bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒02‒05
33 papers selected by
Sofia Costa

  1. Anal Chem. 2023 Jan 30.
      Liquid chromatography-mass spectrometry (LC-MS)-based metabolomics usually combines hydrophilic interaction liquid chromatography (HILIC) and reversed-phase (RP) chromatography to cover a wide range of metabolomes, requiring both significant sample consumption and analysis time for separate workflows. We developed an integrated workflow enabling the coverage of both polar and nonpolar metabolites with only one injection of the sample for each ionization mode using heart-cutting trapping to combine HILIC and RP separations. This approach enables the trapping of some compounds eluted from the first chromatographic dimension for separation later in the second dimension. In our case, we applied heart-cutting to non-retained metabolites in the first dimension. For that purpose, two independent miniaturized one-dimensional HILIC and RP methods were developed by optimizing the chromatographic and ionization conditions using columns with an inner diameter of 1 mm. They were then merged into one two-dimensional micro LC-MS method by optimization of the trapping conditions. Equilibration of the HILIC column during elution on the RP column and vice versa reduced the overall analysis time, and the multidimensionality allows us to avoid signal measurements during the solvent front. To demonstrate the benefits of this approach to metabolomics, it was applied to the analysis of the human plasma standard reference material SRM 1950, enabling the detection of hundreds of metabolites without the significant loss of some of them while requiring an injection volume of only 0.5 μL.
  2. Methods Enzymol. 2023 ;pii: S0076-6879(22)00354-8. [Epub ahead of print]680 247-273
      Plants are expert chemists producing millions of metabolites, only a fraction of which are known to date. Plant metabolomics explores the rationale for highly diverse metabolites evolved and synthesized by plants. Over two-thirds of modern medicines are somehow inspired and/or derived from plants, making the identification of phytochemicals a means of discovering new medicines to challenge existing and emerging diseases. This chapter introduces our established liquid chromatography-tandem mass spectrometry-based untargeted metabolomics approach centered around discovering specialized metabolites (so-called secondary metabolites) across broad lineages of nonmodel plant species. Detecting hundreds to thousands of metabolite peaks, including assigning chemical identity, makes metabolomics data generation and analysis a very complex process. Various mass spectrometry techniques are currently being developed to approach the comprehensive metabolome. Among them, untargeted metabolomics can provide new biological insights by simultaneously and unbiasedly measuring and analyzing all detected metabolites. We have provided a hands-on modular account for untargeted plant metabolomics, from preparing plant biological samples to data analysis and processing using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. The methods described here offer a foundation and expert opinion on plant metabolome analysis.
    Keywords:  Liquid chromatography; Metabolite extraction; Plant metabolome analysis; Quadrupole time-of-flight mass spectrometry; Specialized metabolite analysis; Untargeted metabolomics
  3. Anal Chem. 2023 Jan 30.
      Lipid analysis at the molecular species level represents a valuable opportunity for clinical applications due to the essential roles that lipids play in metabolic health. However, a comprehensive and high-throughput lipid profiling remains challenging given the lipid structural complexity and exceptional diversity. Herein, we present an 'omic-scale targeted LC-MS/MS approach for the straightforward and high-throughput quantification of a broad panel of complex lipid species across 26 lipid (sub)classes. The workflow involves an automated single-step extraction with 2-propanol, followed by lipid analysis using hydrophilic interaction liquid chromatography in a dual-column setup coupled to tandem mass spectrometry with data acquisition in the timed-selective reaction monitoring mode (12 min total run time). The analysis pipeline consists of an initial screen of 1903 lipid species, followed by high-throughput quantification of robustly detected species. Lipid quantification is achieved by a single-point calibration with 75 isotopically labeled standards representative of different lipid classes, covering lipid species with diverse acyl/alkyl chain lengths and unsaturation degrees. When applied to human plasma, 795 lipid species were measured with median intra- and inter-day precisions of 8.5 and 10.9%, respectively, evaluated within a single and across multiple batches. The concentration ranges measured in NIST plasma were in accordance with the consensus intervals determined in previous ring-trials. Finally, to benchmark our workflow, we characterized NIST plasma materials with different clinical and ethnic backgrounds and analyzed a sub-set of sera (n = 81) from a clinically healthy elderly population. Our quantitative lipidomic platform allowed for a clear distinction between different NIST materials and revealed the sex-specificity of the serum lipidome, highlighting numerous statistically significant sex differences.
  4. Mass Spectrom (Tokyo). 2022 ;11(1): A0112
      Proton-transfer-reaction (PTR) mass spectrometry (MS), a widely used method for detecting trace-levels of volatile organic compounds in gaseous samples, can also be used for the analysis of small non-volatile molecules by using supercritical fluid as a transporter for the molecules. Supercritical fluid extraction (SFE) is a method that permits lipophilic compounds to be rapidly and selectively extracted from complex matrices. The combination of the high sensitivity of PTR MS with the SFE is a potentially novel method for analyzing small molecules in a single cell, particularly for the analysis of lipophilic compounds. We preliminarily evaluated this method for analyzing the components of a single HeLa cell that is fixed on a stainless steel frit and is then directly introduces the SFE extracts into the PTR MS. A total of 200/91 ions were observed in positive/negative ion mode time-of-flight mass spectra, and the masses of 11/10 ions could be matched to chemical formulae obtained from the LipidMaps lipids structure database. Using various authentic lipophilic samples, the method could be used to detect free fatty acids in the sub-femtomole to femtomole order in the negative ion mode, the femtomole to sub-picomole order for fat-soluble vitamins, and the picomole order for poly aromatic hydrocarbons in both the positive and negative ion mode.
    Keywords:  proton-transfer-reaction mass spectrometry; single-cell lipid metabolite analysis; supercritical fluid chromatography; supercritical fluid extraction; water ion chemical ionization
  5. Methods Enzymol. 2023 ;pii: S0076-6879(22)00340-8. [Epub ahead of print]680 325-350
      Non-targeted metabolome approaches aim to detect metabolite markers related to stress, disease, developmental or genetic perturbation. In the later context, it is also a powerful means for functional gene annotation. A prerequisite for non-targeted metabolome analyses are methods for comprehensive metabolite extraction. We present three extraction protocols for a highly efficient extraction of metabolites from plant material with a very broad metabolite coverage. The presented metabolite fingerprinting workflow is based on liquid chromatography high resolution accurate mass spectrometry (LC-HRAM-MS), which provides suitable separation of the complex sample matrix for the analysis of compounds of different polarity by positive and negative electrospray ionization and mass spectrometry. The resulting data sets are then analyzed with the software suite MarVis and the web-based interface MetaboAnalyst. MarVis offers a straightforward workflow for statistical analysis, data merging as well as visualization of multivariate data, while MetaboAnalyst is used in our hands as complementary software for statistics, correlation networks and figure generation. Finally, MarVis provides access to species-specific metabolite and pathway data bases like KEGG and BioCyc and to custom data bases tailored by the user to connect the identified markers or features with metabolites. In addition, identified marker candidates can be interactively visualized and inspected in metabolic pathway maps by KEGG pathways for a more detailed functional annotation and confirmed by mass spectrometry fragmentation experiments or coelution with authentic standards. Together this workflow is a valuable toolbox to identify novel metabolites, metabolic steps or regulatory principles and pathways.
    Keywords:  Custom databases; Data mining; Extraction; Metabolite fingerprinting; Metabolome; Non-targeted metabolomics
  6. MethodsX. 2023 ;10 102026
      Hydrophilic interaction liquid chromatography (HILIC), coupled to tandem mass spectrometry, can be used to separate and determine various polar lipid classes. The development of an HILIC chromatographic separation of several molecular species among five phospholipid classes (PC, PE, PG, PI and PS) is reported here. In this method, a gradient with acetonitrile and 40 mM ammonium acetate buffer was employed. The initial composition was 95% of acetonitrile, then this proportion was decreased to 70% in order to elute all the compounds of interest for a total running time of 11 mins. Furthermore, mobile phase pH can affect the ionizable character of the compounds, according to their pKa values, and also the stationary phase charge state. The influence of such a parameter on both retention times and resolution was evaluated. Besides, the response of different kinds of internal standards (post-extraction standard addition) was evaluated in four different biological matrices, two microalgae extracts and two marine fish extracts. This study found that the recovery rates were between 70 and 140% of the expected value, with relative standard deviations between 10 and 35%, and then limited matrix effects.•HILIC approach can be used to separate phospholipid according to their polar head-group, and electrospray ionization in negative mode as well as MS/MS allows further identification of the molecular species within each phospholipid class.•Matrix effects are low and compensated with appropriate internal standards.•The limits of quantifications were ranging from 0.05 to 0.14 µg.mL-1, depending on the analyte.
    Keywords:  HILIC; HPLC-ESI-MS/MS; Hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry method for quantification of five phospholipid classes in various matrices; Mobile phase pH; Phospholipids
  7. bioRxiv. 2023 Jan 04. pii: 2023.01.04.522722. [Epub ahead of print]
      In untargeted metabolomics, multiple ions are often measured for each original metabolite, including isotopic forms and in-source modifications, such as adducts and fragments. Without prior knowledge of the chemical identity or formula, computational organization and interpretation of these ions is challenging, which is the deficit of previous software tools that perform the task using network algorithms. We propose here a generalized tree structure to annotate ions to relationships to the original compound and infer neutral mass. An algorithm is presented to convert mass distance networks to this tree structure with high fidelity. This method is useful for both regular untargeted metabolomics and stable isotope tracing experiments. It is implemented as a Python package (khipu), and provides a JSON format for easy data exchange and software interoperability. By generalized pre-annotation, khipu makes it feasible to connect metabolomics data with common data science tools, and supports flexible experimental designs.
  8. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jan 27. pii: S1570-0232(23)00028-4. [Epub ahead of print]1217 123618
      The gut microbiome produces a range of short chain fatty acids (SCFA) crucially linked with diet and nutrition, metabolism, gastrointestinal health and homeostasis. SCFA are primarily measured using gas or liquid chromatography-mass spectrometry (LC/MS) after undergoing chemical derivatization. Here we assess the merits of a derivatization protocol using aniline and two aniline analogues (3-phenoxyaniline and 4-(benzyloxy)aniline) for the targeted LC-MS/MS quantification of nine SCFA (acetic, propionic, butyric, valeric, caproic acid, isobutyric, isovaleric, 2-methylbutyric, and 2-ethylbutyric acid). Evaluation of product ion spectra and optimization of MS detection conditions, provided superior detection sensitivity for 3-phenoxyaniline and 4-(benzyloxy)aniline compared to aniline. We developed a facile SCFA derivatization method using 3-phenoxyaniline under mild reaction conditions which allows for the simultaneous quantification of these SCFA in human stool samples in under eleven minutes using multiple reaction monitoring LC-MS/MS. The method was successfully validated and demonstrates intra- and inter-day accuracy (88.5-103% and 86.0-109%) and precision (CV of 0.55-7.00% and 0.33-9.55%) with recoveries (80.1-87.2% for LLOQ, 88.5-93.0% for ULOQ) and carry-over of (2.68-17.9%). Selectivity, stability and matrix effects were also assessed and satisfied validation criteria. Method applicability was demonstrated by analysing SCFA profiles in DNA-stabilized human stool samples from newly diagnosed colorectal cancer patients prior to surgery. The development of this improved method and its compatibility to measure SCFAs from DNA-stabilized stool will facilitate studies investigating the gut microbiome in health and disease.
    Keywords:  Derivatization; Liquid chromatography-mass spectrometry (LC-MS); Microbiome; Quantitation; Short chain fatty acids; Stool
  9. PNAS Nexus. 2022 Nov;1(5): pgac257
      Microbial specialized metabolites are an important source of and inspiration for many pharmaceuticals, biotechnological products and play key roles in ecological processes. Untargeted metabolomics using liquid chromatography coupled with tandem mass spectrometry is an efficient technique to access metabolites from fractions and even environmental crude extracts. Nevertheless, metabolomics is limited in predicting structures or bioactivities for cryptic metabolites. Efficiently linking the biosynthetic potential inferred from (meta)genomics to the specialized metabolome would accelerate drug discovery programs by allowing metabolomics to make use of genetic predictions. Here, we present a k-nearest neighbor classifier to systematically connect mass spectrometry fragmentation spectra to their corresponding biosynthetic gene clusters (independent of their chemical class). Our new pattern-based genome mining pipeline links biosynthetic genes to metabolites that they encode for, as detected via mass spectrometry from bacterial cultures or environmental microbiomes. Using paired datasets that include validated genes-mass spectral links from the Paired Omics Data Platform, we demonstrate this approach by automatically linking 18 previously known mass spectra (17 for which the biosynthesis gene clusters can be found at the MIBiG database plus palmyramide A) to their corresponding previously experimentally validated biosynthetic genes (e.g., via nuclear magnetic resonance or genetic engineering). We illustrated a computational example of how to use our Natural Products Mixed Omics (NPOmix) tool for siderophore mining that can be reproduced by the users. We conclude that NPOmix minimizes the need for culturing (it worked well on microbiomes) and facilitates specialized metabolite prioritization based on integrative omics mining.
    Keywords:  biosynthetic gene clusters; genomics; machine learning; mass spectrometry; specialized metabolites
  10. Se Pu. 2023 Feb;41(2): 131-141
      Alcohol intake is an important risk factor for cardiovascular disease, liver disease, and diabetes. The accurate and objective evaluation of alcohol intake is important for disease prevention and intervention, as well as alcohol intake monitoring. Phosphatidylethanol (PEth) is a potential clinical biomarker of alcohol consumption. Monitoring PEth levels can provide an objective and quantitative basis for alcohol intake studies. Unlike other current alcohol biomarkers, PEth can only be produced in the presence of alcohol. Therefore, PEth is highly specific for alcohol intake and not affected by confounding factors, such as age, gender, hypertension, kidney disease, liver disease, and other comorbidities. Because of its long half-life and high specificity for alcohol intake, PEth may be used as a tool for monitoring drinking behavior in the clinical, transportation, and other fields. Given rapid developments in mass spectrometry technology over the past decade, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the preferred method for PEth detection. However, most current LC-MS/MS methods focus on the determination of one or several PEth homologs, and the number of PEth homologs that can be determined simultaneously is relatively limited. Moreover, the detection capacity of the available methods remains insufficient, and their analytical sensitivity for some PEth homologs must be further improved. In this study, a novel LC-MS/MS method based on an intelligent scheduled time-zone negative multiple reaction monitoring acquisition technology (Scheduled-MRM) was developed. The technology monitors two ion channels in each PEth to ensure reliable results and can quantify 18 PEth homologs in human whole blood simultaneously. Methanol-methyl tert-butyl ether-water was used as the extraction system. An XBridge C18 column (100 mm×2.1 mm, 3.5 μm) was selected for gradient elution with 2.5 mmol/L ammonium acetate isopropanol solution and 2.5 mmol/L ammonium acetate aqueous solution-acetonitrile (50∶50, v/v) as the mobile phases. Negative electronic spray ionization in scheduled-MRM mode was applied for MS/MS detection. The method was validated to have a linear range of 10-2500 ng/mL with correlation coefficients greater than 0.9999. The limits of detection and quantification were 0.7-2.8 and 2.2-9.4 ng/mL, respectively, and the spiked recoveries ranged from 91.0% to 102.2%. The method was confirmed to be simple, rapid, and precise, and subsequently validated for the measurement of 18 PEth homologs in human blood. Scheduled-MRM can assign a suitable scan time to each ion channel and effectively reduce the number of concurrent ion pairs monitored per unit time. This technology overcomes the problem of insufficient dwell time caused by an excessive number of ion channels, thereby avoiding the redundant monitoring of non-retention times. Scheduled-MRM significantly improved the detection sensitivity, data acquisition quality, and signal response of the proposed method. Whole blood samples from 359 volunteers with regular drinking habits were analyzed using this method. The total PEth concentrations ranged from 51.13 ng/mL to 2.89 μg/mL, with a mean of 363.16 ng/mL. PEth 16∶0/18∶1 and 16∶0/18∶2 were the two most abundant homologs, with mean concentrations of 74.21 and 48.75 ng/mL, accounting for approximately 20.43% and 13.42%, respectively, of the total PEth. Spearman correlation analyses showed that the PEth homologs correlated well with each other, γ-glutamyltransferase, a clinically available biological marker of alcohol, and other clinical biochemical parameters related to liver and kidney function. Overall, the method was demonstrated to be sensitive, precise, and accurate; thus, it may be an effective tool for monitoring alcohol intake in the clinical and other fields.
    Keywords:  alcohol intake monitoring; high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS); intelligent scheduled time-zone acquisition technology; phosphatidylethanol (PEth)
  11. Sci Adv. 2023 Feb 03. 9(5): eadd0455
      Skeletal muscle myofibers are heterogeneous in their metabolism. However, metabolomic profiling of single myofibers has remained difficult. Mass spectrometry imaging (MSI) is a powerful tool for imaging molecular distributions. In this work, we optimized the workflow of matrix-assisted laser desorption/ionization (MALDI)-based MSI from cryosectioning to metabolomics data analysis to perform high-spatial resolution metabolomic profiling of slow- and fast-twitch myofibers. Combining the advantages of MSI and liquid chromatography-MS (LC-MS), we produced spatial metabolomics results that were more reliable. After the combination of high-spatial resolution MSI and LC-MS metabolomic analysis, we also discovered a new subtype of superfast type 2B myofibers that were enriched for fatty acid oxidative metabolism. Our technological workflow could serve as an engine for metabolomics discoveries, and our approach has the potential to provide critical insights into the metabolic heterogeneity and pathways that underlie fundamental biological processes and disease states.
  12. Anal Chem. 2023 Feb 03.
      Accurate reconstruction of metabolic pathways is an important prerequisite for interpreting metabolomics changes and understanding the diverse biological processes in disease models. A tracer-based metabolomics strategy utilizes stable isotope-labeled precursors to resolve complex pathways by tracing the labeled atom(s) to downstream metabolites through enzymatic reactions. Isotope enrichment analysis is informative and achieved by counting total labeled atoms and acquiring the mass isotopologue distribution (MID) of the intact metabolite. However, quantitative analysis of labeled metabolite substructures/moieties (MS2 fragments) can offer more valuable insights into the reaction connections through measuring metabolite transformation. In order to acquire the isotopic labeling information at the intact metabolite and moiety level simultaneously, we developed a method that couples hydrophilic interaction liquid chromatography (HILIC) with Zeno trap-enabled high-resolution multiple reaction monitoring (MRMHR). The method enabled accurate and reproducible MID quantification for intact metabolites as well as their fragmented moieties, with notably high sensitivity in the MS2 fragmentation mode based on the measurement of 13C- or 15N-labeled cellular samples. The method was applied to human-induced pluripotent stem cell-derived neurons to trace the fate of 13C/15N atoms from D-13C6-glucose/L-15N2-glutamine added to the media. With the MID analysis of both intact metabolites and fragmented moieties, we validated the pathway reconstruction of de novo glutathione synthesis in mid-brain neurons. We discovered increased glutathione oxidization from both basal and newly synthesized glutathione pools under neuronal oxidative stress. Furthermore, the significantly decreased de novo glutathione synthesis was investigated and associated with altered activities of several key enzymes, as evidenced by suppressed glutamate supply via glucose metabolism and a diminished flux of glutathione synthetic reaction in the neuronal model of rotenone-induced neurodegeneration.
  13. Anal Chem. 2023 Jan 31.
      The diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY) experiment allows the calculation of diffusion coefficient values of metabolites in complex mixtures. However, this experiment has not yet been broadly used for metabolic profiling due to lack of a standardized protocol. Here we propose a pipeline for the DOSY experimental setup and data processing in metabolic phenotyping studies. Due to the complexity of biological samples, three experiments (a standard DOSY, a relaxation-edited DOSY, and a diffusion-edited DOSY) have been optimized to provide DOSY metabolic profiles with peak-picked diffusion coefficients for over 90% of signals visible in the one-dimensional 1H general biofluid profile in as little as 3 min 36 s. The developed parameter sets and tools are straightforward to implement and can facilitate the use of DOSY for metabolic profiling of human blood plasma and urine samples.
  14. Mass Spectrom (Tokyo). 2022 ;11(1): A0108
      We have developed a rapid and sensitive analytical method for α-tocopherol and its oxidative products by combining online hyphenation of supercritical fluid extraction-supercritical fluid chromatography (SFC) with proton transfer reaction (PTR) ionization mass spectrometry (MS). α-Tocopherol is a well-known antioxidant that plays a vital role in the antioxidant defense system in plant cells. However, studies on the cellular mechanisms of α-tocopherol have been limited owing to the lack of a rapid analytical method, which limits the comparison of plant cells incubated in various conditions. Additionally, complex sample preparation and long chromatography separation times are required. Moreover, the majority of the involved molecules are a combination of isomers, which must be separated before applying tandem MS. α-Tocopherol produces the α-tocopheroxyl radical in the first step of its antioxidant function; another ion with the same mass may also be generated from the source. SFC separation effectively distinguished the observed ions from their oxidative products in the sample and those produced during the ionization reaction process. This method enabled the measurement of α-tocopherol and its oxidative products such as α-tocopheroxyl radical and α-tocopheryl quinone in approximately 3 min per sample, including the time required for sample preparation.
    Keywords:  antioxidant; chemical ionization; proton-transfer reaction; supercritical fluid extraction-supercritical fluid chromatography; α-tocopherol
  15. Mass Spectrom (Tokyo). 2022 ;11(1): A0106
      In metabolomics studies using high-resolution mass spectrometry (MS), a set of product ion spectra is comprehensively acquired from observed ions using the data-dependent acquisition (DDA) mode of various tandem MS. However, especially for low-intensity signals, it is sometimes difficult to distinguish artifact signals from true fragment ions derived from a precursor ion. Inadequate precision in the measured m/z value is also one of the bottlenecks to narrowing down the candidate compositional formula. In this study, we report that averaging multiple product ion spectra can improve m/z precision as well as the reliability of fragment ions that are observed in such spectra. A graph-based method was applied to cluster a set of similar spectra from multiple DDA data files resulting in creating an averaged product-ion spectrum. The error levels for the m/z values declined following the central limit theorem, which allowed us to reduce the number of candidate compositional formulas. The improved reliability and precision of the averaged spectra will contribute to a more efficient annotation of product ion spectral data.
    Keywords:  data-dependent acquisition mode; elemental composition search; metabolomics; precision
  16. Electrophoresis. 2023 Jan 31.
      Tryptophan (TRP) is an essential amino acid catabolized mainly through the kynurenine pathway (KP), and part of it is catabolized in the brain. The abnormal depletion of TRP and production of kynurenine (KYN) by two enzymes, tryptophan 2, 3-dioxygenase (TDO) and indolamine 2, 3-dioxygenase (IDO), have been linked to various neurological diseases. The ratio of TRP/KYN in plasma is a valuable measure for IDO/TDO activity and the prognosis of disease conditions. The vinylphenyl boronic acid (4-VPBA) was evaluated as a novel stationary phase for OT-CEC-MS/MS. TRP, KYN, and 3-hydroxykynurenine (3-OHKYN) were separated using optimum conditions of 15 mM (NH4 )2 CO3 at pH 8 as a BGE and 25 kV separation voltage on a 90 cm column. The usefulness of the 4-VPBA column for simple, fast, repeatable, and sensitive CEC-ESI-MS/MS application was demonstrated for the quantitation of TRP and KYN in the plasma of healthy human subjects and neuroinflammation subjects. The plasma sample was extracted on a zirconia-based ion-exchange cartridge for simultaneous protein precipita-tion and phospholipid removal. The method of standard addition, in combination with the internal standards approach, was used to prepare the calibration curve to overcome matrix matching and eliminate procedural errors. The developed quantitation method was validated according to FDA guidelines for sensitivity, accuracy, precision, and extraction recovery. The measured plasma level of TRP and KYN in healthy humans is aligned with the human metabolome database for the same two metabolites. This article is protected by copyright. All rights reserved.
    Keywords:  kynurenines; neuroinflammation; open tubular capillary electrochromatography-mass spectrometry; solid-liquid extraction; tryptophan
  17. Anal Chem. 2023 Feb 01.
      First-tier MS-based newborn screening by flow injection analysis can have high presumptive positive rates, often due to isomeric/isobaric compounds or poor biomarker specificity. These presumptive positive samples can be analyzed by second-tier screening assays employing separations such as liquid chromatography-mass spectrometry (LC-MS/MS), which increases test specificity and drastically reduces false positive referrals. The ability to screen for multiple disorders in a single multiplexed test simplifies workflows and maximizes public health laboratories' resources. In this study, we developed and validated a highly multiplexed second-tier method for dried blood spots using a hydrophilic interaction liquid chromatography (HILIC) column coupled to an MS/MS system. The LC-MS/MS method was capable of simultaneously detecting second-tier biomarkers for maple syrup urine disease, homocystinuria, methylmalonic acidemia, propionic acidemia, glutaric acidemia type 1, glutaric acidemia type 2, guanidinoacetate methyltransferase deficiency, short-chain acyl-CoA dehydrogenase deficiency, adrenoleukodystrophy, and Pompe disease.
  18. Front Pharmacol. 2023 ;14 1076046
      Etimicin is a fourth-generation aminoglycoside antibiotic. It has potent activity and low toxicity when employed for the treatment of Gram-negative and Gram-positive bacterial infections. The pharmacokinetics of etimicin in humans have not been elucidated completely. Two liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical methods, without the use of any ion-pairing reagents, were developed and validated for the quantification of etimicin in human samples of serum and urine. Using a deuterated reagent as the internal standard, analytes in serum and urine samples were extracted by protein precipitation and dilution before LC-MS/MS analysis, respectively. For the two methods, chromatographic separations were undertaken under isocratic elution of water-ammonia solution-acetic acid (96:3.6:0.2, v/v/v) and methanol at 50%:50% and a flow rate of 0.35 ml/min within 5 min. A Waters XTerra MS C18 column (2.1 × 150 mm, 3.5 μm) and a column temperature of 40°C were chosen. A Sciex Qtrap 5500 mass spectrometer equipped with an electrospray ion source was used in both methods under multiple-reaction monitoring in positive-ion mode. The two methods showed good linearity, accuracy, and precision with high recovery and a minimal matrix effect in the range of 50.0-20000 ng/ml for serum samples and 50.0-10000 ng/ml for urine samples, respectively. Carry-over effects were not observed. Etimicin remained stable in human samples of serum or urine under the storage, preparation, and analytical conditions of the two methods. These two simple and reliable methods were applied successfully to a dose-escalation, phase I clinical trial of etimicin in Chinese healthy volunteers after intravenous administration of single and multiple doses. Based on these two methods we ascertained, for the first time, the comprehensive pharmacokinetics of etimicin in humans, which will be used for the exploration of the breakpoint research further.
    Keywords:  LC-MS/MS; aminoglycoside antibiotic; etimicin; human pharmacokinetics; human serum and urine; method validation
  19. Front Mol Biosci. 2022 ;9 1070394
      KODAMA is a valuable tool in metabolomics research to perform exploratory analysis. The advanced analytical technologies commonly used for metabolic phenotyping, mass spectrometry, and nuclear magnetic resonance spectroscopy push out a bunch of high-dimensional data. These complex datasets necessitate tailored statistical analysis able to highlight potentially interesting patterns from a noisy background. Hence, the visualization of metabolomics data for exploratory analysis revolves around dimensionality reduction. KODAMA excels at revealing local structures in high-dimensional data, such as metabolomics data. KODAMA has a high capacity to detect different underlying relationships in experimental datasets and correlate extracted features with accompanying metadata. Here, we describe the main application of KODAMA exploratory analysis in metabolomics research.
    Keywords:  KODAMA; clustering; metabolomics; semi-supervised; unsupervised
  20. Se Pu. 2023 Feb;41(2): 160-167
      Capillary electrophoresis-mass spectrometry (CE-MS) combines the advantages of capillary electrophoresis, such as the high separation efficiency and low sample consumption, and the high detection sensitivity of mass spectrometry and the ability for providing the structural information for structure elucidation of unknown components. However, the interface technology for coupling capillary electrophoresis and mass spectrometry is still not well resolved. In the present work, we explored the application of the sheathless CE-MS interface which was prepared by gold foil-wrapped CE separation column tip directly as a spray electrode for the analysis of five tyrosine kinase inhibitors, namely sunitinib, imatinib mesylate, gefitinib, dasatinib and erlotinib. This interface integrates separation and electrospray ionization in one capillary, which is easy to manufacture, low in cost, and can be produced in batches. We found that using the nonaqueous CE separation mode can not only achieve baseline separation of five tyrosine kinase inhibitors, but also obtain stable mass spectrometry signals. First, we investigated the effect of the electrolyte solution composition on the separation. The optimized background electrolyte composition was obtained: 2% (v/v) acetic acid and 5 mmol/L ammonium acetate in acetonitrile-methanol (80∶20, v/v). Under optimized conditions, the five kinase inhibitors could be baseline separated, meantime, the sheathless interface could also maintain stable electrospray for a long time. The relative standard deviation (RSD) values of the intraday and interday repeatability of the analyte retention times were less than 0.5% and 0.8%, respectively, and the RSD value between interface batches is less than 2.6%. Compared with CE-MS with aqueous phase, the separation column efficiency of the five tyrosine kinase inhibitors under nonaqueous phase conditions is higher, the detection sensitivity is higher, and the absolute detection limit reaches amol level. In addition, we evaluated the sheathless interface with various organic acids, such as palaflin A, salvianolic acid C, and rosmarinic acid, as well as hydrophobic macrolide antibiotics, azithromycin, erythromycin, and sporin A, good separation effect and mass spectrometric detection results can be obtained.
    Keywords:  gold foil-wrapped electrospray ionization emitter; nonaqueous capillary electrophoresis-mass spectrometry (NACE-MS); sheathless interface; tyrosine kinase inhibitors
  21. Anal Chim Acta. 2023 Mar 01. pii: S0003-2670(22)01345-9. [Epub ahead of print]1244 340774
      Analytical techniques, such as liquid chromatography coupled to mass spectrometry (LC-MS) or nuclear magnetic resonance (NMR), are widely used for characterization of complex mixtures of (isomeric) proteins, carbohydrates, lipids, and phytochemicals in food. Food can contain isomers that are challenging to separate, but can possess different reactivity and bioactivity. Catechins are the main phenolic compounds in tea; they can be present as various stereoisomers, which differ in their chemical properties. Currently, there is a lack of fast and direct methods to monitor interconversion and individual reactivity of these epimers (e.g. epicatechin (EC) and catechin (C)). In this study, cyclic ion mobility mass spectrometry (cIMS-MS) was explored as a potential tool for the separation of catechin epimers. Formation of sodium and lithium adducts enhanced IMS separation of catechin epimers, compared to deprotonation and protonation. Baseline separation of the sodium adducts of catechin epimers was achieved. Moreover, we developed a fast method for the identification and semi-quantification of cIMS-MS separated catechin epimers. With this method, it is possible to semi-quantify the ratio between EC and C (1:5 to 5:1, within 50-1200 ng mL-1) in food samples, such as tea. Finally, the newly developed approach for cIMS-MS separation of flavonoids was demonstrated to be successful in separation of two sets of positional isomers (i.e. morin, tricetin, and quercetin; and kaempferol, fisetin, luteolin, and scutellarein). To conclude, we showed that both epimers and positional isomers of flavonoids can be separated using cIMS-MS, and established the potential of this method for challenging flavonoid separations.
    Keywords:  Adduct formation; Diastereomers; Flavan-3-ols; Ion mobility; Mass spectrometry; Semi-quantification
  22. bioRxiv. 2023 Jan 14. pii: 2023.01.13.524013. [Epub ahead of print]
      MALDI-TOF MS is a powerful tool to analyze biomolecules owing to its soft ionization nature and generally results in simple spectra of singly charged ions. Moreover, implementation of the technology in imaging mode provides a means to spatially map analytes in situ. Recently, a new matrix, DBDA (N1,N4-dibenzylidenebenzene-1,4-diamine) was reported to facilitate the ionization of free fatty acids in the negative ion mode. Building on this finding, we sought to implement DBDA for MALDI mass spectrometry imaging studies in brain tissue and successfully map oleic acid, palmitic acid, stearic acid, docosahexaenoic acid and arachidonic acid using mouse brain sections. Moreover, we hypothesized that DBDA would provide superior ionization for sulfatides, a class of sulfolipids, with multiple biological functions. Herein we also demonstrate that DBDA is ideal for MALDI mass spectrometry imaging of fatty acids and sulfatides in brain tissue sections. Additionally, we show enhanced ionization of sulfatides using DBDA compared to three different traditionally used MALDI matrices. Together these results provide new opportunities for studies to measure sulfatides by MALDI-TOF MS including in imaging modes.
  23. Ther Drug Monit. 2023 Jan 10.
      BACKGROUND: The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, palbociclib, ribociclib, and abemaciclib, are standard-of-care agents for patients with hormone receptor-positive human epidermal growth factor receptor 2-negative metastatic breast cancer. In support of therapeutic drug monitoring and clinical pharmacokinetic studies, a liquid chromatography coupled with tandem mass spectrometry assay for the simultaneous quantitation of CDK4/6 inhibitors and the major active metabolite M2 of abemaciclib in human plasma has been developed.METHODS: Analytes were extracted from 50 μL of human plasma by precipitating proteins with methanol and then collecting the supernatant. Reversed-phase high-performance liquid chromatography was performed for analyte separation using a biphasic gradient at a flow rate of 0.25-0.5 mL/min. The total run time was 9.5 minutes. The analytes were detected using MS/MS with electrospray ionization operating in positive ion mode.
    RESULTS: Validation according to the US Food and Drug Administration's guidance showed that the new assay produced accurate (94.7%-107%) and precise (within-run: 1.2%-8.2%; between-run: 0.6%-7.5%) measurements of all analytes over a concentration range of 5-2000 ng/mL. Overall, analyte recoveries were consistent (mean values: 110%-129%). The analytes were also stable in human plasma and the final extract under various storage conditions. Finally, the clinical applicability of the assay was confirmed by quantitation of all analytes in plasma samples obtained from patients treated with CDK4/6 inhibitors. Reproducibility of the measured analyte concentrations in study samples was confirmed successfully by incurred sample reanalysis.
    CONCLUSIONS: A sensitive liquid chromatography coupled with tandem mass spectrometry method to measure CDK4/6 inhibitors was developed and validated according to the Food and Drug Administration criteria. Quantitation of all analytes in clinical plasma samples confirmed that the assay is suitable for therapeutic drug monitoring and clinical pharmacokinetic studies of CDK4/6 inhibitors.
  24. Forensic Chem. 2022 Sep;30
      Techniques developed for the screening of forensic samples can be useful for increasing sample throughput and decreasing backlog in forensic laboratories. One such technique, rapid gas chromatography mass spectrometry (GC-MS), allows for fast sample screening (≈1 min) and has gained interest in recent years for forensic applications. This work focuses on the development of a method for ignitable liquid analysis using rapid GC-MS. A sampling protocol and temperature program were developed for the analysis of these volatile samples. Using the optimized method for analysis, the limits of detection for compounds commonly found in ignitable liquids ranged from 0.012 mg/mL to 0.018 mg/mL. Once the method was developed, neat ignitable liquids (i.e., gasoline and diesel fuel) were analyzed, and major components in each liquid were identified. The identification of major compounds in gasoline and diesel fuel in the presence of substrate interferences was then assessed through the analysis of simulated fire debris samples. Three different substrates were spiked with each ignitable liquid, burned, and analyzed. Major compounds in both liquids were identified using the total ion chromatograms, relevant extracted ion profiles, and deconvolution methods.
    Keywords:  GC-MS; fire debris analysis; ignitable liquid residue; screening methods
  25. Anal Chem. 2023 Jan 30.
      Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.
  26. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jan 27. pii: S1570-0232(23)00015-6. [Epub ahead of print]1217 123605
      Pyridoxal-5'-phosphate (PLP), the active form of vitamin B6, is required for numerous enzymatic reactions. Vitamin B6 deficiency or exceptionally high levels of PLP have negative implications, making measurements of PLP imperative for diagnoses and monitoring in many clinical scenarios. Traditional assays are enzymatic, ELISA based, or employ HPLC with various detection modalities; all of these are prone to interferences and crossreactivity with other compounds. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been increasingly used to overcome these issues, but the high polarity of PLP raises chromatographic challenges. Using ion pairing reagents in the mobile phases is a possible solution, but these reagents often have deleterious effects on instrumentation. An alternative strategy is the addition of an ion pairing reagent after extraction, but prior to injection. To prove this, we used 1-octanesulfonic acid (OSA) without changing the LC method or column. With this technique, we observed a 2-4 fold increase in signal-to-noise ratio. Intraday and interday precision of replicate measurements also improved drastically compared to analyses without OSA, while also yielding a dramatic improvement in column life compared to our previous approach and to this point no deleterious effects on instrument hardware commonly associated with traditional ion pairing reagent techniques have been observed.
    Keywords:  Liquid chromatography-tandem mass spectrometry; PLP; Pyridoxal-5′-phosphate; Vitamin B6
  27. J Chromatogr A. 2023 Jan 24. pii: S0021-9673(23)00046-8. [Epub ahead of print]1691 463818
      An analytical method based on isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC‒MS/MS) was developed to accurately determine four representative tetracyclines (tetracycline, chlortetracycline, doxycycline, and oxytetracycline) in chicken meat. Tetracyclines are known to have a great tendency for epimerization and keto-enol tautomerism, which often provoke major challenges in their determination. Since this isomerization was found to be unavoidable during the whole chain of the current analysis, the total content (µg kg‒1) of individual tetracycline was quantified as a sum of each parent compound and its respective isomeric forms. Using this approach in combination with IDMS analysis, more consistent, accurate, and reproducible measurement results for the four tetracyclines in chicken meat were acquired. LC-MS/MS conditions and sample preparation processes were comprehensively optimized to minimize the chelating effect of tetracyclines and possible co-extracted interferences. Details of the sample preparation scheme, LC‒MS/MS detection, calculation equation, and method validation are described in this article. The method provided very good accuracy (97.7-102.6%) for all analytes across the concentration range of 10-200 µg kg‒1, with relative standard deviations for intra-day and inter-day precision of less than 4%. The limits of quantification were below 0.2 µg kg‒1, demonstrating the high sensitivity of the method. Furthermore, the measurement uncertainty was generally below 5.5%. Hence, the established method exhibits high-order metrological quality with superior performance over various existing methodologies. Moreover, this method can provide references for general food testing laboratories close to and far below the established maximum residue limits (100 µg kg‒1) for animal muscle tissues.
    Keywords:  4-epimers; Chicken meat; Isotope dilution mass spectrometry; Metrological quality; Tetracycline antibiotics
  28. Anal Chem. 2023 Feb 03.
      Infrared ion spectroscopy (IRIS) can be used to identify molecular structures detected in mass spectrometry (MS) experiments and has potential applications in a wide range of analytical fields. However, MS-based approaches are often combined with orthogonal separation techniques, in many cases liquid chromatography (LC). The direct coupling of LC and IRIS is challenging due to the mismatching timescales of the two technologies: an IRIS experiment typically takes several minutes, whereas an LC fraction typically elutes in several seconds. To resolve this discrepancy, we present a heartcutting LC-IRIS approach using a setup consisting of two switching valves and two sample loops as an alternative to direct online LC-IRIS coupling. We show that this automated setup enables us to record multiple IR spectra for two LC-features from a single injection without degrading the LC-separation performance. We demonstrate the setup for application in drug metabolism research by recording six m/z-selective IR spectra for two drug metabolites from a single 2 μL sample of cell incubation extract. Additionally, we measure the IR spectra of two closely eluting diastereomeric biomarkers for the inborn error of metabolism pyridoxine-dependent epilepsy (PDE-ALDH7A1), which shows that the heartcutting LC-IRIS setup has good sensitivity (requiring ∼μL injections of ∼μM samples) and that the separation between closely eluting isomers is maintained. We envision applications in a range of research fields, where the identification of molecular structures detected by LC-MS is required.
  29. Anal Methods. 2023 Jan 30.
      This study reports a novel fluorescent chiral derivatization reagent, 4-(N,N-dmethylaminosulfonyl)-2,1,3-benzoxadiazole-(2-succinimidoxy)-trans-2-methyl-L-proline (DBD-S-M-Pro), with a benzoxadiazole structure containing an N-hydroxysuccinimide activation group. DBD-S-M-Pro targets chiral amino-functional compounds under alkaline conditions without a condensation agent. Gradient elution was performed on a BEH C18 (100 × 2.1 mm, 1.7 μm) column with a mobile phase of 0.05% formic acid (FA) in 10 mM ammonium acetate (CH3COONH4) and 0.1% FA in acetonitrile or methanol. The efficiency of the chiral resolution was evaluated under excitation and emission wavelengths of 450 nm and 560 nm, respectively. The 19 chiral amino acids were separated in the range of 1.45-14.84. The resolutions of almost all DL-amino acids exceeded 1.5; the exceptions were serine (Ser) and lysine (Lys), with resolutions of 1.45 and 1.46, respectively. In addition, a new approach was devised for the simultaneous analysis of four chiral amino acids (DL-Glu, DL-Ala, DL-Val, and DL-Phe) in human hair. These amino acids were analyzed in the range of 12.5-400 pmol, with R2 ≥ 0.9990, limits of detection (S/N = 3) of 4-10 pmol, and intraday and interday precisions of 0.57-6.23%. The average spikes in the hair recoveries were 89.76-111.54%, and the matrix effects were 92.47-102.40%. Next, the contents of free chiral amino acids in the hair samples of 10 healthy volunteers (five males and five females) were analyzed with this method, and the differences were compared. The developed DBD-S-M-Pro provides a novel strategy for the sensitive determination of free chiral amino acids in living organisms.
  30. Anal Chem. 2023 Feb 01.
      Mass spectrometry imaging (MSI) is a powerful tool that can be used to simultaneously investigate the spatial distribution of different molecules in samples. However, it is difficult to comprehensively analyze complex biological systems with only a single analytical technique due to different analytical properties and application limitations. Therefore, many analytical methods have been combined to extend data interpretation, evaluate data credibility, and facilitate data mining to explore important temporal and spatial relationships in biological systems. Image registration is an initial and critical step for multimodal imaging data fusion. However, the image registration of multimodal images is not a simple task. The property difference between each data modality may include spatial resolution, image characteristics, or both. The image registrations between MSI and different imaging techniques are often achieved indirectly through histology. Many methods exist for image registration between MSI data and histological images. However, most of them are manual or semiautomatic and have their prerequisites. Here, we built MSI Registrar (MSIr), a web service for automatic registration between MSI and histology. It can help to reduce subjectivity and processing time efficiently. MSIr provides an interface for manually selecting region of interests from histological images; the user selects regions of interest to extract the corresponding spectrum indices in MSI data. In the performance evaluation, MSIr can quickly map MSI data to histological images and help pinpoint molecular components at specific locations in tissues. Most registrations were adequate and were without excessive shifts. MSIr is freely available at and
  31. Se Pu. 2023 Feb;41(2): 195-204
      A two-dimensional ultra performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (2D-UPLC-Q/TOF-MS) method was established for the separation and structural analysis of the components in teicoplanin. This method effectively solved the problems associated with chromatographic systems, such as liquid chromatography-mass spectrometry (LC-MS), which used a non-volatile phosphate buffer as the mobile phase and were not suitable for the rapid identification of impurities. Moreover, this method circumvented the complexities associated with locating and identifying impurities using the original method by re-establishing a chromatographic system suitable for LC-MS. In this study, for one-dimensional (1D) chromatography, the chromatographic separation was performed on an Octadecyl silica (ODS) hypersil column (250 mm×4.6 mm, 5 μm) with gradient elution using 3.0 g/L sodium dihydrogen phosphate buffer (pH 6.0)/acetonitrile=9/1 (v/v) as mobile phase A and 3.0 g/L sodium dihydrogen phosphate buffer (pH 6.0)/acetonitrile=3/7 (v/v) as mobile phase B. The column temperature was maintained at 30 ℃ and an ultraviolet detector was used at 254 nm for analysis. For 2D chromatography, desalting was performed on a Waters ACQUITY UPLC BEH C18 column (50 mm×2.1 mm, 1.7 μm) with gradient elution using ammonium formate buffer (pH 6.0) and acetonitrile as the mobile phases. The column temperature was maintained at 45 ℃. The MS data for the components and impurities were collected by positive ion electrospray ionization (ESI) using the full-information tandem MS mode (MSE). The cone and nebulizer gas flow rates were set at 50 and 900 L/h, respectively. The ion source and nebulizer gas temperatures were set at 120 ℃ and 500 ℃, respectively. The ESI and cone needle voltages were set at 2500 and 60 V, respectively. The collision energy was set at 20-50 eV. The molecular formulas of the components and impurities were determined using their exact masses and isotope distributions, and the structural components and impurities of teicoplanin were deduced from their fragment ions according to the fragmentation pathway of the TA2-2 component. Moreover, the 10 components reported in the European Pharmacopoeia 10.0 were analyzed and 22 impurities of teicoplanin were identified by 2D-UPLC-Q/TOF-MS. Three new impurities and two characteristic fragment ions of the teicoplanin parent nucleus were detected, and the fragmentation pathway of TA2-2 was deduced. Using this method, 1D-UPLC is applicable for the accurate qualification of components based on relative retention times, and 2D-UPLC-Q/TOF-MS is suitable for the rapid identification of the structure of components based on their fragment ions. The results indicate that 2D-UPLC-Q/TOF-MS may be used to analyze the structure of impurities in teicoplanin based on their exact masses, isotope distributions, and fragment ions. The method is rapid, simple, and sensitive, which provides a novel strategy for the quality control and process optimization of teicoplanin.
    Keywords:  analysis; impurity; quadrupole/time-of-flight mass spectrometry (Q/TOF-MS); teicoplanin; two-dimensional ultra performance liquid chromatography (2D-UPLC)
  32. J Anal Toxicol. 2023 Jan 28. pii: bkad006. [Epub ahead of print]
      Numerous methods and techniques have been published for the identification of new psychoactive substances (NPS) and their metabolites in urine. However, there lacks a holistic approach to analyze different groups of NPS and their metabolites with decision points for reporting their use. In this study, Data Dependent Acquisition (DDA) workflow using Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS) was developed and validated for the identification of a total of 94 NPS and metabolites in urine using the established decision points. The limit of identification for all analytes was determined at 25% below their respective decision points. The method was demonstrated to be accurate and precise at their respective decision points with extraction recoveries and ion suppression/enhancement ranging from 51.0% to 103.5% and -81.6% to 159.1%, respectively. There was no observed carryover at up to 200 ng/mL for all analytes and no interferences from urine matrices, internal standards and other common drugs of abuse. The extracted drug analytes were stable at 4 °C and 15 °C for up to 3 days. The validated method was successfully evaluated and applied in the testing of urine samples from NPS users. In conclusion, this validated method can analyze a wide range of NPS and their metabolites with the use of decision points for consistency in reporting.
    Keywords:  LC-QTOF-MS; New psychoactive substances; decision points for reporting NPS use; high-resolution mass spectrometry; supported-liquid extraction; urine
  33. Mass Spectrom (Tokyo). 2022 ;11(1): A0111
      Niemann-Pick disease type C (NPC) is an autosomal recessive disorder that is characterized by progressive neuronal degeneration. Patients with NPC have a wide age of onset and various clinical symptoms. Therefore, the discovery and diagnosis of NPC are very difficult. Conventional laboratory tests are complicated and time consuming. In this context, biomarker searches have recently been performed. Our research group has previously also investigated NPC biomarkers based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) and related techniques. To identify biomarker candidates, nontargeted analysis with high-resolution MS and MS/MS scanning is commonly used. Structural speculation has been performed using LC/MS/MS fragmentation and chemical derivatization, while identification is performed by matching authentic standards and sample specimens. Diagnostic performance evaluation was performed using the validated LC/MS/MS method and analysis of samples from patients and control subjects. NPC biomarkers, which have been identified and evaluated in terms of performance, are various classes of lipid molecules. Oxysterols, cholenoic acids, and conjugates are cholesterol-derived molecules detected in the blood or urine. Plasma lyso-sphingolipids are biomarkers for both NPC and other lysosomal diseases. N-palmitoyl-O-phosphocholine-serine is a novel class of lipid biomarkers for NPC. This article reviews biomarkers for NPC and the analysis methods employed to that end.
    Keywords:  LC/MS/MS; Niemann–Pick disease type C; biomarker; identification; quantification