenylphospine-hydroxytyrosol (TPP-HT)
    DOI:  https://doi.org/10.3390/molecules30091900
  48. Metabolomics. 2025 May 10. 21(3): 66
       BACKGROUND: Metabolic profiling of blood metabolites, particularly in plasma and serum, is vital for studying human diseases, human conditions, drug interventions and toxicology. The clinical significance of blood arises from its close ties to all human cells and facile accessibility. However, patient-specific variables such as age, sex, diet, lifestyle and health status, along with pre-analytical conditions (sample handling, storage, etc.), can significantly affect metabolomic measurements in whole blood, plasma, or serum studies. These factors, referred to as confounders, must be mitigated to reveal genuine metabolic changes due to illness or intervention onset.
    REVIEW OBJECTIVE: This review aims to aid metabolomics researchers in collecting reliable, standardized datasets for NMR-based blood (whole/serum/plasma) metabolomics. The goal is to reduce the impact of confounding factors and enhance inter-laboratory comparability, enabling more meaningful outcomes in metabolomics studies.
    KEY CONCEPTS: This review outlines the main factors affecting blood metabolite levels and offers practical suggestions for what to measure and expect, how to mitigate confounding factors, how to properly prepare, handle and store blood, plasma and serum biosamples and how to report data in targeted NMR-based metabolomics studies of blood, plasma and serum.
    Keywords:  Blood; Metabolites; Metabolomics; NMR; Plasma; Serum; Standardization
    DOI:  https://doi.org/10.1007/s11306-025-02259-7
  49. Methods Mol Biol. 2025 ;2916 25-38
      Jasmonates are a group of plant signaling compounds that control defense mechanisms and modulate growth and development in response to environmental challenges. The protocol in this chapter describes a simple and rapid quantification of the most commonly studied jasmonate, jasmonic acid (JA), and also both its precursor 12-oxo-phytodienoic acid (OPDA) and JA-isoleucine (JA-Ile) the bioactive form, in the abscission zone (AZ) and the mesocarp tissue of oil palm fruits. These tissues are lipid-rich and difficult to analyze because of high levels of triglycerides (TAGs). When TAGs are extracted by apolar solvents, the column and analytical system can be clogged, and the jasmonate quantification is perturbed. In order to determine jasmonate distribution in lipid-rich tissues, we present a protocol to eliminate TAGs to successfully extract and quantify jasmonates, including 12-oxo-phytodienoic acid (OPDA), the most apolar compound, and JA-Ile, the form that binds to the JA co-receptor complex and regulates responses through JA signaling. The protocol is used with the objective of understanding the function of jasmonates in the AZ of ripe oil palm fruit during abscission.
    Keywords:  12-Oxo-phytodienoic acid (OPDA); Jasmonic acid; Lipids; MRM; Organ abscission; UPLC-MS
    DOI:  https://doi.org/10.1007/978-1-0716-4470-6_3
  50. Nat Commun. 2025 May 12. 16(1): 4373
      Metabolites, lipids, and glycans are fundamental but interconnected classes of biomolecules that form the basis of the metabolic network. These molecules are dynamically channeled through multiple pathways that govern cellular physiology and pathology. Here, we present a framework for the simultaneous spatial analysis of the metabolome, lipidome, and glycome from a single tissue section using mass spectrometry imaging. This workflow integrates a computational platform, the Spatial Augmented Multiomics Interface (Sami), which enables multiomics integration, high-dimensional clustering, spatial anatomical mapping of matched molecular features, and metabolic pathway enrichment. To demonstrate the utility of this approach, we applied Sami to evaluate metabolic diversity across distinct brain regions and to compare wild-type and Ps19 Alzheimer's disease (AD) mouse models. Our findings reveal region-specific metabolic demands in the normal brain and highlight metabolic dysregulation in the Ps19 model, providing insights into the biochemical alterations associated with neurodegeneration.
    DOI:  https://doi.org/10.1038/s41467-025-59487-7
  51. Int J Mass Spectrom. 2025 Aug;pii: 117459. [Epub ahead of print]514
      Cerebrosides, a class of biologically important lipids, are comprised of a monosaccharide head group along with their ceramide tail. However, their accurate characterization is challenging because of the isomerism in both the tail, from potential double bond positioning, or in the head from monosaccharide composition and αβ anomericity. In this work, we focused on tackling the identification of the β-monosaccharide head group, as either glucose or galactose, in various cerebroside isomers as well as demonstrating how our methodology could be applied to unknowns found in a porcine extract. To achieve this, we performed collision-induced dissociation prior to cyclic ion mobility separations to generate monosaccharide fragment ions from the starting cerebroside precursor ions. With this pre-cIMS CID approach, we observed that the cIMS separations of the fragment ions were diagnostic of the β-monosaccharide head group composition (i.e., glucose versus galactose), regardless of the ceramide tail length. From there, we demonstrated an example of how this methodology could also be applied to cerebrosides found in a porcine extract and a framework for how this approach could be added to existing workflows in developing collision cross section databases. Overall, we envision that our developed pre-cIMS CID-based approach will be a complementary and orthogonal tool to existing ones in glycolipidomics workflows.
    Keywords:  cerebrosides; ion mobility spectrometry; isomers; lipidomics; tandem mass spectrometry
    DOI:  https://doi.org/10.1016/j.ijms.2025.117459
  52. Bioanalysis. 2025 May 15. 1-9
       AIMS: This study aimed to establish a robust LC-MS/MS method for quantifying trazodone in human plasma and investigate its pharmacokinetic profile in healthy Chinese subjects under fasting and fed conditions.
    MATERIALS & METHODS: A validated LC-MS/MS method using protein precipitation was applied to analyze trazodone in 68 healthy subjects (34 fasting/34 fed), with full method validation performed.
    RESULTS: This method exhibited high selectivity, precision, and accuracy. The results showed good linearity in the range of 5-3000 ng/mL and matrix effect results showed no matrix interference. The pharmacokinetic data revealed notable differences between the fasting and postprandial states.
    CONCLUSIONS: The validated LC-MS/MS method proved reliable for trazodone quantification. The observed food effects on absorption suggest clinical dosing should consider administration conditions to ensure optimal therapeutic outcomes.
    Keywords:  LC-MS/MS; Trazodone hydrochloride; internal standard; isotopic trazodone-d6; methodology validation; pharmacokinetic
    DOI:  https://doi.org/10.1080/17576180.2025.2506349
  53. Clin Lab Med. 2025 Jun;pii: S0272-2712(25)00005-8. [Epub ahead of print]45(2): 187-205
      This review presents current scientific literature focused on chromatography-free/ambient ionization mass spectrometry techniques for clinical toxicology. Traditional methods like gas and liquid chromatography, paired with mass spectrometry, are commonly used in clinical laboratories but can be time-consuming, leading to backlogs. To alleviate workloads, immunoassays are often used for screening before confirmatory methods but are susceptible to false positives/negatives especially with structurally similar compounds. Therefore, chromatography-free ionization mass spectrometry methods could address concerns regarding uncertainty and lengthy separation. This work examines current efforts implementing these techniques along with their capabilities, limitations, and potential for future integration into clinical toxicology.
    Keywords:  Ambient ionization; Chromatography free; Clinical toxicology; Drug screening; Mass spectrometry
    DOI:  https://doi.org/10.1016/j.cll.2025.01.004