bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒08‒27
29 papers selected by
Sofia Costa, Matterworks

  1. Metabolites. 2023 Aug 07. pii: 923. [Epub ahead of print]13(8):
      The untargeted approach to mass spectrometry-based metabolomics has a wide potential to investigate health and disease states, identify new biomarkers for diseases, and elucidate metabolic pathways. All this holds great promise for many applications in biological and chemical research. However, the complexity of instrumental parameters on advanced hybrid mass spectrometers can make the optimization of the analytical method immensely challenging. Here, we report a strategy to optimize the selected settings of a hydrophilic interaction liquid chromatography-tandem mass spectrometry method for untargeted metabolomics studies of human plasma, as a sample matrix. Specifically, we evaluated the effects of the reconstitution solvent in the sample preparation procedure, the injection volume employed, and different mass spectrometry-related operating parameters including mass range, the number of data-dependent fragmentation scans, collision energy mode, duration of dynamic exclusion time, and mass resolution settings on the metabolomics data quality and output. This study highlights key instrumental variables influencing the detection of metabolites along with suggested settings for the IQ-X tribrid system and proposes a new methodological framework to ensure increased metabolome coverage.
    Keywords:  LC-MS; liquid chromatography; mass spectrometry; method optimization; plasma; untargeted metabolomics
  2. Anal Chim Acta. 2023 Oct 09. pii: S0003-2670(23)00876-0. [Epub ahead of print]1277 341655
      Although various metabolomic methods have been reported in recent years, simultaneous detection of hydrophilic and hydrophobic metabolites in a single analysis remains a technical challenge. In this study, based on the combination of hydrophilic interaction liquid chromatography (HILIC) and reversed phase liquid chromatography (RPLC), an online two-dimensional liquid chromatography/triple quadrupole mass spectrometry method (2D-LC/TQMS) was developed for the simultaneous analysis of hydrophilic and hydrophobic metabolites of various biological samples. The method can measure 417 biologically important metabolites (e.g., amino acids and peptides, pyrimidines, purines, monosaccharides, fatty acids and conjugates, organic dicarboxylic acids, and others) with logP values ranging from -10.3 to 21.9. The metabolites are involved in a variety of metabolic pathways (e.g., purine metabolism, pyrimidine metabolism, tyrosine metabolism, galactose metabolism, gluconeogenesis, and TCA cycle). The developed method has good intra- and inter-day reproducibility (RSD of retention time <2%, RSD of peak area <30%), good linearity (R2 > 0.9) and wide linear range (from 0.0025 μg/mL to 5 μg/mL). The applicability of the method was tested using different biological samples (i.e., plasma, serum, urine, fecal, seminal plasma and liver) and it was found that 208 (out of 417) identical metabolites were detected in all biological samples. Furthermore, the metabolomic method was applied to a case/control study of urinary of bladder cancer. Thirty differential metabolites were identified that were involved in carbohydrate and amino acid metabolism.
    Keywords:  Biological sample; Bladder cancer; Targeted metabolomics; Triple quadrupole mass spectrometry; Two-dimensional liquid chromatography
  3. Anal Chem. 2023 Aug 23.
      Peak alignment is a crucial step in liquid chromatography-mass spectrometry (LC-MS)-based large-scale untargeted metabolomics workflows, as it enables the integration of metabolite peaks across multiple samples, which is essential for accurate data interpretation. Slight differences or fluctuations in chromatographic separation conditions, however, can cause the chromatographic retention time (RT) shift between consecutive analyses, ultimately affecting the accuracy of peak alignment between samples. Here, we introduce a novel RT shift correction method based on the retention index (RI) and apply it to peak alignment. We synthesized a series of N-acyl glycine (C2-C23) homologues via the amidation reaction between glycine with normal saturated fatty acids (C2-C23) as calibrants able to respond proficiently in both mass spectrometric positive- and negative-ion modes. Using these calibrants, we established an N-acyl glycine RI system. This RI system is capable of covering a broad chromatographic space and addressing chromatographic RT shift caused by variations in flow rate, gradient elution, instrument systems, and LC separation columns. Moreover, based on the RI system, we developed a peak shift correction model to enhance peak alignment accuracy. Applying the model resulted in a significant improvement in the accuracy of peak alignment from 15.5 to 80.9% across long-term data spanning a period of 157 days. To facilitate practical application, we developed a Python-based program, which is freely available at
  4. Metabolites. 2023 Aug 20. pii: 963. [Epub ahead of print]13(8):
      Approximately 25% of psoriasis patients have an inflammatory arthritis termed psoriatic arthritis (PsA). There is strong interest in identifying and validating biomarkers that can accurately and reliably predict conversion from psoriasis to PsA using novel technologies such as metabolomics. Lipids, in particular, are of key interest in psoriatic disease. We sought to develop a liquid chromatography-mass spectrometry (LC-MS) method to be used in conjunction with solid-phase microextraction (SPME) for analyzing fatty acids and similar molecules. A total of 25 chromatographic methods based on published lipid studies were tested on two LC columns. As a proof of concept, serum samples from psoriatic disease patients (n = 27 psoriasis and n = 26 PsA) were processed using SPME and run on the selected LC-MS method. The method that was best for analyzing fatty acids and fatty acid-like molecules was optimized and applied to serum samples. A total of 18 tentatively annotated features classified as fatty acids and other lipid compounds were statistically significant between psoriasis and PsA groups using both multivariate and univariate approaches. The SPME-LC-MS method developed and optimized was capable of detecting fatty acids and similar lipids that may aid in differentiating psoriasis and PsA patients.
    Keywords:  lipids; liquid chromatography; mass spectrometry; metabolomics; psoriatic disease; solid-phase microextraction
  5. J Pharm Biomed Anal. 2023 Aug 14. pii: S0731-7085(23)00402-8. [Epub ahead of print]235 115633
      Sulfasalazine has been identified as a candidate molecule to be investigated as an intervention to treat preterm pre-eclampsia during pregnancy. However, placental exposure of sulfasalazine and its systemically absorbed metabolite, sulfapyridine, is unknown. A robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to simultaneously quantitate these analytes in human placenta with an application to a pilot clinical trial. The placental tissue was homogenised using a water:methanol (1:1, v/v) mixture, followed by sample extraction using both protein precipitation and solid phase extraction. Sulfasalazine-d4 and sulfapyridine-d4 were used as internal standards. An Agilent Poroshell EC-C18 (3.0 ×100 mm, 2.7 µm) column was used for chromatographic separation, with gradient elution employed at a flow rate of 0.450 mL/min over a total run time of seven minutes. The mobile phases consisted of water with 0.1% formic acid (mobile phase A) and acetonitrile:methanol (90:10, v/v) with 0.1% formic acid (mobile phase B). A Shimadzu-8040 mass spectrometer was operated in multiple reaction monitoring (MRM) mode using positive electrospray ionisation (ESI). For both analytes, the assay was validated over the range 30-30,000 ng/mL, or 150-150,000 ng/g. During inter-day validations (n = 18), the average accuracies of quality controls ranged from 101.6% to 112.7% with corresponding precisions of 4.4-6.7% for sulfasalazine, and from 97.4% to 108.4%, with corresponding precisions of 3.7-10.0% for sulfapyridine. No significant matrix effects were observed, and the method proved to be sensitive and specific for both analytes. This study presents the first validated analytical method for quantifying sulfasalazine and sulfapyridine in human placenta as part of a pilot clinical trial to generate preliminary data on its pharmacokinetics and efficacy as in intervention for preterm pre-eclampsia.
    Keywords:  Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Placenta; Pre-eclampsia; Sulfapyridine; Sulfasalazine
  6. Anal Bioanal Chem. 2023 Aug 25.
      Metabolomics is a biochemical analysis tool for identifying metabolic phenotypes and used to reveal the pathogenic mechanisms of disease and to inform drug-targeted therapies. Carboxyl-containing metabolites (CCMs) account for an important proportion of the metabolome, but because of the diversity of physical and chemical properties of CCMs in biological samples, traditional liquid chromatography-mass spectrometry (LC-MS) targeted metabolome analysis methods cannot achieve simultaneous quantification of multiple types of CCMs. Therefore, we proposed for the first time a targeted metabolomics strategy using isoniazid derivatization combined with LC-MS/MS to simultaneously quantify 39 CCMs of 5 different types (short-chain fatty acids, amino acids, bile acids, phenylalanine and tryptophan metabolic pathway acids) with large polarity differences associated with Alzheimer's disease (AD) and significantly improve the detection coverage and sensitivity. The yields of isoniazid derivative CCMs were high and could guarantee the accuracy of CCM quantification. The LODs of CCMs increased significantly (1.25-2000-fold) after derivatization. The method showed good selectivity, intra-day and inter-day accuracies and precisions, and repeatability. There was no significant effect on the determination of CCMs in terms of matrix effect and recovery. CCMs showed good stability. And CCMs showed good stability under short-term storage and freeze-thaw cycles. At the same time, the regulatory effects of Schisandrae chinensis Fructus and Ginseng Radix et Rhizoma (SG) herb pair on CCM metabolic disorders in feces, urine, serum, and the brain of AD rats were elucidated from the perspective of targeted metabolomics. In combination with pharmacodynamic evaluation and gut microbiota analysis, the mechanism of SG herb pair on AD rats was comprehensively understood. In summary, this innovative isoniazid derivatization combined with a targeted metabolomics method has great potential for trace biological lineage analysis.
    Keywords:  Alzheimer’s disease; Carboxyl-containing metabolites; Isoniazid derivatization; Targeted metabolomics
  7. Anal Chem. 2023 Aug 21.
      The purity of tandem mass spectrometry (MS/MS) is essential to MS/MS-based metabolite annotation and unknown exploration. This work presents a de novo approach to cleaning chimeric MS/MS spectra generated in liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based metabolomics. The assumption is that true fragments and their precursors are well correlated across the samples in a study, while false or contamination fragments are rather independent. Using data simulation, this work starts with an investigation of the negative effects of chimeric MS/MS spectra on spectral similarity analysis and molecular networking. Next, the characteristics of true and false fragments in chimeric MS/MS spectra were investigated using MS/MS of chemical standards. We recognized three fragment peak attributes indicative of whether a peak is a false fragment, including (1) intensity ratio fluctuation, (2) appearance rate, and (3) relative intensity. Using these attributes, we tested three machine learning models and identified XGBoost as the best model to achieve an area under the precision-recall curve of 0.98 for a clear separation between true and false fragments. Based on the trained model, we constructed an automated bioinformatic platform, DNMS2Purifier (short for de novo MS2Purifier), for metabolic features from metabolomics studies. DNMS2Purifier recognizes and processes chimeric MS/MS spectra without additional sample analysis or library confirmation. DNMS2Purifer was evaluated on a metabolomics data set generated with different MS/MS precursor isolation windows. It successfully captured the increase in the number of false fragments from the increased isolation window. DNMS2Purifier was also compared to MS2Purifier, an existing MS/MS spectral cleaning tool based on the addition of data-independent acquisition (DIA) analysis. Results indicated that DNMS2Purifier uniquely recognizes false fragments, which complements the previous DIA-based approach. Finally, DNMS2Purifier was demonstrated using a real experimental metabolomics study, showing improved MS/MS spectral quality and leading to an improved spectral match ratio and molecular networking outcome.
  8. STAR Protoc. 2023 Aug 17. pii: S2666-1667(23)00184-3. [Epub ahead of print]4(3): 102226
      Polyunsaturated fatty acids (PUFAs) and their oxidized products (oxylipins) are important mediators in intra- and extra-cellular signaling. We describe here the simultaneous quantification of 163 PUFAs and oxylipins using liquid chromatography-mass spectrometry (LC-MS). The protocol details steps for PUFA purification from various biological materials, the conditions for LC-MS analysis, as well as quantitative approaches for data evaluation. We provide an example of PUFA quantification in animal tissue along with the bioinformatic protocol, enabling efficient inter-sample comparison and statistical analysis. For complete details on the use and execution of this protocol, please refer to Vila et al.,1 Costanza et al.,2 Blomme et al.,3 and Blomme et al.4.
    Keywords:  Chemistry; Mass Spectrometry; Metabolomics
  9. Metabolites. 2023 Aug 13. pii: 944. [Epub ahead of print]13(8):
      Large-scale metabolomics assays are widely used in epidemiology for biomarker discovery and risk assessments. However, systematic errors introduced by instrumental signal drifting pose a big challenge in large-scale assays, especially for derivatization-based gas chromatography-mass spectrometry (GC-MS). Here, we compare the results of different normalization methods for a study with more than 4000 human plasma samples involved in a type 2 diabetes cohort study, in addition to 413 pooled quality control (QC) samples, 413 commercial pooled plasma samples, and a set of 25 stable isotope-labeled internal standards used for every sample. Data acquisition was conducted across 1.2 years, including seven column changes. In total, 413 pooled QC (training) and 413 BioIVT samples (validation) were used for normalization comparisons. Surprisingly, neither internal standards nor sum-based normalizations yielded median precision of less than 30% across all 563 metabolite annotations. While the machine-learning-based SERRF algorithm gave 19% median precision based on the pooled quality control samples, external cross-validation with BioIVT plasma pools yielded a median 34% relative standard deviation (RSD). We developed a new method: systematic error reduction by denoising autoencoder (SERDA). SERDA lowered the median standard deviations of the training QC samples down to 16% RSD, yielding an overall error of 19% RSD when applied to the independent BioIVT validation QC samples. This is the largest study on GC-MS metabolomics ever reported, demonstrating that technical errors can be normalized and handled effectively for this assay. SERDA was further validated on two additional large-scale GC-MS-based human plasma metabolomics studies, confirming the superior performance of SERDA over SERRF or sum normalizations.
    Keywords:  GC–MS; data normalization; derivatization; primary metabolism; statistics
  10. J Am Soc Mass Spectrom. 2023 Aug 22.
      Mass spectrometry imaging (MSI) is an analytical technique capable of measuring and visualizing the spatial distribution of thousands of ions across a sample. Measured ions can be putatively identified and annotated by comparing their mass-to-charge ratio (m/z) to a database of known compounds. For high-resolution, accurate mass (HRAM) imaging data sets, this is commonly performed by the annotation platform METASPACE. Annotations are reported with a metabolite-signal-match (MSM) score as a measure of the annotation's confidence level. However, the MSM scores reported by METASPACE often do not reflect a reasonable confidence level of an annotation and are not assigned consistently. The metabolite annotation confidence score (MACS) is an alternative scoring system based on fundamental mass spectrometry imaging metrics (mass measurement accuracy, spectral accuracy, and spatial distribution) to generate values that reflect the confidence of a specific annotation in HRAM-MSI data sets. Herein, the MACS system is characterized and compared to MSM scores from ions annotated by METASPACE.
    Keywords:  IR-MALDESI; MATLAB; SSIM; annotation scoring; mass measurement accuracy; spectral accuracy
  11. Front Mol Biosci. 2023 ;10 1230282
      This mini review focuses on the opportunities provided by current and emerging separation techniques for mass spectrometry metabolomics. The purpose of separation technologies in metabolomics is primarily to reduce complexity of the heterogeneous systems studied, and to provide concentration enrichment by increasing sensitivity towards the quantification of low abundance metabolites. For this reason, a wide variety of separation systems, from column chemistries to solvent compositions and multidimensional separations, have been applied in the field. Multidimensional separations are a common method in both proteomics applications and gas chromatography mass spectrometry, allowing orthogonal separations to further reduce analytical complexity and expand peak capacity. These applications contribute to exponential increases in run times concomitant with first dimension fractionation followed by second dimension separations. Multidimensional liquid chromatography to increase peak capacity in metabolomics, when compared to the potential of running additional samples or replicates and increasing statistical confidence, mean that uptake of these methods has been minimal. In contrast, in the last 15 years there have been significant advances in the resolution and sensitivity of ion mobility spectrometry, to the point where high-resolution separation of analytes based on their collision cross section approaches chromatographic separation, with minimal loss in sensitivity. Additionally, ion mobility separations can be performed on a chromatographic timescale with little reduction in instrument duty cycle. In this review, we compare ion mobility separation to liquid chromatographic separation, highlight the history of the use of ion mobility separations in metabolomics, outline the current state-of-the-art in the field, and discuss the future outlook of the technology. "Where there is one, you're bound to divide it. Right in two", James Maynard Keenan.
    Keywords:  chromatography; drift time; drift tube; high-field asymmetric; separation; trapped; travelling wave
  12. J Mass Spectrom. 2023 Aug 24. e4973
      Omics studies such as metabolomics, lipidomics, and proteomics have become important for understanding the mechanisms in living organisms. However, the compounds detected are structurally different and contain isomers, with each structure or isomer leading to a different result in terms of the role they play in the cell or tissue in the organism. Therefore, it is important to detect, characterize, and elucidate the structures of these compounds. Liquid chromatography and mass spectrometry have been utilized for decades in the structure elucidation of key compounds. While prediction models of parameters (such as retention time and fragmentation pattern) have also been developed for these separation techniques, they have some limitations. Moreover, ion mobility has become one of the most promising techniques to give a fingerprint to these compounds by determining their collision cross section (CCS) values, which reflect their shape and size. Obtaining accurate CCS enables its use as a filter for potential analyte structures. These CCS values can be measured experimentally using calibrant-independent and calibrant-dependent approaches. Identification of compounds based on experimental CCS values in untargeted analysis typically requires CCS references from standards, which are currently limited and, if available, would require a large amount of time for experimental measurements. Therefore, researchers use theoretical tools to predict CCS values for untargeted and targeted analysis. In this review, an overview of the different methods for the experimental and theoretical estimation of CCS values is given where theoretical prediction tools include computational and machine modeling type approaches. Moreover, the limitations of the current experimental and theoretical approaches and their potential mitigation methods were discussed.
    Keywords:  CCS; computational methods; ion mobility; machine learning; omics
  13. Metabolites. 2023 Aug 12. pii: 941. [Epub ahead of print]13(8):
      Metabolomics has advanced to an extent where it is desired to standardize and compare data across individual studies. While past work in standardization has focused on data acquisition, data processing, and data storage aspects, metabolomics databases are useless without ontology-based descriptions of biological samples and study designs. We introduce here a user-centric tool to automatically standardize sample metadata. Using such a tool in frontends for metabolomic databases will dramatically increase the FAIRness (Findability, Accessibility, Interoperability, and Reusability) of data, specifically for data reuse and for finding datasets that share comparable sets of metadata, e.g., study meta-analyses, cross-species analyses or large scale metabolomic atlases. SMetaS (Sample Metadata Standardizer) combines a classic database with an API and frontend and is provided in a containerized environment. The tool has two user-centric components. In the first component, the user designs a sample metadata matrix and fills the cells using natural language terminology. In the second component, the tool transforms the completed matrix by replacing freetext terms with terms from fixed vocabularies. This transformation process is designed to maximize simplicity and is guided by, among other strategies, synonym matching and typographical fixing in an n-grams/nearest neighbors model approach. The tool enables downstream analysis of submitted studies and samples via string equality for FAIR retrospective use.
    Keywords:  FAIR; meta-analysis; metadata; repository; standardization
  14. Pak J Pharm Sci. 2023 Jul;36(4(Special)): 1281-1290
      A new method for the determination of rebamipide in human heparin sodium plasma by LC-MS was established and its methodology was validated. In this method, protein precipitation method was used to pretreat the samples and the rebamipide-d4 isotope of rebamipide was used as the internal standard. In the multi reaction monitoring mode, the electrospray ion source was used as the ionization technolog and LC-MS was used for detection and analysis. The liquid chromatographic conditions were: 00B-4605-AN (Kinetex® XB-C18 100A 50mm × 2.1mm, 5μm); mobile phase A: 0.1% FA and 1 mM NH4FA aqueous solution, mobile phase B: 0.1% FA and 1mM NH4FA 90% ACN solution, flow rate: 0.300mL/min, injection volume: 10uL, column temperature: 30oC, collection time: 3 min, injector temperature control: 5oC. The retention time of rebamipide and rebamipide-d4 were 1.32min and 1.31min, respectively. The lower limit of quantification was 1ng/mL and the calibration map of rebamipide in the concentration range of 1 to 800ng/mL was linear (R2 >0.990, n=11). The CV% values of the inter and intra batch precision of the method were both less than 15.0%. This method has been successfully applied to pharmacokinetic studies to evaluate the main pharmacokinetic parameters of rebamipide.
  15. Anal Methods. 2023 Aug 22.
      Anti-obesity drugs, used to suppress appetite and reduce fat absorption, have been circulated and traded illegally worldwide. The traditional methods of liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) for analyzing these drugs in in vivo samples require complex sample pretreatment and time-consuming procedures. To address this issue, a thermal-assisted paper spray mass spectrometry (PS-MS) method was developed in this study to analyze anti-obesity drugs in raw urine. By incorporating a heat source and optimizing the spray solvent and paper substrate, this technique demonstrates reduced matrix effect and higher sensitivity compared to traditional PS-MS methodology for direct analysis of anti-obesity drugs in urine samples. A temperature range of 100-200 °C can be set for screening anti-obesity drugs in urine samples, with the flexibility to adjust the temperature according to the specific drug being analyzed. The limits of detection (LODs) for these 15 anti-obesity drugs in urine ranged between 1 and 500 ng mL-1. Furthermore, the thermal-assisted PS-MS method exhibited good linearities (R2, 0.9903-0.9997) within the range from 10-100 to 1000 ng mL-1 for the direct quantitation of anti-obesity drugs in urine samples with an internal standard. Therefore, the thermal-assisted PS-MS technique may provide a novel approach for the direct analysis of drugs in complex samples.
  16. J Pharm Biomed Anal. 2023 Aug 19. pii: S0731-7085(23)00398-9. [Epub ahead of print]235 115629
      BACKGROUND: Direct acting antiviral (DAA) therapies are effective in the treatment and management of chronic HCV infections. Glecaprevir (GLE) and pibrentasvir (PIB) are pangenotypic DAAs that are delivered alone or as a fixed-dose oral formulation to treat chronic HCV infections with or without cirrhosis. Sensitive and dynamic bioanalytical assays are needed to understand the pharmacology of GLE and PIB.METHODS: Drug free K2EDTA plasma was spiked with GLE, PIB, and their internal standards. Drugs were extracted from plasma via protein precipitation, and subsequently quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated according to regulatory recommendations, and evaluated in remnant plasma samples from individuals prescribed GLE and PIB.
    RESULTS: The analytical measuring ranges for GLE and PIB were 0.25-2000 ng/mL and 0.25-1000 ng/mL, respectively. The method showed acceptable accuracy and precision for both DAAs. GLE and PIB in plasma were stable following six freeze thaw cycles and at room temperature for up to 67 h. All analytes were stable in whole blood incubated at room temperature for 24 h, and at 40 °C and 100% humidity for 2 h. Negligible percent matrix effects were observed for PIB and PIB-IS across the measuring range of the assay. Significant ion suppression was observed for GLE, with an average matrix effects of 27.9%. However, relative matrix effects were < 6.3% between drug and internal standard, and deemed acceptable. Assay validation assessments in alternative matrices also met acceptance criteria. Both DAAs were successfully measured in remnant plasma samples from individuals administered GLE and PIB.
    CONCLUSIONS: An LC-MS/MS method for GLE and PIB quantification in plasma has been developed and validated. The assay met acceptable performance criteria and may be used in downstream applications to characterize DAA pharmacology for HCV treatment.
    Keywords:  Direct acting antivirals; Glecaprevir; Hepatitis C; LC-MS/MS; Mass spectrometry; Pibrentasvir
  17. Mass Spectrom Rev. 2023 Aug 19.
      Epigenetic modifications are closely related to certain disorders of the organism, including the development of tumors. One of the main epigenetic modifications is the methylation of DNA cytosines, 5-methyl-2'-deoxycycytidine. Furthermore, 5-mdC can be oxidized to form three new modifications, 5-(hydroxymethyl)-2'-deoxycytidine, 5-formyl-2'-deoxycytidine, and 5-carboxy-2'-deoxycytidine. The coupling of liquid chromatography with tandem mass spectrometry has been widely used for the total determination of methylated DNA cytosines in samples of biological and clinical interest. These methods are based on the measurement of the free compounds (e.g., urine) or after complete hydrolysis of the DNA (e.g., tissues) followed by a preconcentration, derivatization, and/or clean-up step. This review highlights the main advances in the quantification of modified nucleotides and nucleosides by isotope dilution using isotopically labeled analogs combined with liquid or gas chromatography coupled to mass spectrometry reported in the last 20 years. The different possible sources of labeled compounds are indicated. Special emphasis has been placed on the different types of chromatography commonly used (reverse phase and hydrophilic interaction liquid chromatography) and the derivatization methods developed to enhance chromatographic resolution and ionization efficiency. We have also revised the application of bidimensional chromatography and indicated significant biological and clinical applications of these determinations.
    Keywords:  gas and liquid chromatography; isotope dilution; mass spectrometry; methylated and modified nucleotides and nucleosides
  18. Metabolites. 2023 Aug 19. pii: 962. [Epub ahead of print]13(8):
      Gas chromatography-mass spectrometry (GC-MS) usually employs hard electron ionization, leading to extensive fragmentations that are suitable to identify compounds based on library matches. However, such spectra are less useful to structurally characterize unknown compounds that are absent from libraries, due to the lack of readily recognizable molecular ion species. We tested methane chemical ionization on 369 trimethylsilylated (TMS) derivatized metabolites using a quadrupole time-of-flight detector (QTOF). We developed an algorithm to automatically detect molecular ion species and tested SIRIUS software on how accurate the determination of molecular formulas was. The automatic workflow correctly recognized 289 (84%) of all 345 detected derivatized standards. Specifically, strong [M - CH3]+ fragments were observed in 290 of 345 derivatized chemicals, which enabled the automatic recognition of molecular adduct patterns. Using Sirius software, correct elemental formulas were retrieved in 87% of cases within the top three hits. When investigating the cases for which the automatic pattern analysis failed, we found that several metabolites showed a previously unknown [M + TMS]+ adduct formed by rearrangement. Methane chemical ionization with GC-QTOF mass spectrometry is a suitable avenue to identify molecular formulas for abundant unknown peaks.
    Keywords:  chemical ionization; compound identification; quadrupole time-of-flight
  19. J Sep Sci. 2023 Aug 21. e2300343
      The analysis of organic acids in complex mixtures by LC-MS can often prove challenging, especially due to the poor sensitivity of negative ionization mode required for detection of these compounds in their native (i.e., underivatized or untagged) form. These compounds have also been difficult to measure using supercritical fluid chromatography (SFC)-MS, a technique of growing importance for metabolomic analysis, with similar limitations based on negative ionization. In this report, the use of a high proton affinity N-(4-aminophenyl)piperidine derivatization tag is explored for the improvement of organic acid detection by SFC-MS. Four organic acids (lactic, succinic, malic, and citric acids) with varying numbers of carboxylate groups were derivatized with N-(4-aminophenyl)piperidine to achieve detection limits down to 0.5 ppb, with overall improvements in detection limit ranging from 25-to-2100-fold. The effect of the derivatization group on sensitivity, which increased by at least 200-fold for compounds that were detectable in their native form, and mass spectrometric detection are also described. Preliminary investigations into the separation of these derivatized compounds identified multiple stationary phases that could be used for complete separation of all four compounds by SFC. This derivatization technique provides an improved approach for the analysis of organic acids by SFC-MS, especially for those that are undetectable in their native form.
    Keywords:  SFC-MS; chemical derivatization; organic acids; supercritical fluid chromatography
  20. Metabolites. 2023 Aug 15. pii: 948. [Epub ahead of print]13(8):
      Metabolomics is an analytical approach that involves profiling and comparing the metabolites present in biological samples. This scoping review article offers an overview of current metabolomics approaches and their utilization in evaluating metabolic changes in biological fluids that occur in response to viral infections. Here, we provide an overview of metabolomics methods including high-throughput analytical chemistry and multivariate data analysis to identify the specific metabolites associated with viral infections. This review also focuses on data interpretation and applications designed to improve our understanding of the pathogenesis of these viral diseases.
    Keywords:  COVID-19; HBV; HCMV; HCV; HIV; LC-MS; NMR; influenza; metabolomics; viral infections
  21. Metabolites. 2023 Aug 03. pii: 908. [Epub ahead of print]13(8):
      To represent the composition of small molecules circulating in HepG2 cells and the formation of the "core" of characteristic metabolites that often attract researchers' attention, we conducted a meta-analysis of 56 datasets obtained through metabolomic profiling via mass spectrometry and NMR. We highlighted the 288 most commonly studied compounds of diverse chemical nature and analyzed metabolic processes involving these small molecules. Building a complete map of the metabolome of a cell, which encompasses the diversity of possible impacts on it, is a severe challenge for the scientific community, which is faced not only with natural limitations of experimental technologies, but also with the absence of transparent and widely accepted standards for processing and presenting the obtained metabolomic data. Formulating our research design, we aimed to reveal metabolites crucial to the Hepg2 cell line, regardless of all chemical and/or physical impact factors. Unfortunately, the existing paradigm of data policy leads to a streetlight effect. When analyzing and reporting only target metabolites of interest, the community ignores the changes in the metabolomic landscape that hide many molecular secrets.
    Keywords:  GC-MS; HepG2; LC-MS; NMR; data availability; data submission guidelines; meta-analysis; metabolic profile; panoramic metabolomics
  22. Anal Chim Acta. 2023 Oct 09. pii: S0003-2670(23)00889-9. [Epub ahead of print]1277 341668
      Indoxyl sulfate (INDS) and p-cresol sulfate (pCS) are two of the most relevant uremic toxins that are recognized to have an essential role in chronic kidney disease (CKD) progression and associated cardiovascular risk. Thus, it is crucial to accurately assess their circulating levels in the body. Aiming at establishing an analytical strategy for quantification of INDS and pCS in human plasma, an automatic on-line micro-solid-phase extraction (μSPE) procedure hyphenated to tandem mass spectrometry (MS/MS) detection without previous chromatographic separation was herein developed. The bead injection (BI) concept was used to implement the μSPE procedure in the lab-on-valve (LOV) format. After studying the extraction conditions, the anion-exchange OASIS WAX sorbent beads (10 mg) and 99% ACN-H2O (15:85, v/v)-1% (v/v) NH4OH were chosen as sorbent and eluent, respectively, as they provided the highest analyte recoveries. Subsequently, the μSPE-BI-LOV system was hyphenated on-line to a MS/MS detector and the full analytical cycle, comprising sample preparation and analytes detection, was completed in <20 min. The developed μSPE-BI-LOV-MS methodology presented good linearity (r2 > 0.999) for quantification of the target analytes at concentrations ranging from 18 to 360 μg mL-1 in plasma. LOQ values were 2 μg mL-1 for INDS and 7 μg mL-1 for pCS in plasma. Human plasma samples from healthy subjects and individuals with CKD were successfully analyzed using the developed approach. The proposed automatic methodology can be described as an eco-friendly strategy, with a favorable score of 0.64 after greenness evaluation using the AGREE metric.
    Keywords:  Automation; Indoxyl sulfate; Lab-on-valve; Mass spectrometry; On-line hyphenation; p-Cresol sulfate
  23. Biomed Chromatogr. 2023 Aug 22. e5708
      Dolutegravir (DTG) has been the first-line drug in many human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) guidelines for the treatment of naïve and experienced HIV-infected individuals, which calls for cost-effective and convenient methods for quantitative detection of DTG in human plasma for pharmacokinetic studies and patient adherence evaluation. Here, an HPLC-ultraviolet method in combination with liquid-liquid extraction with isocratic elution was developed for the first time. The analysis was performed on a CLC-ODS column (6 mm internal diameter × 15 cm, 5 μm) using a mixture of acetonitrile and phosphate buffer (40:60, v/v) as the mobile phase at the flow rate of 1 mL/min. Using triamcinolone as the internal standard, 100 μL of plasma sample was extracted by methyl tert-butyl ether, followed by evaporating under nitrogen stream, re-dissolving with 100 μL mobile phase, and injection of 20-40 μL of supernatant into the chromatographic system. The linearity of DTG was good in the range of 0.05-10 μg/mL (r = 0.9995), and the inter- and intra-day variabilities were 0.4%-4.3% (n = 10) and 1.2%-6.2% (n = 10) for the lower limit of quantification, low-, medium-, and high-concentration quality control samples (0.05, 0.1, 0.8, and 8 μg/mL), respectively, while the methodological and extraction recoveries were 98.0%-103.0% (n = 20) and 65.2%-75.7% (n = 3), respectively. This method was successfully applied to analyze DTG plasma concentration in 84 Chinese patients with HIV.
    Keywords:  HPLC-UV; dolutegravir; human plasma; integrase inhibitor; quantification
  24. Front Pharmacol. 2023 ;14 1227354
      Introduction: The aim of the present study was to establish a simple method for the determination of riluzole in human plasma by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and apply it for the determination of riluzole in amyotrophic lateral sclerosis (ALS) patients. Methods: Samples were prepared by protein precipitation and were then gradient-eluted on a column of ACQUITY UPLC® HSS T3 by using 0.1% formic acid acetonitrile and 0.1% formic acid water as the mobile phase. Detection was performed on a Xevo TQ-S tandem mass spectrometer in the multiple-reaction monitoring mode using positive electrospray ionization. Validation was performed in the range of 5-800 ng/mL. Results and discussion: Three batches of precision accuracy, selectivity, matrix effects, extraction recovery, and stability were also verified and met the requirements. The results showed that the method was reliable and successfully applied to the pharmacokinetics study of riluzole in Chinese amyotrophic lateral sclerosis patients. Meanwhile, in comparison with other prior published methods, our method has the advantages of simple sample preparation, relatively short running time, and small plasma sample consumption, which represented a high-throughput sample determination potential.
    Keywords:  amyotrophic lateral sclerosis; clobazam; pharmacokinetics; riluzole; ultraperformance liquid chromatography-tandem mass spectrometry
  25. Environ Sci Pollut Res Int. 2023 Aug 19.
      Human biomonitoring (HBM) frameworks assess human exposure to hazardous chemicals. In this review, we discuss and summarize sample preparation procedures and analytical methodology for six groups of chemicals of emerging concern (CECs), namely diisocyanates, benzotriazoles, benzothiazoles, 4-methylbenzylidene camphor, isothiazolinones, fragrances, and non-phthalate plasticizers, which are increasingly detected in urine, however, are not yet widely included in HBM schemes, despite posing a risk to human health. The sample preparation procedures depend largely on the chemical group; however, solid-phase extraction (SPE) is most often used due to the minimized sample handling, lower sample volume, and generally achieving lower limits of quantification (LOQs) compared to other extraction techniques. In terms of sample analysis, LC-based methods generally achieve lower limits of quantification (LOQs) compared to GC-based methods for the selected six groups of chemicals owing to their broader chemical coverage. In conclusion, since these chemicals are expected to be more frequently included in future HBM studies, it becomes evident that there is a pressing need for rigorous quality assurance programs to ensure better comparability of data. These programs should include the reporting of measurement uncertainty and facilitate inter-laboratory comparisons among the reporting laboratories. In addition, high-resolution mass spectrometry should be more commonly employed to enhance the specificity and selectivity of the applied analytical methodology since it is underrepresented in HBM. Furthermore, due to the scarcity of data on the levels of these CECs in urine, large population HBM studies are necessary to gain a deeper understanding of the associated risks.
    Keywords:  Biomarker; Exposome; Exposure; Human biomonitoring; Mass spectrometry; Sample preparation
  26. J Chromatogr A. 2023 Aug 14. pii: S0021-9673(23)00519-8. [Epub ahead of print]1707 464294
      For confirmation and/or screening purposes, rapid, selective, and precise chromatographic methods are required. In this vein, the utility of SiH columns (C18, UDC Cholesterol, and Diamond Hydride) with photodiode array UV absorption and single quadrupole MS detection for multi-modal separation of representative drugs from different drug classes on a single column using the same solvent reservoirs was investigated. For a conventional two column approach employing a combination of conventional C18 and silica columns operating in both the reversed phase chromatographic and hydrophilic interaction chromatographic modes, gradient analysis is required for the first column and there is a lack of retention on the second column for non-amine analytes. In comparison, all analytes are retained for two relatively rapid (< 10 min), precise (% RSD <0.4%), and non-correlated isocratic separations (R2=0.2115) when using a UDC Cholesterol column.
    Keywords:  Dual mode chromatography; Photodiode array UV; Seized drugs; Silica hydride columns; Single Quad MS; Ultra high performance liquid chromatography (UHPLC)
  27. Yonago Acta Med. 2023 Aug;66(3): 365-374
      Background: Voriconazole therapy for fungal infections usually continues for several years and is often administered on an outpatient basis. Maintaining the voriconazole plasma concentration in the therapeutic range is highly important for effective therapy; however, it is difficult to obtain sufficient information to assess the voriconazole concentration in outpatients. Therefore, we developed a method to simultaneously measure the plasma concentrations of voriconazole and its major metabolite, voriconazole N-oxide, to obtain rapid results after outpatient blood collection and before medical consultation and to attain a better understanding of adherence and the drug-drug interactions of voriconazole.Methods: Fifty microliters of patient plasma was deproteinized with methanol, injected into the liquid chromatography-tandem mass spectrometry system, and purified using an online column. Separation was achieved on an InertSustain C18 column (2.1 mm id × 50 mm, 2 μm) with a mobile phase of 30:70 (0.1% formic acid in water:methanol) at a flow rate of 0.2 mL/min. Detection was performed using electrospray ionization in positive ion multiple reaction monitoring mode.
    Results: The analysis time was 4 min. The calibration curve was linear, in the range of 0.1 μg/mL to 20 μg/mL for voriconazole and 0.05 μg/mL to 10 μg/mL for voriconazole N-oxide, with a coefficient of determination at R2 > 0.999.
    Conclusion: There is no need to dilute the patient's plasma even if the concentration of voriconazole is near the upper limit of measurement. Furthermore, the short measurement-time could immediately inform physicians of the patient's voriconazole concentration during ambulatory medical care. Simultaneous measurement of voriconazole and voriconazole N-oxide may also be useful for the immediate adjustment of voriconazole dosage in outpatients and would help us to understand adherence or drug-drug interactions in plasma voriconazole concentrations.
    Keywords:  liquid chromatography; pharmacokinetics; tandem mass spectrometry; therapeutic drug monitoring; voriconazole
  28. Pak J Pharm Sci. 2023 Jul;36(4(Special)): 1271-1279
      Liquid chromatography-tandem mass (LC MS/MS) was used for the determination of therapeutic drug monitoring (TDM) of the three antipsychotics (aripiprazole, quetiapine and olanzapine) and three antidepressants (paroxetine, Escitalopram and sertraline) drugs simultaneously. Both groups of drugs can be concurrently used to treat behavioral disorders. It appears that there is no test for the rapid detection of all six compounds simultaneously using LC MS/M, despite the fact that several analysis publications found these drugs individually. 50µl of taken from finger pricks as dried blood spots (DBS) spiked with sample solution containing the six understudied drugs was extracted. A C18-BEH column with a mobile phase made up of gradient elution ammonium acetate with acetonitrile in methanol. The total run time of this method is about 5.5 min. LC MS/MS showed an excellent linearity in the range of 5-100ng ml-1 with a correlation coefficient (r) >0.992. The values of the intra- and inter-day precision of the tested drugs satisfy the regulatory requirements' acceptance criteria. The test was approved in accordance with accepted standards for bioanalytical procedures and it can be successfully applied for therapeutic drug monitoring studies for the tested drugs if they administered concurrently or individually.
  29. J Chromatogr A. 2023 Aug 15. pii: S0021-9673(23)00528-9. [Epub ahead of print]1707 464303
      The herein presented work aims to the development of an easy method for the quantitative determination of parabens and bisphenols in human salivabased on the use of methyl chloroformate as a derivatizing agent, followed by solid-phase microextraction (SPME) and gas chromatography-triple quadrupole mass spectrometry (GC-QqQ-MS) analysis with selected reaction monitoring (SRM). Using multivariate analysis, two derivatization strategies were compared and optimized, demonstrating that the use of methyl chloroformate led to better sensitivity than the classical derivatization by acetic anhydride. Good performance in the sorption process of the derivatized target analytes was obtained using the most recent commercialized overcoated fiber (PDMS/DVB/PDMS). The validation procedure of the final protocol led to satisfactory results in terms of linearity, limit of quantitation, accuracy, and precision. All parabens were quantified from 10 ng/L using the developed method, except for methylparaben, which was quantified from 100 ng/L along with all bisphenols. Intra- and inter-day accuracy and intra- and inter-day precision can be considered satisfactory for all analytes (values between 73% and 118%), except for the inter-day accuracy of BPF. Quite good results also in terms of matrix effect were obtained for the target compounds (range 71% to 118%, RSD% less than 13.6%), except for BPA at the middle concentration and MeP at the lowest concentration. The greenness of the method was evaluated and the results indicated that our approach is more eco-friendly than previously published methods. Based on its characteristics, the presented method can be considered a suitable approach to determine parabens and bisphenols in routine analysis for biomonitoring purposes.
    Keywords:  Bisphenols; Derivatization; Gas chromatography; Parabens; Solid phase microextraction SPME