bims-metlip 2024-01-07 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–01–07
twenty-six papers selected by
Sofia Costa, Matterworks

Increasing coverage of the urinary polar metabolome using ultra high-performance hydrophobic interaction liquid chromatography combined with linear and cyclic travelling wave ion mobility and mass spectrometry.
Quantification of glucocorticoid and progestogen metabolites in bovine plasma, skimmed milk and saliva by UHPLC-HR-MS with polarity switching.
The first LC-MS/MS stereoselective bioanalytical methods to quantitatively detect 9R- and 9S-hexahydrocannabinols and their metabolites in human blood, oral fluid and urine.
COLMARppm: A Web Server Tool for the Accurate and Rapid Prediction of 1H and 13C NMR Chemical Shifts of Organic Molecules and Metabolites.
Simultaneous determination of free biliverdin and free bilirubin in serum: A comprehensive LC-MS approach.
Optimised liquid chromatography tandem mass spectrometry method for the simultaneous quantification of serum vitamin D analogues while also accounting for epimers and isobars.
Improved quantification of carbonyl sub-metabolome by liquid chromatography mass spectrometry using a fragment controlled multiplexed isotopic tag.
Absolute quantification of nicotinamide mononucleotide in biological samples by double isotope-mediated liquid chromatography-tandem mass spectrometry (dimeLC-MS/MS).
Comparison of LC-MS-based methods for the determination of carboxylic acids in animal matrices.
Absolute Quantification of Human Serum Albumin Isoforms by Internal Calibration Based on a Top-Down LC-MS Approach.
A chemical derivatization-based pseudotargeted liquid chromatography-tandem mass spectrometry method for sensitive and high coverage determination of bile acids in human serum.
Comparison between ammonium formate and ammonium fluoride in the analysis of stratum corneum lipids by reversed phase chromatography coupled with high resolution mass spectrometry.
Quantitative analysis of tetrahydrocannabinol isomers and other toxicologically relevant drugs in blood.
Investigating the Metabolic Heterogeneity of Cancer Cells Using Functional Single-Cell Selection and nLC Combined with Multinozzle Emitter Mass Spectrometry.
Comprehensive profiling of ceramides in human serum by liquid chromatography coupled to tandem mass spectrometry combining data independent/dependent acquisition modes.
A fast and reliable LC-MS/MS method for the quantification of saxitoxin in blood plasma samples.
Rapid Analysis of Fentanyl and Fentanyl Analogues from Whole Blood Using SPME Coupled to the Microfluidic Open Interface.
Evaluation of lipid extraction methods for fatty acid quantification and compound-specific δ13C and δ2Hn analyses.
Fast mass spectrometry search and clustering of untargeted metabolomics data.
LiLA: lipid lung-based ATLAS built through a comprehensive workflow designed for an accurate lipid annotation.
Comparison of high-resolution mass spectrometry acquisition methods for the simultaneous quantification and identification of per- and polyfluoroalkyl substances (PFAS).
QComics: Recommendations and Guidelines for Robust, Easily Implementable and Reportable Quality Control of Metabolomics Data.
Polarity-regulated derivatization-assisted LC-MS method for amino-containing metabolites profiling in gastric cancer.
Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution Mass Spectrometry.
Chromatographic speed classification for liquid chromatography using average theoretical peak time (ATPT).
DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells.

J Chromatogr A. 2024 Jan 11. pii: S0021-9673(23)00762-8. [Epub ahead of print]1714 464537

Increasing coverage of the urinary polar metabolome using ultra high-performance hydrophobic interaction liquid chromatography combined with linear and cyclic travelling wave ion mobility and mass spectrometry.

Adam King, Lee A Gethings, Johannes P C Vissers, Robert S Plumb, Ian D Wilson.

  The use of HILIC-based separations for the analysis of polar metabolites in metabolic phenotyping studies is well established. Here, we demonstrate the increased coverage of the polar metabolome obtained by travelling wave (TW) ion mobility (IM) instruments combined with HILIC and mass spectrometry (MS) for metabotyping rat and mouse urine samples. Profiling was performed using either a linear TW IM-MS based instrument with a path length of 40 cm or an instrument with a cyclic travelling wave analyser (cIM) with a path length of 95 cm. Due to the added resolution afforded by using both the linear and cyclic IM geometries with MS detection (IM-MS) significant increases in feature count (m/z-tR pairs) were generally obtained compared to HILIC-MS alone. In addition, the use of both linear and cyclic IM-MS improved the quality of the mass spectra obtained as a result of the separation of co-eluting analytes. As would be expected from the increased path length of the cyclic IM-MS instrument compared to the linear device, the largest gains in feature detection were obtained for the HILIC-cIM-MS combination. By increasing the resolution of coeluting components, the cyclic IM-MS instrumentation also provided the largest improvement in the quality of the mass spectral data obtained. When applied to mouse urines obtained from both control and gefitinib-dosed mice, time-related changes were detected in those obtained from the treated animals that were not seen in the controls. Polar metabolites affected by drug administration included, but were not limited to, hypoxanthine, 1,3-dimethyluracil and acetylcarnitine. The changes seen in the relative concentrations of these endogenous metabolites appeared to be related to drug concentrations in the plasma and urine suggesting a pharmacometabodynamic link.

Keywords:  Ion mobility; Metabolic phenotyping; Metabolite identification; Polar metabolites; Urine

DOI:  https://doi.org/10.1016/j.chroma.2023.464537
Anal Chim Acta. 2024 Jan 25. pii: S0003-2670(23)01339-9. [Epub ahead of print]1287 342118

Quantification of glucocorticoid and progestogen metabolites in bovine plasma, skimmed milk and saliva by UHPLC-HR-MS with polarity switching.

Karoline Rehm, Anna-Katharina Hankele, Susanne E Ulbrich, Laurent Bigler.

  Steroid metabolites are increasingly in focus when searching for novel biomarkers in physiological mechanisms and their disorders. While major steroids such as progesterone and cortisol are well-researched and routinely determined to assess the health, particularly the reproductive status of mammals, the function of potentially biologically active progestogen and glucocorticoid metabolites is widely unexplored. One of the main reasons for this is the lack of comprehensive, sensitive, and specific analytical methods. This is particularly the case when analyzing matrices like milk or saliva obtained by non-invasive sampling with steroid concentrations often below those present in plasma. Therefore, a new UHPLC-HR-MS method based on an Ultimate UHPLC system equipped with an Acquity HSS T3 reversed-phase column and a Q Exactive™ mass spectrometer was developed, enabling the simultaneous chromatographic separation, detection and quantification of eleven isobaric glucocorticoids (11-dehydrocorticosterone (A), corticosterone (B), cortisol (F), cortisone (E), the tetrahydrocortisols (THF): 3α,5α-THF, 3α,5β-THF, 3β,5α-THF, 3β,5β-THF, and the tetrahydrocortisones (THE): 3α,5α-THE, 3α,5β-THE, 3β,5α-THE) and twelve progestogens (progesterone (P4), pregnenolone (P5), the dihydroprogesterones (DHP): 20α-DHP, 20β-DHP, 3α-DHP, 3β-DHP, 5α-DHP, 5β-DHP, and the tetrahydroprogesterones (THP): 3α,5α-THP, 3α,5β-THP, 3β,5α-THP, 3β,5β-THP) in bovine plasma, skimmed milk, and saliva. A simple liquid-liquid extraction (LLE) with MTBE (methyl tert-butyl ether) was used for sample preparation of 500 μL plasma, skimmed milk, and saliva. Heated electrospray ionization (HESI) with polarity switching was applied to analyze steroids in high-resolution full scan mode (HR-FS). The method validation covered the investigation of sensitivity, selectivity, curve fitting, carry-over, accuracy, precision, recovery, matrix effects and applicability. A high sensitivity in the range of pg mL-1 was achieved for all steroids suitable for the analysis of authentic samples.

Keywords:  Cattle; Endogenous; LC-MS; Liquid-liquid extraction; Steroids; Surrogate matrix

DOI:  https://doi.org/10.1016/j.aca.2023.342118
J Pharm Biomed Anal. 2023 Dec 13. pii: S0731-7085(23)00687-8. [Epub ahead of print]240 115918

The first LC-MS/MS stereoselective bioanalytical methods to quantitatively detect 9R- and 9S-hexahydrocannabinols and their metabolites in human blood, oral fluid and urine.

Giorgi Kobidze, Giorgia Sprega, Eva Montanari, Omayema Taoussi, Giulia Bambagiotti, Maria Sofia Fede, Annagiulia Di Trana, Simona Pichini, Francesco Paolo Busardò, Anastasio Tini, Bezhan Chankvetadze, Alfredo Fabrizio Lo Faro.

  A sensitive LC-MS/MS method for the simultaneous quantification of the (9 R)- and (9 S)- hexahydrocannabinols (HHCs), and their metabolites, in human urine, oral fluid (OF) and blood samples were developed, validated and used to the biological samples of volunteers. The analytes were extracted from 100 μL human samples. An isocratic elution mode with methanol was used for chromatographic separation of (9 R)- and (9 S)-HHC on an immobilized amylose tris(3-chloro-5-methylphenylcarbamate)-based chiral column Lux i-Amylose-3. The flow-rate of the mobile phase was 0.5 mL/min. An isocratic elution mode of methanol and water (80/20, v/v) was used for chromatographic separation of metabolites of (9 R)- and (9 S)-HHC on a Lux AMP chiral column (with a proprietary chiral selector) at a flow rate of 0.5 mL/min. MS/MS analysis was performed in positive ionization mode for HHC epimers, while in negative ionization mode was used for metabolites of HHCs. The calibration curves for HHCs and their metabolites in human samples ranged from 0.25- 240 ng mL-1 and 1 - 100 ng mL-1, respectively, with determination coefficients (r2) of ≥ 0.99. All analytes were stable at room temperature, 4 °C, in the autosampler (+10 °C) and -20 °C for 24 h, after three freeze/thaw cycles, and when stored at -20 °C up to one week after quality control (QC) sample preparation (concentration differences less than 20% with respect to time zero response), in blood, urine and OF.

Keywords:  HPLC; Hexahydrocannabinol metabolites; Hexahydrocannabinols; Human samples; Stereoisomers

DOI:  https://doi.org/10.1016/j.jpba.2023.115918
Anal Chem. 2023 Dec 29.

COLMARppm: A Web Server Tool for the Accurate and Rapid Prediction of 1H and 13C NMR Chemical Shifts of Organic Molecules and Metabolites.

Nick Rigel, Da-Wei Li, Rafael Brüschweiler.

Despite rapid progress in metabolomics research, a major bottleneck is the large number of metabolites whose chemical structures are unknown or whose spectra have not been deposited in metabolomics databases. Nuclear magnetic resonance (NMR) spectroscopy has a long history of elucidating chemical structures from experimentally measured 1H and 13C chemical shifts. One approach to characterizing the chemical structures of an unknown metabolite is to predict the 1H and 13C chemical shifts of candidate compounds (e.g., metabolites from the Human Metabolome Database (HMDB)) and compare them with chemical shifts of the unknown. However, accurate prediction of NMR chemical shifts in aqueous solution is challenging due to limitations of experimental chemical shift libraries and the high computational cost of quantum chemical methods. To improve NMR prediction accuracy and applicability, an empirical prediction strategy is introduced here to provide an accurately predicted chemical shift for organic molecules and metabolites within seconds. Unique features of COLMARppm include (i) the training library exclusively consisting of high quality NMR spectra measured under standard conditions in aqueous solution, (ii) utilization of NMR motif information, and (iii) leveraging of the improved prediction accuracy for the automated assignment of experimental chemical shifts for candidate structures. COLMARppm is demonstrated in terms of accuracy and speed for a set of 20 compounds taken from the HMDB for chemical shift prediction and resonance assignment. COLMARppm is applicable to a wide range of small molecules and can be directly incorporated into metabolomics workflows.

DOI: https://doi.org/10.1021/acs.analchem.3c03677
Anal Chim Acta. 2024 Jan 25. pii: S0003-2670(23)01294-1. [Epub ahead of print]1287 342073

Simultaneous determination of free biliverdin and free bilirubin in serum: A comprehensive LC-MS approach.

Alen Albreht, Mitja Martelanc, Lovro Žiberna.

   BACKGROUND: Prognosis, diagnosis, and treatment of several diseases strongly rely on the sensitive, selective, and accurate determination of specific biomarkers in relevant biological samples. Free biliverdin and free bilirubin represent important new biomarkers of oxidative stress, however, the lack of suitable analytical methods for their determination has hindered progress in biomedical and clinical research.
RESULTS: Here, we introduce a first comprehensive approach for robust and simultaneous determination of these bilins in serum using liquid chromatography - mass spectrometry (LC-MS). The developed analytical method exhibits linearity for both analytes within the concentration range of 0.5-100 nM, with limits of detection and quantitation determined at 0.1 nM and 0.5 nM, respectively. Moreover, several analytical pitfalls related to the intrinsic molecular structures of free bilirubin and free biliverdin and their trace concentration levels in biological samples are discussed here in detail for the first time. We have shown that the solubility, chemical stability, and affinity of these bilins to various materials strongly depend on the solvent, pH, and addition of stabilizing and chelating agents. Finally, the validated LC-MS method was successfully applied to the analysis of both bilins in fetus bovine serums, yielding higher free bilirubin/biliverdin ratios compared with previously reported values for human serum.
SIGNIFICANCE: Failure to recognize and address the challenges presented here often leads to substantial analytical errors and consequently biased interpretation of the obtained results. This pertains not only to LC-MS, but also to many other analytical platforms due to the compound-derived sources of error.

Keywords:  Biomedical research; Matrix effects; Robustness; Sensitivity; Serum; Stability

DOI:  https://doi.org/10.1016/j.aca.2023.342073
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Dec 15. pii: S1570-0232(23)00382-3. [Epub ahead of print]1233 123972

Optimised liquid chromatography tandem mass spectrometry method for the simultaneous quantification of serum vitamin D analogues while also accounting for epimers and isobars.

Jacques van der Westhuizen, Barend Christiaan Vorster, Monique Opperman, Peet J Jansen van Rensburg.

  The accurate quantification of multiple vitamin D analogues simultaneously is challenging. This study set out to use liquid chromatography-tandem mass spectrometry (LC-MS/MS) to develop a method capable of measuring a comprehensive vitamin D profile, encompassing twelve vitamin D analogues (vitamin D2, D3, 25(OH)D2, 25(OH)D3, 1,25(OH)2D2, 1,25(OH)2D3, 24,25(OH)2D2, 24,25(OH)2D3, 3-epi-25(OH)D2, 3-epi-25(OH)D3, 7αC4 and1α(OH)D3) in a single run. Serum samples were prepared using double liquid-liquid extraction and analysed on an Agilent 6460 QQQ LC-MS/MS equipped with a Pursuit 3 Pentafluorophenyl (4.6 x 100 mm, 3 μm) column. Recovery rates for all analytes were above 95 % with a coefficient of variation (CV) below 10 %. The method exhibited good linearity (r > 0.995) and had a range of detection limits between 0.01 and 0.35 ng/mL and quantification limits between 0.15 and 0.96 ng/mL. Repeatability and within-lab precision were acceptable, with CV values below 10 % and 15 %, respectively. Method accuracy was excellent, with a systematic error below 6.60 %. additionally, all analytes-maintained stability for 48 h following sample preparation, and no interferences were observed among co-eluting analytes. Lastly, this method achieved "world-class" status according to the Sigma metric scale specifications, requiring minimal quality control to ensure data quality. This successfully validated method has the potential not only for improving vitamin D profiling procedures but also for aiding in the diagnosis of other genetic disorders where measuring beyond 25(OH)D is crucial.

Keywords:  LC-MS/MS; Vitamin D; Vitamin D deficiency; Vitamin D profiling

DOI:  https://doi.org/10.1016/j.jchromb.2023.123972
Anal Chim Acta. 2024 Jan 25. pii: S0003-2670(23)01338-7. [Epub ahead of print]1287 342117

Improved quantification of carbonyl sub-metabolome by liquid chromatography mass spectrometry using a fragment controlled multiplexed isotopic tag.

Xiaobo Tian, Gérard Hopfgartner.

   BACKGROUND: Carbonyl-containing metabolites are a class of key intermediate in metabolism, which has potentials to be biomarkers. Since their poor ionization, derivatization reagents, such as dansylhydrazine, are usually used to improve the sensitivity and/or to facilitate quantification. However, most current carbonyl derivatization reagents only have two channels, one is isotopically labeled and the other one is non-labeled. To quantify more samples in a run and using data-independent acquisition (DIA) mode to get comprehensive and unbiased mass fragmentation, we proposed a fragment-controlled isotopic tag, called DiMe-FP-NHNH2 (FP) which has five channels: Δ0, Δ3, Δ6, Δ9, and Δ12, thus up to 5 samples can be analyzed in a run.
RESULTS: The most important improvement is that the FP tag can produce multiple characteristic signals in tandem mass, diagnostic ions and neutral losses, which helps to selectively detect aldehydes/ketones for targeted and untargeted analysis. To exhibit all capabilities of the FP tag, we mimicked an untargeted metabolomics experiment, which comprises two steps. First, discovery step, using Data-Independent Analysis (SWATH-MS) and the labeling of two channels (Δ0 and Δ3), we picked out aldehyde/ketone from the pooled urine samples based on three characteristic signals, including isotope patterns, diagnostic ions, and neutral losses. Second, five-plex quantification, relative and absolute quantification were achieved in a single LC-MS analysis. Notably, because of different nominal masses, the FP tag can be used on any low or high resolution mass spectrometers.
SIGNIFICANCE: The benefits and performance of the FP tag are demonstrated by the analysis of urine samples collected from patients from a prostate cancer study, in which more than a thousand features were found based on MS1 fingerprint, but only around 120 aldehyde/ketone candidates were confirmed with characteristic signals and nine of which were quantified showing significant differences from healthy and reference urine samples.

Keywords:  Carbonyl sub-metabolome; Derivatization; LC-MS; Metabolomics; Multiplexing; QUAL/QUANT

DOI:  https://doi.org/10.1016/j.aca.2023.342117
NPJ Aging. 2024 Jan 02. 10(1): 2

Absolute quantification of nicotinamide mononucleotide in biological samples by double isotope-mediated liquid chromatography-tandem mass spectrometry (dimeLC-MS/MS).

Junya Unno, Kathryn F Mills, Tairo Ogura, Masayuki Nishimura, Shin-Ichiro Imai.

Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite for fundamental biological phenomena, including aging. Nicotinamide mononucleotide (NMN) is a key NAD+ intermediate that has been extensively tested as an effective NAD+-boosting compound in mice and humans. However, the accurate measurement of NMN in biological samples has long been a challenge in the field. Here, we have established an accurate, quantitative methodology for measuring NMN by using liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS) with double isotopic NMN standards. In this new methodology, the matrix effects of biological samples were properly adjusted, and the fate of NMN could be traced during sample processing. We have demonstrated that this methodology can accurately quantitate NMN levels in mouse plasma and confirmed quick, direct NMN uptake into blood circulation and cells. This double isotope-mediated LC-MS/MS (dimeLC-MS/MS) can easily be expanded to other NAD+-related metabolites as a reliable standard methodology for NAD+ biology.

DOI: https://doi.org/10.1038/s41514-023-00133-1
Anal Bioanal Chem. 2024 Jan 05.

Comparison of LC-MS-based methods for the determination of carboxylic acids in animal matrices.

Heidi E Schwartz-Zimmermann, Manuel Hündler, Nicole Reiterer, Sara Ricci, Raul Rivera-Chacon, Ezequias Castillo-Lopez, Qendrim Zebeli, Franz Berthiller.

  Carboxylic acids (CAs) are key players in human and animal metabolism. As they are hardly retained under reversed-phase liquid chromatography (RP-LC) conditions in their native form, derivatization is an option to make them accessible to RP-LC and simultaneously increase their response for mass spectrometric detection. In this work, two RP-LC tandem mass spectrometry-based methods using aniline or 3-nitrophenylhydrazine (3-NPH) as derivatization agents were compared with respect to several factors including completeness of derivatization, apparent recoveries (RAs) in both cow feces and ruminal fluid, and concentrations obtained in feces and ruminal fluid of cows. Anion exchange chromatography coupled to high-resolution mass spectrometry (AIC-HR-MS) served as reference method. Derivatization efficiencies were close to 100% for 3-NPH derivatization but variable (20-100%) and different in solvent solutions and matrix extracts for aniline derivatization. Likewise, average RAs of 13C-labeled short-chain fatty acids as internal standards were around 100% for 3-NPH derivatization but only 45% for aniline derivatization. Quantification of CAs in feces and ruminal fluid of cows initially fed a forage-only diet and then transitioned to a 65% high-grain diet which yielded similar concentrations for 3-NPH derivatization and AIC-HR-MS, but concentrations determined by aniline derivatization were on average five times lower. For these reasons, derivatization with aniline is not recommended for the quantitative analysis of CAs in animal samples.

Keywords:  Anion exchange chromatography; Derivatization; Mass spectrometry; Reversed-phase high-performance liquid chromatography; Short-chain fatty acids

DOI:  https://doi.org/10.1007/s00216-023-05113-8
Anal Chem. 2024 Jan 02.

Absolute Quantification of Human Serum Albumin Isoforms by Internal Calibration Based on a Top-Down LC-MS Approach.

Roy Lakis, François-Ludovic Sauvage, Emilie Pinault, Pierre Marquet, Franck Saint-Marcoux, Souleiman El Balkhi.

Well-characterized biomarkers using reliable quantitative methods are essential for the management of various pathologies such as diabetes, kidney, and liver diseases. Human serum albumin (HSA) isoforms are gaining interest as biomarkers of advanced liver pathologies. In view of the structural alterations observed for HSA, insights into its isoforms are required to establish them as reliable biomarkers. Therefore, a robust absolute quantification method seems necessary. In this study, we developed and validated a far more advanced top-down liquid chromatography-mass spectrometry (LC-MS) method for the absolute quantification of HSA isoforms, using myoglobin (Mb) as an internal standard for quantification and for mass recalibration. Two different quantification approaches were investigated based on peak integration from the deconvoluted spectrum and extracted ion chromatogram (XIC). The protein mixture human serum albumin/myoglobin eluted in well-shaped separated peaks. Mb allowed a systematic mass recalibration for every sample, resulting in extremely low mass deviations compared to conventional deconvolution-based methods. In total, eight HSA isoforms of interest were quantified. Specific-isoform calibration curves showing good linearity were obtained by using the deconvoluted peaks. Noticeably, the HSA ionization behavior appeared to be isoform-dependent, suggesting that the use of an enriched isoform solution as a calibration standard for absolute quantification studies of HSA isoforms is necessary. Good repeatability, reproducibility, and accuracy were observed, with better sensitivity for samples with low albumin concentrations compared to routine biochemical assays. With a relatively simple workflow, the application of this method for absolute quantification shows great potential, especially for HSA isoform studies in a clinical context, where a high-throughput method and sensitivity are needed.

DOI: https://doi.org/10.1021/acs.analchem.3c03933
Anal Chim Acta. 2024 Jan 25. pii: S0003-2670(23)01340-5. [Epub ahead of print]1287 342119

A chemical derivatization-based pseudotargeted liquid chromatography-tandem mass spectrometry method for sensitive and high coverage determination of bile acids in human serum.

Xin Xiong, Xunke Gu, Xiaona Li, Keke Jia, Yuan Wei, Rongsheng Zhao.

  Global profiling of bile acids (BAs) is imperative for understand their function and disease pathogenesis. But it is still a challenging task, as the collision-induced dissociation (CID) fragment ions of unconjugated BAs showed low ion intensities to insufficient analysis. Herein, we developed a highly sensitive method for pseudotargeted profiling of BAs by chemical derivatization. In the developed method, a labeling reagent, 2-dimethylaminoethylamine (DMED), was adopted to label the carboxyl group of BAs. The results demonstrated that the detection sensitivities of unconjugated BAs were increased by 4-200 folds after DMED-labeling. Moreover, to profile other potential BAs not included in the 91 known targets, diverse survey experiments were performed on Qtrap-MS to search BAs for both precursor and fragment ion species, and retention index (RI) strategy was adopted to facilitate the identification of isomers. Finally, MRM-based LC-MS/MS method was validated for the pseudotargeted profiling of the BAs submetabolome with good linearity (r2 ≥ 0.990 for 89 known BAs) and high sensitivity (0.05-0.5 ng/mL for unconjugated BAs), covering unconjugated, glycine, taurine, sulfuric acid, glucuronic acid, and as well as those doubly-conjugated with above types. With this method, a total of 107 BAs, covering 54 BAs identified by authentic standards and 53 BAs candidates, were successfully determined in human serum of women with intrahepatic cholestasis of pregnancy (ICP). Multivariate analysis revealed deferentially expressed BAs. ICP disease altered the BAs profile with a reduced proportion of unconjugated, sulfate- and doubly-conjugated BAs and an increased proportion of glycine and taurine conjugates. Altered proportion and profile of BAs in ICP groups were gradually recovered during the ursodeoxycholic acid (UDCA) therapy. Overall, the strategy of DMED-labeling technique combined with diverse survey experiments is sufficiently sensitive and robust to comprehensively analysis of metabolic profiling of BAs in human serum.

Keywords:  Bile acids; Chemical labeling; Intrahepatic cholestasis of pregnancy; Mass spectrometry; Pseudotargeted metabolomics

DOI:  https://doi.org/10.1016/j.aca.2023.342119
Sci Rep. 2024 01 02. 14(1): 40

Comparison between ammonium formate and ammonium fluoride in the analysis of stratum corneum lipids by reversed phase chromatography coupled with high resolution mass spectrometry.

Miriam Maiellaro, Grazia Bottillo, Alessia Cavallo, Emanuela Camera.

Lipids are key constituents of the barrier function in the human stratum corneum (SC), which is the outermost layer of the epidermis and amenable to non-invasive sampling by tape stripping. The three major lipid classes in the SC, i.e., ceramides, fatty acids, and cholesterol, present equimolar concentration. Liquid chromatography coupled with mass spectrometry (LCMS) is elective in profiling lipids in the SC in both positive and negative ion modes. Nevertheless, the latter one allows for the simultaneous detection of the three major epidermal components of the SC. Determination of ceramides in the SC poses analytical challenges due to their wide range of structures and concentrations especially in the case of limited sample amounts. Ammonium formate is a commonly used modifier added to the mobile phase to assist ionization. However, it introduces uncertainty in the identification of ceramides when operating in negative ion mode, even with high resolution MS. We tested the advantages of using fluoride in the lipid profiling of SC and unambiguous identification of ceramides subclasses. The use of fluoride enhanced the ionization of ceramides, regardless the specific substructure, solved misidentification issues, and was successfully applied to the simultaneous detection of all three lipid classes in the human SC.

DOI: https://doi.org/10.1038/s41598-023-50051-1
Drug Test Anal. 2023 Dec 30.

Quantitative analysis of tetrahydrocannabinol isomers and other toxicologically relevant drugs in blood.

Dylan Mantinieks, Matthew Di Rago, Olaf H Drummer, Linda Glowacki, Jennifer Schumann, Dimitri Gerostamoulos.

  A multi-analyte liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described, involving the separation of delta-9-tetrahydrocannabinol (delta-9-THC) and delta-8-THC in addition to other commonly encountered drugs and metabolites. Briefly, sample preparation involved an alkaline liquid-liquid extraction (methyl tert-butyl ether) of blood (100 μl). The solvent layer was transferred, evaporated to dryness, reconstituted, and samples then separated on an Agilent Poroshell 120 EC-C18 100 Å (50 mm × 3.0 mm, 2.7 μm) analytical column using a multi-step gradient elution of 50 mM ammonium formate in water (pH 3.5) and 0.1% formic acid in methanol over 14 min. A SCIEX Triple Quad 6500+ system operating in scheduled multiple reaction monitoring and positive electrospray ionization was used for detection. There were no interferences, and matrix effects were generally acceptable (±20% of neat response). Linearity was achieved within the calibration range, including methylamphetamine (MA) (10-1000 ng/ml), 3,4-methylenedioxy-N-methylamphetamine (MDMA) (10-1,000 ng/ml), cocaine (10-1000 ng/ml), and two THC isomers (1-100 ng/ml). Accuracies of MA, MDMA, cocaine, and two THC isomers were 3.6 to 8.9%, -1.2 to 4%, -5.3 to 5.8%, and -11 to 14%, respectively; while precision estimates of the same were 1.6 to 5.4%, 1.7 to 5.3%, 1.2 to 4.5%, and 2 to 10%, respectively. Autosampler stability and dilution integrity were within acceptable limits, and no carryover was detected at the limit of detection. This validated LC-MS/MS method made the routine identification of both delta-9-THC and delta-8-THC in blood possible.

Keywords:  amphetamines; cannabinoids; cocaine; driving under the influence; forensic toxicology

DOI:  https://doi.org/10.1002/dta.3632
Anal Chem. 2023 Dec 29.

Investigating the Metabolic Heterogeneity of Cancer Cells Using Functional Single-Cell Selection and nLC Combined with Multinozzle Emitter Mass Spectrometry.

Kai-Wen Cheng, Pin-Rui Su, Kate Jo-Ann Feller, Miao-Ping Chien, Cheng-Chih Hsu.

Tumor metastasis and cancer recurrence are often a result of cell heterogeneity, where specific subpopulations of tumor cells may be resistant to radio- or chemotherapy. To investigate this physiological and phenotypic diversity, single-cell metabolomics provides a powerful approach at the chemical level, where distinct lipid profiles can be found in different tumor cells. Here, we established a highly sensitive platform using nanoflow liquid chromatography (nLC) combined with multinozzle emitter electrospray ionization mass spectrometry for more in-depth metabolomics profiling. Our platform identified 15 and 17 lipids from individual osteosarcoma (U2OS) and glioblastoma (GBM) cells when analyzing single-cell samples. Additionally, we used the functional single-cell selection (fSCS) pipeline to analyze the subpopulations of cells with a DNA damage response (DDR) in U2OS cells and fast migration in GBM cells. Specifically, we observed a down-regulation of polyunsaturated fatty acids (PUFAs) in U2OS cells undergoing DDR, such as fatty acids FA 20:3; O2 and FA 17:4; O3. Furthermore, ceramides (Cer 38:0; O3) and triglycerides (TG 36:0) were found to be down-regulated in fast-migrating GBM cells compared to the slow-migrating subpopulation. These findings suggest the potential roles of these metabolites and/or lipids in the cellular behavior of the subpopulations.

DOI: https://doi.org/10.1021/acs.analchem.3c03688
Anal Chim Acta. 2024 Jan 25. pii: S0003-2670(23)01336-3. [Epub ahead of print]1287 342115

Comprehensive profiling of ceramides in human serum by liquid chromatography coupled to tandem mass spectrometry combining data independent/dependent acquisition modes.

D Luque-Córdoba, M Calderón-Santiago, O A Rangel-Zúñiga, A Camargo, J López-Miranda, F Priego-Capote.

  Ceramides are sphingolipids with a structural function in the cell membrane and are involved in cell differentiation, proliferation and apoptosis. Recently, these chemical species have been pointed out as potential biomarkers in different diseases, due to their abnormal levels in blood. In this research, we present an overall strategy combining data-independent and dependent acquisitions (DIA and DDA, respectively) for identification, confirmation, and quantitative determination of ceramides in human serum. By application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in DIA mode we identified 49 ceramides including d18:1, d18:0, d18:2, d16:1, d17:1 and t18:0 species. Complementary, quantitative determination of ceramides was based on a high-throughput and fully automated method consisting of solid-phase extraction on-line coupled to LC-MS/MS in DDA to improve analytical features avoiding the errors associated to sample processing. Quantitation limits were at pg mL-1 level, the intra-day and between-days variability were below 20 and 25 %, respectively; and the accuracy, expressed as bias, was always within ±25 %. The proposed method was tested with the CORDIOPREV cohort in order to obtain a qualitative and quantitative profiling of ceramides in human serum. This characterization allowed identifying d18:1 ceramides as the most concentrated with 70.8% of total concentration followed by d18:2 and d18:0 with 13.0 % and 8.8 %, respectively. Less concentrated ceramides, d16:1, d17:1 and t18:0, reported a 7.1 % of the total content. Combination of DIA and DDA LC-MS/MS analysis enabled to profile qualitative and quantitatively ceramides in human serum.

Keywords:  Ceramide; Data-dependent acquisition; Data-independent acquisition; LC–MS/MS; Multiple reaction monitoring; Serum

DOI:  https://doi.org/10.1016/j.aca.2023.342115
J Anal Toxicol. 2024 Jan 04. pii: bkad092. [Epub ahead of print]

A fast and reliable LC-MS/MS method for the quantification of saxitoxin in blood plasma samples.

Klára Odehnalová, Petra Přibilová, Blahoslav Maršálek, Pavel Babica.

  Saxitoxins (STXs) are potent neurotoxins produced by marine dinoflagellates or freshwater cyanobacteria, known to cause acute and eventually fatal human intoxications, which are classified as Paralytic Shellfish Poisonings (PSPs). Rapid analysis of STXs in blood plasma can be used for a timely diagnosis and confirmation of PSPs. We developed a fast and simple method of STX extraction based on plasma sample acidification and precipitation by acetonitrile, followed by quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our approach provides the results ≤30 min, with a limit of detection (LOD) of 2.8 ng/mL and a lower limit of quantification (LLOQ) of 5.0 ng/mL. Within-run and between-run precision experiments showed good reproducibility with ≤15 % values. Standard curves for calibration were linear with correlation coefficients ≥0.98 across the assay calibration range (5-200 ng/mL). In an interlaboratory analytical exercise, the method was found to be 100% accurate in determining the presence or absence of STX in human plasma specimens, with recovery values of 86-99 %. This simple method for STX determination in animal or human plasma can quickly and reliably diagnose STX exposures and confirm suspected PSPs cases to facilitate patient treatment or expedite necessary public health or security actions.

Keywords:  LC-MS/MS; biotoxin; human blood plasma; paralytic shellfish poisonings; saxitoxin

DOI:  https://doi.org/10.1093/jat/bkad092
Anal Chem. 2023 Dec 29.

Rapid Analysis of Fentanyl and Fentanyl Analogues from Whole Blood Using SPME Coupled to the Microfluidic Open Interface.

Emir Nazdrajić, Daniel A Rickert, Janusz Pawliszyn.

Fentanyl and its analogues are potent opioids that pose a significant threat to society. Over the last several years, considerable focus has been on the concerning trend of increasing fentanyl usage among drug users. Fentanyl analogues are mainly synthesized to evade analytical detection or increase their potency; thus, very low concentrations are sufficient to achieve a therapeutic effect. In an effort to help combat the synthetic opioid epidemic, developing targeted mass spectrometric methods for quantifying fentanyl and its analogues at ultralow concentrations is incredibly important. Most methods used to analyze fentanyl and its analogues from whole blood require manual sample preparation protocols (solid-phase extraction or liquid-liquid extraction), followed by chromatographic separation and mass spectrometric detection. The main disadvantages of these methods are the tedious sample preparation workflows, resulting in lengthy analysis times. To mitigate these issues, we present a targeted method capable of analyzing 96 samples containing fentanyl, several fentanyl analogues, and a common fentanyl (analogue) precursor simultaneously in 2.4 min per sample. This is possible by using a high-throughput solid phase microextraction workflow on the Concept96 autosampler followed by manual coupling of solid-phase microextraction fibers to the microfluidic open interface for tandem mass spectrometry analysis. Our quantitative method is capable of extremely sensitive analysis, with limits of quantification ranging from 0.002 to 0.031 ng mL-1 and linearity ranging from 0.010 to 25.0 ng mL-1. The method shows very good reproducibility (1-18%), accuracy (81-100%) of calibration and validation points, and good interday reproducibility (6-15%).

DOI: https://doi.org/10.1021/acs.analchem.3c04354
Anal Biochem. 2023 Dec 30. pii: S0003-2697(23)00420-7. [Epub ahead of print]687 115455

Evaluation of lipid extraction methods for fatty acid quantification and compound-specific δ13C and δ2Hn analyses.

Matthias Pilecky, Martin J Kainz, Leonard I Wassenaar.

  Lipids, with fatty acids (FA) as a crucial subset, have become a focal point for diverse medical, physiological, and ecological studies. However, a comprehensive assessment of the various pre-analytical FA extraction methods published in the scientific literature remains lacking. In this study, we examined the efficacy of seven well-established sample preparation methods, specifically focusing on their effectiveness in total lipid and fatty acid extraction and their impact on compound-specific stable hydrogen (δ2H) and carbon (δ13C) isotope values. We also considered the repercussions of FA removal efficacy on residual bulk tissue δ2Hn analysis, because lipids typically have low δ2H values. Our findings showed that in most cases chloroform-based extraction methods outperformed those without chloroform. While discrepancies were not as evident for smaller organisms, such as plankton, marked variations were discernible in the extraction efficiencies for muscle and liver samples, which was also manifested in the residual bulk tissue δ2Hn results. Notably, most extraction methods had little effect on specific δ13C or δ2H isotope values of FA; instead, an emphasis should be on using an extraction method that achieves optimal baseline peak separation of the chromatograms for C and H isotope measurements.

Keywords:  Compound-specific stable isotopes; Deuterium; Fatty acids; GC-IRMS; Lipids

DOI:  https://doi.org/10.1016/j.ab.2023.115455
Nat Biotechnol. 2024 Jan 02.

Fast mass spectrometry search and clustering of untargeted metabolomics data.

Mihir Mongia, Tyler M Yasaka, Yudong Liu, Mustafa Guler, Liang Lu, Aditya Bhagwat, Bahar Behsaz, Mingxun Wang, Pieter C Dorrestein, Hosein Mohimani.

The throughput of mass spectrometers and the amount of publicly available metabolomics data are growing rapidly, but analysis tools such as molecular networking and Mass Spectrometry Search Tool do not scale to searching and clustering billions of mass spectral data in metabolomics repositories. To address this limitation, we designed MASST+ and Networking+, which can process datasets that are up to three orders of magnitude larger than those processed by state-of-the-art tools.

DOI: https://doi.org/10.1038/s41587-023-01985-4
Commun Biol. 2024 Jan 05. 7(1): 45

LiLA: lipid lung-based ATLAS built through a comprehensive workflow designed for an accurate lipid annotation.

Belén Fernández Requena, Sajid Nadeem, Vineel P Reddy, Vanessa Naidoo, Joel N Glasgow, Adrie J C Steyn, Coral Barbas, Carolina Gonzalez-Riano.

Accurate lipid annotation is crucial for understanding the role of lipids in health and disease and identifying therapeutic targets. However, annotating the wide variety of lipid species in biological samples remains challenging in untargeted lipidomic studies. In this work, we present a lipid annotation workflow based on LC-MS and MS/MS strategies, the combination of four bioinformatic tools, and a decision tree to support the accurate annotation and semi-quantification of the lipid species present in lung tissue from control mice. The proposed workflow allowed us to generate a lipid lung-based ATLAS (LiLA), which was then employed to unveil the lipidomic signatures of the Mycobacterium tuberculosis infection at two different time points for a deeper understanding of the disease progression. This workflow, combined with manual inspection strategies of MS/MS data, can enhance the annotation process for lipidomic studies and guide the generation of sample-specific lipidome maps. LiLA serves as a freely available data resource that can be employed in future studies to address lipidomic alterations in mice lung tissue.

DOI: https://doi.org/10.1038/s42003-023-05680-7
Anal Bioanal Chem. 2023 Dec 30.

Comparison of high-resolution mass spectrometry acquisition methods for the simultaneous quantification and identification of per- and polyfluoroalkyl substances (PFAS).

Jordan M Partington, Sahil Rana, Drew Szabo, Tarun Anumol, Bradley O Clarke.

  Simultaneous identification and quantification of per- and polyfluoroalkyl substances (PFAS) were evaluated for three quadrupole time-of-flight mass spectrometry (QTOF) acquisition methods. The acquisition methods investigated were MS-Only, all ion fragmentation (All-Ions), and automated tandem mass spectrometry (Auto-MS/MS). Target analytes were the 25 PFAS of US EPA Method 533 and the acquisition methods were evaluated by analyte response, limit of quantification (LOQ), accuracy, precision, and target-suspect screening identification limit (IL). PFAS LOQs were consistent across acquisition methods, with individual PFAS LOQs within an order of magnitude. The mean and range for MS-Only, All-Ions, and Auto-MS/MS are 1.3 (0.34-5.1), 2.1 (0.49-5.1), and 1.5 (0.20-5.1) pg on column. For fast data processing and tentative identification with lower confidence, MS-Only is recommended; however, this can lead to false-positives. Where high-confidence identification, structural characterisation, and quantification are desired, Auto-MS/MS is recommended; however, cycle time should be considered where many compounds are anticipated to be present. For comprehensive screening workflows and sample archiving, All-Ions is recommended, facilitating both quantification and retrospective analysis. This study validated HRMS acquisition approaches for quantification (based upon precursor data) and exploration of identification workflows for a range of PFAS compounds.

Keywords:  Analytical chemistry; EPA Method 533; Emerging contaminants; Quadrupole time-of-flight mass spectrometry (QTOF); Suspect screening

DOI:  https://doi.org/10.1007/s00216-023-05075-x
Anal Chem. 2024 Jan 05.

QComics: Recommendations and Guidelines for Robust, Easily Implementable and Reportable Quality Control of Metabolomics Data.

Álvaro González-Domínguez, Núria Estanyol-Torres, Carl Brunius, Rikard Landberg, Raúl González-Domínguez.

The implementation of quality control strategies is crucial to ensure the reproducibility, accuracy, and meaningfulness of metabolomics data. However, this pivotal step is often overlooked within the metabolomics workflow and frequently relies on the use of nonstandardized and poorly reported protocols. To address current limitations in this respect, we have developed QComics, a robust, easily implementable and reportable method for monitoring and controlling data quality. The protocol operates in various sequential steps aimed to (i) correct for background noise and carryover, (ii) detect signal drifts and "out-of-control" observations, (iii) deal with missing data, (iv) remove outliers, (v) monitor quality markers to identify samples affected by improper collection, preprocessing, or storage, and (vi) assess overall data quality in terms of precision and accuracy. Notably, this tool considers important issues often neglected along quality control, such as the need of separately handling missing values and truly absent data to avoid losing relevant biological information, as well as the large impact that preanalytical factors may elicit on metabolomics results. Altogether, the guidelines compiled in QComics might contribute to establishing gold standard recommendations and best practices for quality control within the metabolomics community.

DOI: https://doi.org/10.1021/acs.analchem.3c03660
J Pharm Anal. 2023 Nov;13(11): 1353-1364

Polarity-regulated derivatization-assisted LC-MS method for amino-containing metabolites profiling in gastric cancer.

Jie Han, Shilin Gong, Xiqing Bian, Yun Qian, Guilan Wang, Na Li, Jian-Lin Wu.

  Amino-containing compounds, including amino acids, aliphatic amines, aromatic amines, small peptides and catecholamines, are involved in various biological processes and play vital roles in multiple metabolic pathways. Previous studies indicated that some amino-containing metabolites are significant diagnostic and prognostic biomarkers of gastric cancer. However, the discovery of precise biomarkers for the preoperative diagnosis of gastric cancer is still in an urgent need. Herein, we established a polarity-regulated derivatization method coupled with liquid chromatography-mass spectrometry (LC-MS) for amino-containing metabolites profiling in the serum samples of patients with gastric cancer and healthy controls, based on our newly designed and synthesized derivatization reagent (S)-3-(1-(diisopropoxyphosphoryl) pyrrolidine-2-carboxamido)-N-hydroxysuccinimidyl ester (3-DP-NHS). Enhanced separation efficiency and detection sensitivity for amino-containing metabolites were achieved after derivatization. This method exhibited good linearity, recovery, intra- and inter-day precision and accuracy. Only 5 μL serum is needed for untargeted analysis, enabling 202 amino-containing metabolites to be detected. Statistical analysis revealed altered amino acid metabolisms in patients with gastric cancer. Furthermore, ultra high performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS) analysis quantification revealed increased serum levels of tryptamine and decreased concentrations of arginine and tryptophan in patients with gastric cancer. Receiver operating characteristic (ROC) curves indicated that an increased tryptamine/tryptophan ratio could serve as a potential biomarker for gastric cancer diagnosis. This study demostrated the possibility of using serum amino acid biomarkers for gastric cancer diagnosis, providing new avenues for the treatment of gastric cancer.

Keywords:  Amino-containing metabolites; Gastric cancer; Liquid chromatography-mass spectrometry; Polarity-regulated derivatization

DOI:  https://doi.org/10.1016/j.jpha.2023.06.009
Anal Chem. 2023 Dec 29.

Decoding Complexity in Synthetic Oligonucleotides: Unraveling Coeluting Isobaric Impurity Ions by High Resolution Mass Spectrometry.

A M Abdullah, Cynthia Sommers, Jason D Rodriguez, Deyi Zhang, Darby Kozak, Jessica Hawes, Mohan Sapru, Kui Yang.

Analyzing coeluting impurities with similar masses in synthetic oligonucleotides by liquid chromatography-mass spectrometry (LC-MS) poses challenges due to inadequate separation in either dimension. Herein, we present a direct method employing fully resolved isotopic envelopes, enabled by high resolution mass spectrometry (HRMS), to identify and quantify isobaric impurity ions resulting from the deletion or addition of a uracil (U) or cytosine (C) nucleotide from or to the full-length sequence. These impurities may each encompass multiple sequence variants arising from various deletion or addition sites. The method utilizes a full or targeted MS analysis to measure accurate isotopic distributions that are chemical formula dependent but nucleotide sequence independent. This characteristic enables the quantification of isobaric impurity ions involving sequence variants, a capability typically unavailable in sequence-dependent MS/MS methods. Notably, this approach does not rely on standard curves to determine isobaric impurity compositions in test samples; instead, it utilizes the individual isotopic distributions measured for each impurity standard. Moreover, in cases where specific impurity standards are unavailable, the measured isotopic distributions can be adequately replaced with the theoretical distributions (calculated based on chemical formulas of standards) adjusted using experiment-specific correction factors. In summary, this streamlined approach overcomes the limitations of LC-MS analysis for coeluting isobaric impurity ions, offering a promising solution for the in-depth profiling of complex impurity mixtures in synthetic oligonucleotide therapeutics.

DOI: https://doi.org/10.1021/acs.analchem.3c05016
Anal Chim Acta. 2024 Jan 25. pii: S0003-2670(23)01313-2. [Epub ahead of print]1287 342092

Chromatographic speed classification for liquid chromatography using average theoretical peak time (ATPT).

Tiago Schena, Carin von Mühlen.

   BACKGROUND: The development of analytical techniques in the field of liquid chromatography has brought new frontiers in performance and analytical speed for the technique. The proper evaluation of the analytical boundaries achieved with those developments was not addressed in the literature, since different liquid chromatography (LC) techniques have not yet received any classification regarding their chromatographic speed. Defining chromatographic analysis speed based simply on analysis time is an outdated concept since it is sample and analyte-dependent. In this context, the application of the Average Theoretical Peak Time concept (ATPT) is proposed as a unified metric for chromatographic speed classification.
RESULTS: This metric was evaluated using PCA analysis in a group of more than 50 publications, which generated the classification of LC methods in normal, high, hyper, and ultra-high-speed separations using ATPT. Normal speed (ATPT values greater than 18000 ms/peak) was found in HPLC, nano-LC, SFC, and CEC methods. Therefore, high-speed methods (ATPT values between 4000 and 18000 ms/peak) were found in UHPLC techniques, while LC × LC methods presented higher ATPT values between 1000 and 4000 ms/peak being classified as hyper-speed separations. ATPT can also be used as an optimization parameter, since older methods show higher ATPT values, while recent published papers show lower values of this metric. This behavior is justified due to the improvement of the LC methods over the years.
SIGNIFICANCE: This work fulfills the gap in chromatographic definitions and metrics, regarding analytical speed in one-dimensional and multidimensional liquid chromatographic techniques and shows that ATPT metrics is a robust parameter that can be used to classify the separation speed as well as a metric to evaluate the LC Method optimization. It also corrects the historical application of separation time as a metric for chromatographic speed.

Keywords:  Average theoretical peak time; Chromatographic speed; Liquid chromatography methods; Optimization parameter; PCA

DOI:  https://doi.org/10.1016/j.aca.2023.342092
J Pharm Anal. 2023 Nov;13(11): 1365-1373

DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells.

Li Liu, Yu-Han Lu, Min-Dan Wang, Qun-Fei Zhao, Xiu-Ping Chen, Hang Yin, Chen-Guo Feng, Fang Zhang.

  In this work, a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolite profiling of the glutathione anabolic pathway (GAP) in cancer tissues and cells. The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate (DMMIC) was used to label the amino group of metabolites, and a reductant of dithiothreitol (DTT) was employed to stabilize the thiol group. By combining DMMIC derivatization with LC-MS, it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples, which had good linearity (R2 = 0.9981-0.9999), precision (interday precision of 1.6%-19.0% and intraday precision of 1.4%-19.8%) and accuracy (83.4%-115.7%). Moreover, the recovery assessments in tissues (82.5%-107.3%) and in cells (98.1%-118.9%) with GSH-13C2, 15N, and Cys-15N demonstrated the reliability of the method in detecting tissues and cells. Following a methodological evaluation, the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma (ESCC) and the effect of p-hydroxycinnamaldehyde (CMSP) on the GAP in KYSE-150 esophageal cancer cells. The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes, which can contribute to research on drugs and diseases.

Keywords:  DMMIC derivatization; Esophageal squamous cell carcinoma; Glutathione anabolic pathway; KYSE-150cell; LC-MS; Metabolite profiling; p-Hydroxycinnamaldehyde

DOI:  https://doi.org/10.1016/j.jpha.2023.08.016