bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2025–01–26
thirty papers selected by
Sofia Costa, Matterworks



  1. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jan 13. pii: S1570-0232(25)00021-2. [Epub ahead of print]1252 124469
      The integrated stress response (ISR) is a cellular defense mechanism activated under stress conditions. When the ISR is activated, it slows the production of proteins, the building blocks that cells need to function. Trans-integrated stress response inhibitor (trans-ISRIB) is a compound that can reverse the effects of ISR activation, showing promise for treating neurodegenerative diseases. The preclinical and clinical evaluation of trans-ISRIB necessitates a reliable analytical method. This study presents the development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the quantitative analysis of trans-ISRIB in human plasma, conforming to the U.S. FDA's guidelines for bioanalytical method validation. The method developed utilizes a liquid-liquid extraction procedure to prepare plasma samples with a spiked internal standard (IS). The extracts containing trans-ISRIB and the IS were dried under nitrogen, reconstituted in the mobile phase, and separated on a Waters XSelect HSS T3 column under isocratic conditions with a mobile phase containing 0.1 % acetic acid in 70 % methanol aqueous solution at a flow rate of 0.500 mL/min. Detection and quantification were accomplished using a positive electrospray ionization tandem mass spectrometer (ESI+-MS/MS) operated in multiple-reaction-monitoring (MRM) mode. The method demonstrated a linear calibration range for trans-ISRIB concentrations from 0.500 to 1.00 x 103 nM, with high specificity, precision, accuracy, and recovery. This method addresses a significant analytical gap, offering a robust tool for quantifying trans-ISRIB in human plasma. Chemical compounds studied in this article: 2-(4-chlorophenoxy)-N-[4-[[2-(4-chlorophenoxy)acetyl]amino]cyclohexyl]acetamide (trans-ISRIB) (CAS # 1597403-47-8); 2-(4-chlorophenoxy)-N-(2-{[(4-chlorophenoxy)acetyl]amino}ethyl)acetamide (CAS # 327071-30-7).
    Keywords:  Human plasma; ISRIB; Mass spectrometric detection; Method validation; Reverse-phase chromatography
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124469
  2. Anal Bioanal Chem. 2025 Jan 23.
      Free fatty acids (FFAs) are important energy sources and significant for energy transport in the body. They also play a crucial role in cellular oxidative stress responses, following cell membrane depolarization, making accurate quantification of FFAs essential. This study presents a novel supercritical fluid chromatography-mass spectrometry (SFC-MS) method using selected ion recording in negative electrospray ionization mode, enabling rapid quantification of 31 FFAs within 6 min without derivatization. FFAs are identified and quantified using an HSS C18 SB column and a secondary mobile phase consisting of methanol with formic acid by detecting their [M - H]- ions. Calibration curves showed strong linearity (R2 ≥ 0.9910), spanning 1000-12,000 ng/mL for short-chain FFAs and 50-1200 ng/mL for medium- and long-chain FFAs. The method achieves detection limits as low as 1 ng/µL for short-chain FFAs and 0.05 pg/µL for other FFAs per on-column injection. The method demonstrated high accuracy and precision, with bias and coefficients of variation maintained below 15% across five quality control levels. Freeze-thaw and autosampler stability studies confirmed the behavior of matrix-matched standards under optimal storage conditions. The validated method was applied to the analysis of pharmaceutical-grade egg yolk powders, using 13 deuterated FFAs as internal standards (IS) in comparison with heptadecanoic acid (C17:0). Significant variations in FFA quantification using two different IS approaches underscore the importance of selecting an appropriate IS. In summary, this study introduces a reliable and validated SFC-MS method for analyzing FFAs ranging from C4 to C26, requiring minimal sample preparation.
    Keywords:  Analytical method development; FFA analysis; Pharmaceutical-grade egg yolk powders; SFC-MS; Total parenteral nutrition
    DOI:  https://doi.org/10.1007/s00216-025-05732-3
  3. Anal Bioanal Chem. 2025 Jan 24.
      Quantitative measurement of metabolites is essential to understand biological and disease processes. Absolute quantification by spiking heavy isotope-labeled internal standards and analyzing on mass spectrometry (MS) platform is a key method to determine the concentration of metabolites in biological samples. However, MS-based absolute quantification is often challenged by the commercial availability and high costs of isotope-labeled internal standards. Here, we establish an absolute quantification method for amine metabolites utilizing isotopic N,N-dimethyl leucine (iDiLeu) tagging on the LC-MS/MS platform. Absolute quantification of metabolites with excellent accuracy and precision can be achieved with five-plex iDiLeu labeling without the need of isotope-labeled internal standards. We demonstrated that iDiLeu labeling improved the separation and detection limits of polar metabolites. Particularly, detection limits for glycine, GABA, and serotonin have been improved by more than 20 folds, and valine by more than 2000 folds. With iDiLeu tagging, 87 amine-containing metabolites were identified and quantified in human cerebrospinal fluid (CSF) samples, revealing potential metabolic changes in Alzheimer's disease patients.
    Keywords:  Absolute quantification; Alzheimer’s disease; Human cerebrospinal fluid; IDiLeu; Mass spectrometry; Metabolites
    DOI:  https://doi.org/10.1007/s00216-025-05748-9
  4. Se Pu. 2025 Feb;43(2): 155-163
      Glufosinate (GLUF) and glyphosate (GLY) are nonselective phosphorus-containing amino acid herbicides that are widely used in agricultural gardens and noncultivated areas. These herbicides give rise to a number of key metabolites, with 3-methyl phosphinicopropionic acid (MPPA), N-acetyl glufosinate (N-acetyl GLUF), aminomethyl phosphonic acid (AMPA), N-acetyl aminomethyl phosphonic acid (N-acetyl AMPA), N-acetyl glyphosate (N-acetyl GLY), N-methyl glyphosate (N-methyl GLY) as the major metabolites obtained from GLUF and GLY. Extensive use of these herbicides may lead to their increased presence in the environment, especially aquatic ecosystems. An increasing number of research studies into the toxicities of GLUF, GLY, and their metabolites have shown that these herbicides are potentially toxic to aquatic biota. GLUF and GLY, as well as their metabolites, are extremely polar and water-soluble, and they lack chromogenic and fluorescent groups; therefore, their concentrations are difficult to determine using conventional methods. Most analytical methods used to date have largely depended on derivatization procedures, leading to overall determination processes that are tedious and time-consuming. Therefore, establishing a quick and sensitive method that does not require derivatives for determining GLUF, GLY, and their metabolites in water environments, including surface water, sediments, and aquatic organisms, is an important endeavor. In this study, a new approach was developed based on pass-through solid-phase extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to determine GLUF, GLY, and their metabolites, including MPPA, N-acetyl GLUF, AMPA, N-acetyl AMPA, N-acetyl GLY, and N-methyl GLY, in sediments. Samples were extracted with 4% (v/v) ammonia water and purified using PRiME HLB pass-through solid-phase extraction columns. The extracts were filtered through a polyethersulfone microfiltration membrane and analyzed by UHPLC-MS/MS. Compounds were separated on a Metrosep A Supp 5 column (150 mm×4.0 mm, 5 μm) using gradient elution with water and 200 mmol/L ammonium hydrogen carbonate solution containing 0.05% (v/v) ammonia water as the mobile phases. Analytes were detected using MS/MS with a negative electrospray ionization (ESI-) source in the multiple reaction monitoring (MRM) mode. A matrix-matched external-standard approach was used for quantitative analysis. GLUF, GLY, and their metabolites were detected within 15 min with good peak shapes and high responses. Calibration curves were linear in the range of 2.0-200 μg/L, with correlation coefficients exceeding 0.995. This method delivered limits of detection (LODs) and limits of quantification (LOQs) of 5 μg/kg and 20 μg/kg, respectively, for GLUF, MPPA, N-acetyl-GLUF, N-acetyl AMPA, N-acetyl GLY, and N-methyl GLY, and 10 μg/kg and 30 μg/kg, for GLY and AMPA, respectively. Average spiked recoveries at three levels (LOQ, 5LOQ, 10LOQ) in sediment with low organic matter content were in the range of 78.5%-107%, and the relative standard deviations (RSDs) were in the range of 1.32%-14.7% (n=6). Average spiked recoveries of 76.4%-113% were determined for three levels (LOQ, 5LOQ, and 10LOQ) in sediments with high organic matter contents, with RSDs of 2.60%-11.2% (n=6). The developed method was used to analyze GLUF, GLY, and their metabolites in ponds, lakes, reservoirs, and river sediments. No target compounds were detected in any sediment sample obtained from a lake, reservoir, or river; however, GLY and AMPA were detected in one pond-sediment sample at levels of 31.7 and 52.3 μg/kg, respectively. The developed approach is simple, fast, and green; moreover, it offers advantages, including high accuracies, high sensitivities, and good reproducibilities. Accordingly, the developed method is suitable for determining GLUF, GLY, and their metabolites in sediments and can provide technical support for studying residue characteristics and environmental behavior in sediments.
    Keywords:  glufosinate; glyphosate; metabolites; non-derivatization; pass-through solid-phase extraction; sediment; ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS)
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.03008
  5. Anal Chim Acta. 2025 Feb 08. pii: S0003-2670(24)01395-3. [Epub ahead of print]1338 343594
       BACKGROUND: Considering the large diversity of chemicals present in the environment and the need to study their effects (alone or as mixtures), the development of high-throughput in vitro assays in line with the Replacement, Reduction, Refinement (3R) strategy is essential for chemical risk assessments.
    RESULTS: We developed a robust analytical workflow based on both low resolution tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry (HRMS) to quantify 13 steroids in NCI-H295R cell culture medium, human plasma and serum. The workflow was validated by screening media from the NCI-H295R cell line exposed in dose-response experiments to 5 endocrine disruptors (EDs) such as bisphenol A, prochloraz, ketoconazole, atrazine and forskolin. Absolute quantifications of the 13 steroids performed on a triple quadrupole (QqQ) MS/MS demonstrated that the performances obtained were in line with OECD recommendations. HRMS (MS1-HRMS) provided measurements nearly as sensitive and as reproducible as those obtained using multiple reaction monitoring (MRM) and ELISA. A bioinformatics workflow, using HRMS, was implemented to detect and annotate disrupted metabolites. HRMS allowed to detect disruptions in pathways associated to fatty acids, purines and amino acids metabolisms after exposure to the EDs tested, in addition to that linked to steroidogenesis.
    SIGNIFICANCE: We developed a robust MS1-HRMS workflow, from sample preparation to compound quantification or annotation, compatible with absolute steroid quantification, to screen NCI-H295R cell media exposed to potential EDs. Using only 200 μL of medium, the method integrates MS/MS and HRMS analyses, 96-well plate solid-phase extraction for high throughput, and automated pre-annotation for cost efficiency. This optimized workflow identifies EDs in cell assays by detecting disruptions in steroidogenesis and other biological pathways.
    Keywords:  Chemical safety; ELISA; Environmental exposure; High-resolution mass spectrometry; Human plasma and serum; MRM; NCI–H295R; Steroidogenesis
    DOI:  https://doi.org/10.1016/j.aca.2024.343594
  6. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jan 16. pii: S1570-0232(25)00022-4. [Epub ahead of print]1252 124470
      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.
    Keywords:  Anti-TB drugs; Bedaquiline; Clofazimine; DBS; LC-MS/MS; Levofloxacin; Linezolid; N-Desmethyl bedaquiline
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124470
  7. Front Pharmacol. 2024 ;15 1506931
       Background: Ripretinib, a broad-spectrum tyrosine kinase inhibitor, has been approved for the treatment of advanced gastrointestinal stromal tumors in adult patients. Clinical studies have shown that higher in vivo exposure of ripretinib correlates with improved efficacy, highlighting the potential clinical significance of therapeutic drug monitoring. In this study, a simple and stable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was attempted to be established and validated for pharmacokinetic studies of ripretinib and its metabolite DP-5439 and therapeutic drug monitoring in human plasma.
    Method: Ripretinib and DP-5439 were separated by chromatography using a Thermofisher Hypersil GOLDTM C18 HPLC column. The mobile phase for gradient elution is composed of 0.1% formic acid in water and acetonitrile. Multiple reaction monitoring was implemented along with electrospray ionization positive mode for detection. The ion pairs of ripretinib, DP-5439 and internal standard D8-ripretinib were m/z 510.1→m/z 417, m/z 496.11→m/z 402.9 and m/z 518.15→m/z 420, respectively. Plasma samples from ripretinib-treated patients of our hospital were collected for pharmacokinetic analysis.
    Results: Ripretinib and DP-5439 demonstrated a strong linear relationship over 10-5,000 μg/L (R 2 > 0.99). Accuracy, precision, specificity, recoveries, matrix effect, stability, and dilution effect were all validated and found to meet the required criteria. Following validation, the method was utilized to determine plasma samples from patients treated with ripretinib. The median steady-state trough concentrations (Cmin, range) were 398.50 (66.98 ∼ 1,458.91) μg/L for ripretinib and 654.74 (30.71 ∼ 1,522.48) μg/L for DP-5439, with a total median concentration of 1,129.46 (140.95 ∼ 2,981.39) μg/L in patients receiving ripretinib at 150 mg once daily. Meanwhile, using the established methods, the study conducted pharmacokinetics studies on four patients with ripretinib and DP-5439.
    Conclusion: This study developed and validated a robust LC-MS/MS method for determining ripretinib and its metabolite DP-5439 in human plasma. Furthermore, the practicality of this method in clinical sample analysis was demonstrated. This approach can serve as an effective tool for the pharmacokinetics analysis and therapeutic drug monitoring in patients treated with ripretinib.
    Keywords:  LC-MS/MS; gastrointestinal stromal tumor; pharmacokinetics; ripretinib; therapeutic drug monitoring (TDM)
    DOI:  https://doi.org/10.3389/fphar.2024.1506931
  8. Se Pu. 2025 Feb;43(2): 164-176
      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 MS2 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 MS2 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 (R2) 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.
    Keywords:  blood; indazole-type; liquid chromatography-linear ion trap-mass spectrometry (LC-LIT-MS); online solid-phase extraction (online SPE); synthetic cannabinoids (SCs); urine
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.02008
  9. Se Pu. 2025 Feb;43(2): 148-154
      Thromboxane A2 (TXA2), a prothrombotic factor that induces platelet aggregation and thrombosis, acts as a vasoconstrictor by activating TXA2 receptors (TP receptors). TXA2 is extremely unstable and metabolizes into three major metabolites: 2,3-dinor thromboxane B2 (2,3-dinor-TXB2), 11-dehydro TXB2(11-dh-TXB2), and 11-dehydro-2,3-dinor TXB2(11-dh-2,3-dinor-TXB2). 8-Iso-prostaglandin F2α(8-iso-PGF2α), a prostaglandin-like compound widely considered the best biomarker of oxidative stress, can also activate TP receptors. The accurate quantification of TXA2 metabolites and 8-iso-PGF2α is critical in cardiovascular disease (CVD). In this study, a method based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous determination of 2,3-dinor-TXB2,11-dh-2,3-dinor-TXB2, 11-dh-TXB2, and 8-iso-PGF2α in human urine. Urine samples were collected from healthy volunteers and patients with CT- or MRI-confirmed ischemic stroke occurring less than 48 h earlier, and cryopreserved at -80 ℃ within 1 h after collection. The urine samples were thawed at room temperature and acidified to pH 2.0-4.0 using hydrochloric acid. The supernatant was collected after centrifugation. A total of 1 mL of each urine sample was added with 100 μL of the internal standard working solution and mixed well. The samples were loaded onto a C18 SPE column (50 mg). The SPE cartridges were preconditioned with 500 μL of methanol and then equilibrated with 500 μL of water. After sample loading, the SPE cartridges were washed with 500 μL of water, 500 μL of 5% methanol aqueous solution containing 0.5% (v/v) ammonia, and 500 μL of 5% methanol aqueous solution containing 2% (v/v) formic acid. The cartridges were dried, and the analytes were eluted with 400 μL of methanol. The eluents were dried and subsequently reconstituted with 50 μL of 13% acetonitrile aqueous solution. After filtration through a filter membrane, the samples were analyzed on an ACQUITY UPLC® BEH phenyl column (50 mm×2.1 mm, 1.7 μm) via gradient elution using 2 mmol/L ammonium acetate aqueous solution containing 0.002% (v/v) ammonia and acetonitrile as the mobile phases. The flow rate was 0.3 mL/min, and the column temperature was 40 ℃. The analytes were determined in negative electrospray ionization and multiple-reaction monitoring modes. The four target compounds showed satisfactory linearity within the relevant ranges, with linear correlation coefficients (R2) greater than 0.99. The limits of detection of the method were 0.02 ng/mL for 2,3-dinor-TXB2 and 0.01 ng/mL for 11-dh-2,3-dinor-TXB2, 11-dh-TXB2, and 8-iso-PGF2α. The limits of quantification were 0.1 ng/mL for 2,3-dinor-TXB2 and 0.05 ng/mL for 11-dh-2,3-dinor-TXB2, 11-dh-TXB2, and 8-iso-PGF2α. In actual urine, the recovery rates at the LOQ level were in the range of 91.48%-104.87%. The recovery rates at low, medium, and high levels were in the range of 92.95%-104.90%. The intra- and inter-day precisions were in the range of 2.79%-13.01% and 4.45%-13.67%, respectively. The relative error (RE) between the average peak area of the mixed matrix and the sum of the ratios of the pure solution and urine matrices was within ±20%. The samples were stable at 4 ℃ for 24 h and at -70 ℃ for 10 d. The developed method is the first to realize the simultaneous determination of 2,3-dinor-TXB2, 11-dh-2,3-dinor-TXB2, 11-dh-TXB2, and 8-iso-PGF2α in urine. The method was used to determine the concentrations of 2,3-dinor-TXB2, 11-dh-2,3-dinor-TXB2, 11-dh-TXB2, and 8-iso-PGF2α in healthy controls and patients with ischemic stroke, and the results were corrected using creatinine. Binary logistic regression analysis was used to construct the prediction model, and a receiver operating characteristic (ROC) curve was drawn to evaluate the clinical diagnostic ability of the method for the target compounds. TXA2 was calculated as the sum of its three metabolites. The area under the curve (AUC) of TXA2 was 0.849, and the method sensitivity and specificity were 69.2% and 92.3%, respectively. The AUC of 8-iso-PGF2α was 0.775, and the method sensitivity and specificity were 84.6% and 76.9%, respectively. The proposed method has good clinical value and is expected to assist in the early screening and diagnosis of ischemic stroke.
    Keywords:  11-dehydro thromboxane B2(11-dh-TXB2); 11-dehydro-2,3-dinor thromboxane B2(11-dh-2,3-dinor-TXB2); 2,3-dinor thromboxane B2(2,3-dinor-TXB2); 8-iso-prostaglandin F2α(8-iso-PGF2α); ischemic stroke; ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS); urine
    DOI:  https://doi.org/10.3724/SP.J.1123.2024.02004
  10. ACS Omega. 2025 Jan 14. 10(1): 885-891
      Water-soluble vitamins play essential roles in normal body functions and metabolic activities. However, few methods have simultaneously measured all nine water-soluble vitamins in biological matrices. In this study, we developed a sensitive and accurate method for the simultaneous measurement of thiamine (B1), riboflavin (B2), nicotinamide (B3), pantothenic acid (B5), 4-pyridoxic acid (B6), biotin (B7), 5-methyltetrahydrofolic acid (B9), ascorbic acid (VC), and methylmalonic acid (MMA) in human serum. Samples were analyzed using ultrahigh-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with Waters HSS T3 and BEH C18 columns, each measuring 2.1 × 50 mm with a particle size of 1.7 μm. The mass spectrometer was equipped with an electrospray ionization source, and optimized multiple reaction monitoring was employed to detect all compounds in positive ionization mode. The capillary voltage was set at 0.5 kV. Nitrogen was used as the desolvation gas, with a flow rate of 1000 L/h at 500 °C. Argon was used as the collision gas. The method's performance was validated according to the Clinical and Laboratory Standards Institute guidelines assessing linearity, limit of quantitation, precision, accuracy, carryover, matrix effects, recovery, and dilution. The results confirmed the successful validation of this method for water-soluble vitamins in serum. However, the presence of common endogenous interferences and the type of blood collection tube influenced the results for certain water-soluble vitamins. The results showed that this method was satisfactory, offering significant improvements in analytical performance, including shorter analysis time, higher sensitivity, and the requirement of a smaller sample volume.
    DOI:  https://doi.org/10.1021/acsomega.4c07968
  11. Carbohydr Polym. 2025 Mar 15. pii: S0144-8617(24)01380-8. [Epub ahead of print]352 123154
      Human milk oligosaccharides (HMOs) are now the principal component of the latest infant formula generation. However, it is challenging to separate and quantify highly heterogeneous isomers when analyzing HMOs. Here, we developed a high-throughput isomer-resolved quantification method for 21 native HMOs based on ultrahigh-performance liquid chromatography-mass spectrometry-multiple reaction monitoring (UPLC-MS-MRM) technology. High-resolution cyclic ion mobility-mass spectrometry technology was applied to distinguish HMO isomers and identify specific ion pairs for quantification to solve the measurement challenge of HMOs without available standards. Simultaneously, nine HMO standards were used to create universal calibration curves to quantify those without standards. This MRM approach features wide isomer coverage (seven series of isomers), short analysis time (12 min), high sensitivity (ng/L level detection limit), and a broad 5-order-of-magnitude quantitation range. After that, the technique was used on 58 Chinese human milk samples, which showed that the three genotype milk groups had different HMO profiles at the isomer level. Four crucial fucosylated HMOs were identified as markers to rapidly assign secretor status. Moreover, correlations analysis unveiled a co-regulatory relationship among Fuc-(α1-3/4), Fuc-(α1-2), and sialylated glycanforms. Our research offers a comprehensive solution for isomeric glycome profiling, and provides a crucial reference for designing precise nutrition formula for infants.
    Keywords:  Cyclic ion mobility-mass spectrometry; Human milk oligosaccharides; LC-MS; Multiple reaction monitoring; Secretor
    DOI:  https://doi.org/10.1016/j.carbpol.2024.123154
  12. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jan 10. pii: S1570-0232(25)00015-7. [Epub ahead of print]1252 124463
      Human exposure to volatile organic compounds (VOCs) poses significant health risks, contributing to cardiovascular disease, pulmonary disease, and cancer. Measurement of VOC metabolites (VOCm) in urine by liquid chromatography-mass spectrometry (LC-MS) is a preferred method for VOCm analysis; however, existing methods encounter challenges related to sensitivity, throughput, and analyte coverage. In addition to VOCm, the measurement of tobacco alkaloids (TAm) is critical to account for tobacco use in population-based studies. A method is needed that is highly sensitive, offers higher throughput, and can analyze VOCm and TAm in a single run. Herein, we present a robust dilute-and-shoot method aimed at overcoming these analytical challenges and expanding the targeted analysis to include 35 urinary VOCm and TAm and their metabolites. By leveraging high-speed polarity switching and optimized chromatographic parameters, our method achieved comprehensive analyte coverage and enhanced sensitivity, enabling reliable individual level VOC exposure assessment. Validation demonstrates robust linearity, sensitivity, accuracy, and precision, with minimal matrix effects. This high-throughput UPLC-MS/MS method significantly enhances VOC exposure assessment by enabling simultaneous measurement of 35 urinary VOC and TAm with high sensitivity and efficiency. Multiple metabolites from single parent xenobiotics are included in one run, expanding biomarker specificity. Our data indicate the method effectively accounts for tobacco consumption as a confounder in population-based studies, ensuring accurate VOC exposure assessment.
    Keywords:  Biomarkers; Tobacco; Tobacco exposure; VOCs; Volatile organic compounds
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124463
  13. Metabolites. 2025 Jan 14. pii: 48. [Epub ahead of print]15(1):
      Introduction: NMR spectroscopy is a powerful technique for studying metabolism, either in metabolomics settings or through tracing with stable isotope-enriched metabolic precursors. MetaboLabPy (version 0.9.66) is a free and open-source software package used to process 1D- and 2D-NMR spectra. The software implements a complete workflow for NMR data pre-processing to prepare a series of 1D-NMR spectra for multi-variate statistical data analysis. This includes a choice of algorithms for automated phase correction, segmental alignment, spectral scaling, variance stabilisation, export to various software platforms, and analysis of metabolic tracing data. The software has an integrated help system with tutorials that demonstrate standard workflows and explain the capabilities of MetaboLabPy. Materials and Methods: The software is implemented in Python and uses numerous Python toolboxes, such as numpy, scipy, pandas, etc. The software is implemented in three different packages: metabolabpy, qtmetabolabpy, and metabolabpytools. The metabolabpy package contains classes to handle NMR data and all the numerical routines necessary to process and pre-process 1D NMR data and perform multiplet analysis on 2D-1H, 13C HSQC NMR data. The qtmetabolabpy package contains routines related to the graphical user interface. Results: PySide6 is used to produce a modern and user-friendly graphical user interface. The metabolabpytools package contains routines which are not specific to just handling NMR data, for example, routines to derive isotopomer distributions from the combination of NMR multiplet and GC-MS data. A deep-learning approach for the latter is currently under development. MetaboLabPy is available via the Python Package Index or via GitHub.
    Keywords:  NMR; Python; deep learning; metabolomics; stable isotope; tracing
    DOI:  https://doi.org/10.3390/metabo15010048
  14. J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Jan 10. pii: S1570-0232(25)00017-0. [Epub ahead of print]1252 124465
       OBJECTIVE: To develop a rapid, convenient, accurate, and low-residual-effect ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the determination of polymyxin B sulfate and colistin sulfate in the blood of patients with multidrug-resistant bacterial infections, as well as caspofungin acetate in the blood of patients with fungal infections, thus facilitating the rational use of antibiotics in clinical applications.
    METHODS: All analytes were diluted with 0.2 % aqueous formic acid, and plasma proteins were precipitated using acetonitrile. The selected reaction monitoring (SRM) mode was used for measurement. Separation of all analytes was completed on a Hypersil GOLD C18 column (100 × 2.1 mm, 3.0 µm). They were quantitatively analyzed using electrospray ionization on a triple quadrupole mass spectrometer in the positive ion mode. The mobile phase consisted of water (containing 0.1 % formic acid) and acetonitrile, which was delivered by gradient elution at a flow rate of 0.3 ml/min. The internal standard was bacitracin zinc (BcZn), and the column temperature was maintained at 25 °C. The runtime for each analysis was 3.5 min.
    RESULTS: The procedure was validated following the recommendations of the U.S. Food and Drug Administration, which included measurements of accuracy (ranging from 83.27 % to 105.86 % for within-run and between-run accuracy), precision (with coefficients of variation from 2.50 % to 16.51 % for within-run precision and between-run precision), and matrix effects (ranging from 88.65 % to 103.94 %). The extraction recoveries ranged from 38.01 % to 42.76 for polymyxin B1 (PMB1), polymyxin B2 (PMB2), polymyxin E1 (PME1), polymyxin E2 (PME2), and 88.65 % to 89.84 % for caspofungin (CPF). Plasma samples were stable under various storage conditions, including three freeze-thaw cycles at -80 °C, 24-hour periods at room temperature and 4 °C, and 30 days of freezing at both -20 °C and -80 °C, with relative standard deviations (RSD) of less than 15 %.
    CONCLUSION: In this study, a UPLC-MS/MS method was developed to simultaneously quantify PMB1, PMB2, PME1, PME2, and CPF in human plasma. The method was validated in blood samples from patients with multidrug-resistant bacteria combined with fungal infections and is suitable for therapeutic drug monitoring.
    Keywords:  Caspofungin acetate; Colistin sulfate; Polymyxin B sulfate; Therapeutic drug monitoring; UPLC–MS/MS
    DOI:  https://doi.org/10.1016/j.jchromb.2025.124465
  15. Anal Bioanal Chem. 2025 Jan 20.
      The wide range of mass spectrometry imaging (MSI) technologies enables the spatial distributions of many analyte classes to be investigated. However, as each approach is best suited to certain analytes, combinations of different MSI techniques are increasingly being explored to obtain more chemical information from a sample. In many cases, performing a sequential analysis of the same tissue section is ideal to enable a direct correlation of multimodal data. In this work, we explored different workflows that allow sequential lipid and elemental imaging on the same tissue section using atmospheric pressure laser desorption/ionisation-plasma post-ionisation-MSI (AP-MALDI-PPI-MSI) and laser ablation-inductively coupled plasma-MSI (LA-ICP-MSI), respectively. It is found that performing lipid imaging first using matrix-coated samples, followed by elemental imaging on matrix-coated samples, provides high-quality MSI datasets for both lipids and elements, with the resulting distributions being similar to those obtained when each is performed in isolation. The effect of matrix removal prior to elemental imaging, and of performing elemental imaging first were also investigated but found to generally yield lower quality elemental imaging data but comparable lipid imaging data. Finally, we used the ability to acquire both elemental and lipid imaging data from the same section to investigate the spatial correlations between different lipids (including ceramides, phosphatidylethanolamine, and hexosylceramides) and elements within mouse brain tissue.
    Keywords:  Elemental analysis; Lipid analysis; Mass spectrometry imaging; Multimodal imaging; Single tissue section
    DOI:  https://doi.org/10.1007/s00216-024-05696-w
  16. Shokuhin Eiseigaku Zasshi. 2024 ;65(6): 178-184
      Thiouracil (2-thiouracil) is a thyrostat used to promote weight gain in cattle. However, its use is prohibited within the European Union (EU), necessitating the monitoring of its presence in bovine urine for beef exports to the EU. In this study, we present the development and validation of a quantitative method for the determination of 2-thiouracil, 4-thiouracil, and 6-methyl-2-thiouracil in bovine urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method involves stabilizing the analytes by adding hydrochloric acid and ethylenediaminetetraacetic acid to the sample, followed by derivatization with 3-iodobenzyl bromide, cleanup with a divinylbenzene-N-vinylpyrrolidone copolymer cartridge, and subsequent LC-MS/MS analysis. The developed method was validated for determination of 2-thiouracil, 4-thiouracil, and 6-methyl-2-thiouracil in bovine urine at a concentration of 10 μg/L. The trueness ranged from 94 to 97%, with intra-day precisions below 5% and inter-day precisions below 8%. No chromatographic interference was observed near the analytes' retention times. This analytical method is particularly valuable because it can determine whether 2-thiouracil was illicitly administered or ingested via feed containing plants of the Brassicaceae family, by confirming the presence of 6-methyl-2-thiouracil or 4-thiouracil alongside 2-thiouracil in bovine urine.
    Keywords:  2-thiouracil; 4-thiouracil; 6-methyl-2-thiouracil; bovine urine
    DOI:  https://doi.org/10.3358/shokueishi.65.178
  17. Food Res Int. 2025 Feb;pii: S0963-9969(24)01682-X. [Epub ahead of print]201 115611
      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 (R2). 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.
    Keywords:  Authentication; Bovine; Collagen; Gelatin; LC-MS/MS; Porcine
    DOI:  https://doi.org/10.1016/j.foodres.2024.115611
  18. Metabolites. 2025 Jan 08. pii: 30. [Epub ahead of print]15(1):
      Background: Quantifying urinary catecholamines and metanephrines is essential for the clinical screening and diagnosis of neuroendocrine tumours. HPLC with electrochemical detection (HPLC-ECD) is commonly used for this type of analysis but requires extensive sample cleanup. Simple and rapid dilute-and-shoot LC-multiple-reaction monitoring (MRM)-MS assays have been developed for quantitating these analytes in urine but have not yet been validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Methods: A simple dilute-and-shoot sample preparation without derivatization was used. C18 RP-UPLC-MRM-MS and positive-ion ESI were used, usually with two transitions per analyte being monitored. Certified deuterated internal standards were used for each analyte. Results: This assay was validated according to the CLSI C62-A guidelines, including accuracy/trueness, imprecision, sensitivity, specificity, carryover, stability, and linearity. The final MRM-MS method was compared to the established HPLC-ECD clinical chemistry reference method. The run time was reduced from 25 min to 5 min. Conclusions: A simple, robust, rapid, and cost-effective LC-MRM-MS assay for measuring urinary catecholamines and metanephrines was developed and validated according to the CLSI guidelines. This validated method requires minimal sample manipulation before analysis and provides sensitivity, specificity, and improved precision. The implementation of this assay in clinical laboratories will facilitate early and accurate diagnosis.
    Keywords:  adrenal gland; catecholamines; dilute-and-shoot; liquid chromatography–multiple-reaction monitoring mass spectrometry (LC-MRM-MS); metanephrines; paraganglioma; pheochromocytoma; urine
    DOI:  https://doi.org/10.3390/metabo15010030
  19. J Pharm Biomed Anal. 2025 Jan 20. pii: S0731-7085(25)00025-1. [Epub ahead of print]256 116684
      Alkaptonuria (AKU) is a rare autosomal-recessive disease which is characterized through black urine and ochronosis. It is caused by deficiency of the enzyme Homogentisate 1,2-dioxygenase in the Phenylalanine/Tyrosine degradation pathway which leads to the accumulation of Homogentisic acid (HGA). Urine was provided by AKU patients and healthy controls. Several different methods were developed in this study each with a specific goal. Firstly, a simple and inexpensive RP-UHPLC-UV method for routine monitoring of HGA as a key metabolite employing a Phenylhexyl stationary phase chemistry. Validation was performed in accordance to FDA guidelines and method selectivity was further evaluated via on-line high-resolution sampling 2D-LC-QToF-MS, coupling the Phenylhexyl phase in the first dimension with a C18 phase in the second dimension. Secondly, a targeted and accurate RP-UHPLC-MRM-QTRAP assay, providing quantitative analysis of the relevant pathway metabolites based on a Phenylhexyl stationary phase, and lastly an untargeted HILIC-UHPLC-QToF-MS/MS method with SWATH (sequential window acquisition of all theoretical mass spectra) acquisition employing a sulfobetaine-type HILIC-Z superficially porous particle column, with the aim of uncovering more details about the metabolic profile of this genetic disorder. By untargeted analysis 204 metabolites could be detected and annotated in positive and negative ESI mode in total. Two separate LC methods were employed, differing in their conditions depending on the ionization mode (20 mM ammonium formate as buffer additive adjusted to a pH = 3.5 with formic acid in ESI+ mode and 20 mM ammonium acetate adjusted to a pH = 7.5 with acetic acid in ESI- mode). By effectively combining the aforementioned methods, a comprehensive workflow was developed, allowing the effective analysis of both patient and control urine samples.
    Keywords:  Data-independent acquisition; HILIC; Inherited metabolic disease; Mass spectrometry; Phenylhexyl-column; Sulfobetaine-column
    DOI:  https://doi.org/10.1016/j.jpba.2025.116684
  20. Analyst. 2025 Jan 23.
      Various technical methodologies are required to accurately detect substances of different chemical and pharmacological properties in biological samples, which are increasing in number and variety daily. Therefore, laboratories where many samples and different factors are analyzed simultaneously need methods with easy sample preparation, short analysis times and low analysis costs. In this study, the objective was to scan substances susceptible to chemical degradation, amenable to analysis without hydrolysis, and exhibiting short-term stability by employing a straightforward, expeditious, and cost-efficient method. For this purpose, a high-throughput dilute and shoot screening protocol was developed and validated utilizing high-performance liquid chromatography coupled with high-resolution mass spectrometry to analyze various pharmacological compounds in horse urine and plasma. Over 200 prohibited substances across multiple categories were scanned within a 13 minute run. Chromatographic separation was performed on a C18 column using an elution gradient of mobile phase A, 5 mM ammonium bicarbonate at pH 9, and mobile phase B, methanol, at a flow rate of 0.3 mL min-1. The method was validated according to the specifications of 2002/657/EC multi-screening requirements. The detection capability ranged from ≤1 to 200 ng mL-1 for prohibited substances. The implementation of the screening method in doping analysis, and the analysis of real positive case samples served to underscore the practical applicability of the developed method. To the best of our knowledge, this is a rare method that can be applied to both urine and plasma samples and provides a rapid, practical, broad-spectrum, and high-throughput analysis of prohibited substances in horse plasma and urine cost-effectively.
    DOI:  https://doi.org/10.1039/d4an01501k
  21. Vet Med Sci. 2025 Jan;11(1): e70212
       BACKGROUND: Amino acid supplements are crucial for animal health and productivity. Traditional analysis methods face limitations like complexity, long testing times and toxic reagents. Therefore, a more efficient and reliable method is needed.
    OBJECTIVES: This study aimed to develop and validate an efficient method for the simultaneous analysis of eight amino acids commonly used in veterinary medicine: alanine, arginine, glutathione, lysine, ornithine, methionine, threonine and tryptophan.
    METHODS: We analysed eight veterinary amino acid preparations. From 100 registered products, we selected 35. After confirming ingredients, we diluted them to 1 mg/L with 50% acetonitrile (ACN) and filtered them using a 0.2 µm RC filter for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis.
    RESULTS: All analytes showed excellent linearity (r2 > 0.99) within 0-10 mg/L. The limits of detection (LOD) ranged from 0.04 to 0.83 mg/L, and the limits of quantification (LOQ) ranged from 0.12 to 2.52 mg/L. Average recovery ranged from 92.96% to 105.61%, with relative standard deviations (RSD) from 0.27% to 3.50%, meeting CD 2002/657/EC standards. Six out of the 35 products (17.14%) did not meet regulations.
    CONCLUSIONS: The method developed in this study offers an efficient and reliable approach for the simultaneous analysis of essential amino acids in veterinary medicine. Implementing this method can improve the quality control of amino acid products, enhancing animal health and productivity. This study also highlights the need for stringent domestic management and continuous monitoring. By overcoming traditional technique limitations, this validated method ensures the quality and efficacy of amino acid supplements in the veterinary industry.
    Keywords:  amino acid analyse; eco‐friendly; green technology; liquid chromatography with tandem mass spectrometry (LC–MS/MS); quality control; veterinary medicine
    DOI:  https://doi.org/10.1002/vms3.70212
  22. STAR Protoc. 2025 Jan 21. pii: S2666-1667(24)00747-0. [Epub ahead of print]6(1): 103582
      Gut-microbiome-combined metabolomics studies in cerebrovascular disease highlight the microbiota-gut-brain axis in neurological disorders. Here, we present a protocol for correlating the gut microbiome and metabolomics in patients with intracranial aneurysms. We describe steps for sample collection, fecal genomic DNA extraction, rRNA PCR amplification, sequencing library construction, and rRNA sequencing. We then detail procedures for metabolite extraction, liquid chromatography-tandem mass spectrometry (LC-MS/MS) non-targeted metabolomics sequencing, and ELISA for cerebrospinal fluid and plasma samples. Finally, we perform combined multi-omics analysis. For complete details on the use and execution of this protocol, please refer to Xu et al.1.
    Keywords:  Clinical Protocol; Metabolomics; Microbiology; Sequencing
    DOI:  https://doi.org/10.1016/j.xpro.2024.103582
  23. Rapid Commun Mass Spectrom. 2025 Jan 23. e9985
       RATIONALE: Teaching mass spectrometry essentials is usually connected with one of the basic courses for undergrads. Thus, specific previous knowledge is required from students. However, the necessity of teaching mass spectrometry essentials to students of different academic specializations and multidisciplinary groups can arise in every academic group. A specific workshop is needed to address such a demand.
    METHODS: The presented workshop consisted of several thematic parts: assembling an ambient ionization ion source using improvised materials, preparing biological samples for analysis, data acquisition, and interpretation of data to solve a simple problem from the real world.
    RESULTS: The first part of the work consisted of assembling an ambient ionization setup and obtaining mass spectra of substances from standard solutions, natural mixtures, and biological fluids such as saliva. The second half of the workshop consists of analyzing the composition of fatty acids of natural and artificial fats using the same ion source. The identification of oils is a simple model problem that makes the workshop attractive for attendees with different backgrounds.
    CONCLUSIONS: The workshop provides students with practical skills that are highly valuable in fundamental and applied mass spectrometry. Students familiarize themselves with the basic concepts, instrument use, and mass spectra interpretation. They achieve basic hands-on experience in experimentation procedures and the practice of using mass spectrometry to solve problems related to real life.
    Keywords:  education; fatty acids methyl esters; paper spray; touch spray
    DOI:  https://doi.org/10.1002/rcm.9985
  24. J Pharm Biomed Anal. 2025 Jan 14. pii: S0731-7085(25)00019-6. [Epub ahead of print]256 116678
      Accurate measurement of serum parathyroid hormone (PTH) is crucial for diagnosing and managing endocrine and osteological diseases. Conventional immunoassay methods struggle with cross reactivity issues between full-length PTH and truncated fragments or post-translationally modified forms. Both the standardization of PTH assays and the peptide's stability are concerning. This study addresses these issues by establishing an immunocapture coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to precisely quantify PTH1-84. PTH1-84 was isolated from one mL serum samples by immunocapture on a polystyrene bead and eluted from matrix, then quantitated by LC-MS/MS. The results from 268 serum samples were compared to an intact PTH immunoassay. The assay's linear range was 5.0-1000.0 pg/mL. The intra-assay coefficients of variation (CVs) ranged from 3.2 % to 6.8 %, and the inter-assay CVs ranged from 4.6 % to 9.5 %. The extraction efficiencies were 98.0 %-100.5 %, with no significant matrix effects observed after internal standard correction. The correlation coefficient between LC-MS/MS and immunoassay was 0.989, but the bias between the methods was substantial. Nevertheless, the immunocapture purification coupled LC-MS/MS method offers a promising approach for accurate PTH measurement.
    Keywords:  Immunoassay; Immunocapture purification; LC–MS/MS; Parathyroid hormone
    DOI:  https://doi.org/10.1016/j.jpba.2025.116678
  25. Anal Chem. 2025 Jan 20.
      The identification of polar and neutral lipid species as biomarkers in complex biological samples is a key task in clinical and life sciences. Electrospray and plasma-based ionization techniques are necessary to cover the full range of lipidomes, owing to their limited molecular polarity ranges. However, combining both to generate hybrid spectra is difficult without averaging spectra, as electrospray and plasma sources operate under vastly different conditions. Their electric fields also interfere, resulting in a mutual destabilization of the ionization processes. Herein, a heated nanoelectrospray is combined with a flexible microtube plasma using a rapid (kHz range) switching process (heated nESI-sFμTP) to generate quasi-simultaneous ionization. This approach loads the ion trap with polar and less-polar ions during each injection cycle, generating hybrid spectra without averaging it. The performance of the quasi-simultaneous approach is investigated in positive ion mode, comparing it with conventional ion sources through the analysis of complex lipid liver and heart samples. While no improvements are observed in negative ion mode, the novel quasi-simultaneous approach shows great potential for analyzing complex samples in positive ion mode. The combined heated nESI-sFμTP exceeds the molecular polarity range of individual sources, offering excellent ionization efficiency and MS2 capabilities.
    DOI:  https://doi.org/10.1021/acs.analchem.4c03621
  26. Talanta. 2025 Jan 08. pii: S0039-9140(25)00027-X. [Epub ahead of print]287 127541
      Chromatographic separations in combination with spectroscopic detectors are a main pillar of today's analytical chemistry. The recorded spectroscopic data is usually not shown in a typical chromatogram, therefore the contained additional information cannot be accessed readily by the analyst and is inspected in tedious additional routines, such as separate database searches. We developed a method to add colors to gas chromatograms with mass spectral detection. The colors make the structural similarity and thus the substance class of the detected analytes visible. The assigned color is stable for a given spectrum and independent of the analytical setup and the sample composition. For "staining" (color coding), the recorded mass spectra are first sorted according to similarity on a self-organizing map that was prepared from a large mass spectral database, and the location of a mass spectrum on this substance map is then converted into a color using the hue, saturation, and lightness color-space. The generated substance maps are retention-time independent summaries of the recorded chromatograms. They represent the sample composition, and they are independent of the analytical setup. They can be used to directly compare separations obtained with different analytical setups. Furthermore, the maps allow straightforward quantification of structurally similar compounds that elute at different retention times, since they are grouped in the same location on the substance map. By calculating the difference between a sample and a reference measurement, the presence of an additional compound, for example an adulterant, or the absence of a typical component becomes readily apparent. Staining (color coding) gas chromatograms and the simple conversion of time-based chromatograms to time-independent substance maps exemplarily demonstrate how chemistry, where medium-sized data sets are commonly inspected visually, can benefit from purposefully designed visualizations and targeted data conversion.
    Keywords:  Classification; Comprehensive GCxGC; Mass spectrometry; Quality control; Self-organizing maps; Visualization
    DOI:  https://doi.org/10.1016/j.talanta.2025.127541
  27. Metabolites. 2025 Jan 03. pii: 14. [Epub ahead of print]15(1):
      Background/Objectives: Metabolic profiling of human milk (HM) is indispensable for elucidating mother-milk-infant relationships. Methods: We evaluated the Biocrates MxP® Quant 500 assay for HM-targeted metabolomics (106 small molecules, 524 lipids) and analyzed in a feasibility test HM from apparently healthy Brazilian mothers (A: 2-8, B: 28-50, C: 88-119 days postpartum, ntotal = 25). Results: Of the 630 possible signatures detectable with this assay, 506 were above the limits of detection in an HM-pool (10 µL) used for assay evaluation, 12 of them above the upper limit of quantitation. Analyzing five different HM-pool volumes (2-20 µL) revealed acceptable linearity for 458 metabolites. Intraday accuracy of 80-120% was attained by 469 metabolites after spiking and for 342 after a 1:2 dilution. Analyzing HM from Brazilian mothers revealed significantly lower concentrations in colostrum vs. mature milk for many flow-injection analyses (FIA) and only a few LC-MS metabolites, including triglycerides, sphingomyelins, and phosphatidylcholines. Higher concentrations at the later lactation stages were found predominantly for amino acids and related compounds. Conclusions: The MxP Quant® 500 assay is a useful tool for HM metabolic profiling, minimizing analytical bias between matrices, and enhancing our ability to study milk as a biological system.
    Keywords:  FIA; LC-MS; colostrum; human milk; lipids; small molecules; targeted metabolites
    DOI:  https://doi.org/10.3390/metabo15010014
  28. Shokuhin Eiseigaku Zasshi. 2024 ;65(6): 154-159
      A method for analyzing tetrodotoxin (TTX) in miso soup samples was proposed. The samples were purified using strong cation exchange solid-phase extraction and analyzed by liquid chromatography-tandem mass spectrometry. The recovery of TTX was considerably influenced by the salt concentration in the loading solution during purification. It was observed that diluting the loading solution to reduce the salt concentration helped to maintain the recovery rate during the washing step. However, during the loading step, the benefit of dilution in improving recovery was not evident, as the enhanced retention on the solid phase caused by dilution was counteracted by losing TTX with the increased volume of the loading solution, which led to enhanced elution. This suggests that the volume of extraction solution used in the loading process is crucial in determining the recovery rate. Additionally, the use of volatile ammonium acetate as the elution solvent was explored to optimize conditions for effective recovery. Testing this method on miso soup samples with varying miso types, salt concentrations, and TTX levels resulted in consistently high recovery rates of>80%.
    Keywords:  LC-MS/MS; miso soup; pufferfish; strong cation exchange; tetrodotoxin
    DOI:  https://doi.org/10.3358/shokueishi.65.154
  29. Scand J Clin Lab Invest. 2025 Jan 23. 1-6
      Analytical errors related to endogenous or exogenous substances are a cause of unnecessary investigation, intervention, and patient concern especially in immunoassay platforms. In this report, we systematically screened for estradiol interference using a practical algorithm. For extended research in interference screening, repeated estradiol measurements for control and case samples were carried out for method comparison (three immunoassay platforms and one liquid chromatography-mass spectrometry (LC-MS/MS) measurement), dilution test, polyethylene glycol (PEG) precipitation, and heterophile antibody blocking tube. When serial dilutions were applied to the sample, a deviation from linearity was observed when compared to the control sample. With PEG precipitation, the recovery rate of 63%. While the patient's serum estradiol value was 389.02 pmol/L using the Siemens Atellica immunoassay method, the same sample was <36.7 pmol/L in the Abbott immunoassay method and 34.9 pmol/L in the Roche immunoassay platform. Applying the LC-MS/MS method, the patient had a serum estradiol of 3.33 pmol/L. Immunoassays are susceptible to interferences that can cause high estimates due to cross-reactivity. Therefore, LC-MS/MS may be more suitable than immunoassays for preventing misdiagnosis and inappropriate treatments, especially for children.
    Keywords:  Estradiol; Liquid Chromatography-Mass Spectrometry; endocrinology; precocious puberty; ımmunoassay
    DOI:  https://doi.org/10.1080/00365513.2025.2457113
  30. Drug Test Anal. 2025 Jan 22.
      Propofol is a widely used anesthetic. Although considered safe, propofol-related deaths occur, as it is sometimes abused recreationally or used to commit suicide. A simple, rapid, and reliable method for its analysis in various biological samples is needed. Sample clean-up is a critical step in the analysis, both in terms of time and cost, indeed. Dispersive liquid-liquid microextraction (DLLME) is a simple and fast extraction based on ternary solvent mixtures that uses small volumes of solvent and sample. A DLLME extraction followed by gas chromatography-mass spectrometry (GC-MS) analysis was developed and validated for the analysis of propofol in blood, urine, and hair. The same extraction mixture of 2.5:1 methanol/chloroform was used for the different biological samples. Validation for linearity, LOD, LOQ, precision, accuracy, and recovery gave satisfactory results for the three types of biological samples included in the study, with limits of quantification of 1 μg/mL for urine, 0.2 μg/mL for blood, and 0.1 ng/mg for hair. The DLLME procedure for purification involves a small amount of solvent, thus reducing the cost and the environmental impact. In addition, a high enrichment factor is obtained, and the time for analysis is short. The method was applied to authentic post-mortem samples for the determination of propofol in blood, urine, and hair. Also, segmental hair analysis was performed to assess chronic propofol abuse. The developed method proved to be rapid, simple, and cost-effective for blood, urine, and hair extract clean-up for the determination of propofol by GC/MS.
    Keywords:  DLLME; microextraction; post‐mortem; propofol; sample preparation
    DOI:  https://doi.org/10.1002/dta.3856