bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒09‒25
fifty-four papers selected by
Sofia Costa

  1. Methods Mol Biol. 2023 ;2571 189-206
      Metabolites represent the most downstream level of the cellular organization. Hence, an in vitro untargeted metabolomics approach is extremely valuable to deepen the understanding of how endogenous metabolites in cells are altered under a given biological condition. This chapter describes a robust liquid chromatography-high-resolution mass spectrometry-based metabolomics and lipidomics platform applied to cell culture extracts. The analytical workflow includes an optimized sample preparation procedure to cover a wide range of metabolites using liquid-liquid extraction and validated instrumental operation procedures with the implementation of comprehensive quality assurance and quality control measures to ensure high reproducibility. The lipidomics platform is based on reversed-phase liquid chromatography for the separation of slightly polar to apolar metabolites and covers a broad range of lipid classes, while the metabolomics platform makes use of two hydrophilic interaction liquid chromatography methods for the separation of polar metabolites, such as organic acids, amino acids, and sugars. The chapter focuses on the analysis of cultured HepaRG cells that are derived from a human hepatocellular carcinoma; however, the sample preparation and analytical platforms can easily be adapted for other types of cells.
    Keywords:  Cell culture extracts; HepaRG cells; High-resolution mass spectrometry; Hydrophilic interaction liquid chromatography; Quality assurance and quality control; Reversed-phase liquid chromatography; Untargeted lipidomics; Untargeted metabolomics
  2. Methods Mol Biol. 2023 ;2571 105-114
      The simultaneous analysis of cationic and anionic metabolites using capillary electrophoresis-mass spectrometry (CE-MS) has been considered challenging, as often two different analytical methods are required. Although CE-MS methods for cationic metabolite profiling have already shown good performance metrics, the profiling of anionic metabolites often results in relatively low sensitivity and poor repeatability caused by problems related to unstable electrospray and corona discharge when using reversed CE polarity and detection by MS in negative ionization mode. In this protocol, we describe a chemical derivatization procedure that provides a permanent positive charge to acidic metabolites, thereby allowing us to profile anionic metabolites by CE-MS using exactly the same separation conditions as employed for the analysis of basic metabolites. The utility of the overall approach is demonstrated for the analysis of energy metabolism-related metabolites in low numbers of HepG2 cells.
    Keywords:  Capillary electrophoresis; Carboxylic acid metabolites; Chemical derivatization; HepG2 cells; Metabolomics
  3. Methods Mol Biol. 2023 ;2571 133-142
      Metabolomics continues to progress, but obstacles remain. The preservation of metabolites in the target tissue and gathering information on the current metabolic state of the organism of interest proves challenging. Robustness, reproducibility, and reliable quantification are necessary for confident metabolite identification and should always be considered for effective biomarker discovery. Recent advancements in analytical platforms, techniques, and data analysis make metabolomics a promising omics for significant research. However, there is no single approach to effectively capturing the metabolome. Coupling separation techniques may improve the power of the analysis and facilitate confident metabolite identification, especially when performing untargeted metabolomics. In this chapter, we will present an untargeted metabolomic analysis of brain tissue from C57BL/6 mice using two UHPLC-MS methods based on reversed-phase and HILIC chromatography.
    Keywords:  HILIC chromatography; High-resolution mass spectrometry; LC–MS/MS; Reversed-phase chromatography; Ultrahigh-performance liquid chromatography; Untargeted metabolomics
  4. Food Sci Anim Resour. 2022 Sep;42(5): 744-761
      The liquid chromatography mass spectrometry (LC-MS)-based metabolomic and lipidomic methodology has great sensitivity and can describe the fingerprint of metabolites and lipids in pork and beef. This approach is commonly used to identify and characterize small molecules such as metabolites and lipids, in meat products with high accuracy. Since the metabolites and lipids can be used as markers for many properties of a food, they can provide further evidence of the foods authenticity claim. Chromatography coupled to mass spectrometry is used to separate lipids and metabolites from meat samples. The research data usually is compared to lipid and metabolite databases and evaluated using multivariate statistics. LC-MS instruments directly connected to the metabolite and lipid databases software can be used to assess the authenticity of meat products. LC-MS has good selectivity and sensitivity for metabolomic and lipidomic analysis. This review highlighted the combination of metabolomics and lipidomics can be used as a reference for analyzing authentication meat products.
    Keywords:  authentication; lipidomics; liquid chromatography mass spectrometry (LC-MS); meat products; metabolomics
  5. Methods Mol Biol. 2023 ;2571 207-239
      Metabolomics is the latest of the omics sciences. It attempts to measure and characterize metabolites-small chemical compounds <1500 Da-on cells, tissue, or biofluids, which are usually products of biological reactions. As metabolic reactions are closer to the phenotype, metabolomics has emerged as an attractive science for various areas of research, including personalized medicine. However, due to the complexity of data obtained and the absence of curated databases for metabolite identification, data processing is the major bottleneck in this area since most technicians lack the required bioinformatics expertise to process datasets in a reliable and fast manner. The aim of this chapter is to describe the available tools for data processing that makes an inexperienced researcher capable of obtaining reliable results without having to undergo through huge parametrization steps.
    Keywords:  Data processing; Liquid chromatography; Mass spectrometry; Untargeted metabolomics
  6. Methods Mol Biol. 2023 ;2571 241-255
      Mass spectrometry (MS)-based metabolomics provides high-dimensional datasets; that is, the data include various metabolite features. Data analysis begins by converting the raw data obtained from the MS to produce a data matrix (metabolite × concentrations). This is followed by several steps, such as peak integration, alignment of multiple data, metabolite identification, and calculation of metabolite concentrations. Each step yields the analytical results and the accompanying information used for the quality assessment of the anterior steps. Thus, the measurement quality can be analyzed through data processing. Here, we introduce a typical data processing procedure and describe a method to utilize the intermediate data as quality control. Subsequently, commonly used data analysis methods for metabolomics data, such as statistical analyses, are also introduced.
    Keywords:  Data processing; Mass spectrometry; Multivariate analysis; Statistical analysis
  7. Methods Mol Biol. 2022 ;2546 295-309
      Measurement of methylmalonic acid (MMA) plays an important role in the diagnosis of vitamin B12 deficiency. Vitamin B12 is an essential cofactor for the enzymatic carbon rearrangement of methylmalonyl-CoA (MMA-CoA) to succinyl-CoA (SA-CoA), and the lack of vitamin B12 leads to elevated concentrations of MMA. Measurement of MMA in biological samples is complicated because of the presence of succinic acid (SA), isomer of MMA. We developed a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for MMA. The method utilizes derivatization and positive ion mode ionization, which is specific to polycarboxylic acids (MMA and SA are dicarboxylic acids), while derivatives of monocarboxylic acids at these conditions are not ionizable and not detectable. The only organic acid, other than MMA, that is detected in this method is SA. The described method does not require chromatographic resolution of the peaks of MMA and SA; quantitative measurement of MMA is performed using a deconvolution algorithm, which mathematically resolves signal corresponding to MMA, from the combined signal of MMA/SA. Because of the high selectivity of detection, this method utilizes isocratic chromatographic separation; reconditioning and re-equilibration of the chromatographic column between injections is unnecessary. The above features allow high-throughput analysis of MMA with injection-to-injection cycle time of approximately 1 minute.
    Keywords:  Data analysis; Deconvolution; Derivatization; Isomers; Liquid chromatography; Methylmalonic acid; Succinic acid; Tandem mass spectrometry
  8. Methods Mol Biol. 2023 ;2571 57-69
      Metabolomics, alone or in combination with other omics sciences, has shown great relevance in a large number of investigations in different branches of biomedicine, often providing novel discoveries and helping to expand the knowledge. Metabolomics analyses are carried out using different techniques, but in this chapter, we focus on liquid chromatography coupled to high-resolution mass spectrometry. The designated methodology consists of an untargeted approach for the analysis of plasma samples. The use of this method, with a reverse-phase column and electrospray ionization in positive mode, covers the detection of a broad range of metabolites, mainly of nonpolar and of intermediate polarity. This chapter also reviews the mass fragmentation spectra for the identification of bile acids, acylcarnitines, and glycerophospholipids.
    Keywords:  Acylcarnitines; Bile acids; Glycerophospolipids; Lipids; Mass spectrometry; Plasma; Reverse phase; Untargeted metabolomics
  9. Methods Mol Biol. 2022 ;2546 35-43
      We describe a simple stable isotope dilution method for accurate determination of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma as a clinical diagnostic test. Determination of SAM/SAH in plasma (20 μL) was performed by high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Calibrators (SAM and SAH) and internal standards (2H3-SAM and 2H4-SAH) were included in each analytical run for calibration. Sample preparation involved combining 20 μL sample with 180 μL of internal standard solution consisting of heavy-isotope-labeled internal standards in mobile phase A and filtering by ultracentrifugation through a 10 kd MW cutoff membrane. Sample filtrate (3 μL) was injected by a Shimadzu Nexera LC System interfaced with a 5500 QTRAP® (Sciex). Chromatographic separation was achieved on a 250 mm × 2.0 mm EZ-faast column from Phenomenex. Samples were eluted at a flow rate of 0.20 mL/min with a binary gradient with a total run time of 10 min. The source operated in positive ion mode at an ion spray voltage of +5000 V. SAM and SAH resolved by a gradient to 100% methanol with retention times of 5.8 and 5.5 min, respectively. HPLC chromatographic conditions did not produce complete separation of SAM and SAH, but they were completely discerned by their different fragmentation pattern in the mass spectrometer working in the MS-MS mode. The observed m/z values of the fragment ions were m/z 399→250 for SAM, m/z 385→136 for SAH, m/z 402→250 for 2H3-SAM, and m/z 203→46. The calibration curve was linear over the range of 12.5-5000 nmol/L for SAM and SAH.
    Keywords:  Mass spectrometry; Methylation; S-adenosylhomocysteine; S-adenosylmethionine
  10. Methods Mol Biol. 2023 ;2571 83-94
      Capillary electrophoresis-mass spectrometry (CE-MS) is an ideal method for analyzing various metabolites in biological samples. CE-MS can simultaneously identify and quantify hundreds of charged metabolites using only two acquisition methods for positively and negatively charged metabolites. Furthermore, CE-MS is commonly used for analyzing biological samples to understand the pathology of diseases at the metabolic level and biofluid samples, such as blood and urine, to explore biomarkers. Here, we introduce a protocol that delineates the handling of clinical samples to ensure that the CE-MS analysis yields reproducible quantified data. We have focused on sample collection, storage, processing, and measurement. Although the implementation of rigorous standard operating protocols is preferred for enhancing the quality of the samples, various limitations in an actual clinical setting make it difficult to adhere to strict rules. Therefore, the effect of each process on the quantified metabolites needs to be evaluated to design a protocol with acceptable tolerances. Furthermore, quality controls and assessments to handle clinical samples are introduced.
    Keywords:  Biofluid; Capillary electrophoresis–mass spectrometry; Clinical samples
  11. Methods Mol Biol. 2022 ;2546 141-148
      Adult and pediatric endocrinology and oncology often requires measuring serum estrogens and testosterone at very low concentrations. Conventional immunoassay methods often lack the required performance to meet this analytical need, and mass spectrometry techniques must be employed. Our aim was to develop a sensitive HPLC-MS/MS assay for both estradiol (E2) and testosterone (Te) in serum, utilizing commercially available calibrators and without the need for chemical derivatization. Serum samples, after the addition of an internal standard, are combined with a hexane:ethyl acetate extraction solution. The samples are vortexed, and the organic layer is decanted into a clean sample tube and evaporated to dryness under a stream of nitrogen. The samples are reconstituted in a water:methanol solution and separated chromatographically using a reversed-phase HPLC column. Subsequent mass spectrometry is performed using both positive ion mode for Te and negative ion mode for E2.
    Keywords:  Commercial calibrators; Estradiol; Liquid chromatography; Mass spectrometry; Serum; Testosterone
  12. Anal Chem. 2022 Sep 22.
      Untargeted liquid chromatography/high-resolution mass spectrometry (LC/HRMS) assays in metabolomics and exposomics aim to characterize the small molecule chemical space in a biospecimen. To gain maximum biological insights from these data sets, LC/HRMS peaks should be annotated with chemical and functional information including molecular formula, structure, chemical class, and metabolic pathways. Among these, molecular formulas may be assigned to LC/HRMS peaks through matching theoretical and observed isotopic profiles (MS1) of the underlying ionized compound. For this, we have developed the Integrated Data Science Laboratory for Metabolomics and Exposomics-United Formula Annotation (IDSL.UFA) R package. In the untargeted metabolomics validation tests, IDSL.UFA assigned 54.31-85.51% molecular formula for true positive annotations as the top hit and 90.58-100% within the top five hits. Molecular formula annotations were also supported by tandem mass spectrometry data. We have implemented new strategies to (1) generate formula sources and their theoretical isotopic profiles, (2) optimize the formula hits ranking for the individual and aligned peak lists, and (3) scale IDSL.UFA-based workflows for studies with larger sample sizes. Annotating the raw data for a publicly available pregnancy metabolome study using IDSL.UFA highlighted hundreds of new pregnancy-related compounds and also suggested the presence of chlorinated perfluorotriether alcohols (Cl-PFTrEAs) in human specimens. IDSL.UFA is useful for human metabolomics and exposomics studies where we need to minimize the loss of biological insights in untargeted LC/HRMS data sets. The IDSL.UFA package is available in the R CRAN repository Detailed documentation and tutorials are also provided at
  13. Methods Mol Biol. 2022 ;2546 95-104
      L-carnitine is a crucial component for transporting long-chained fatty acids from the cytosol into the mitochondrial matrix for fatty acid oxidation. During this process, carnitine forms numerous acylcarnitines before being recycled into the cytosol. Abnormal levels of free carnitine, total carnitine, and acylcarnitines in serum can be indicative of a metabolic disorder before symptoms are present. A liquid chromatography tandem mass spectrometry (LC-MS/MS) method is described for the determination of free and total carnitine in serum. To measure total carnitine, samples are spiked with deuterated carnitine (internal standard) and hydrolyzed with potassium hydroxide to convert acylcarnitines to carnitine. The reaction is quenched by the addition of hydrochloric acid. Carnitine is extracted via a methanolic protein precipitation. The solution is then injected on LC-MS/MS for analysis to determine the carnitine concentration using multiple-reaction monitoring.
    Keywords:  Acylcarnitine; Carnitine; Fatty acid oxidation; Tandem mass spectrometry
  14. Methods Mol Biol. 2022 ;2546 451-457
      Congenital adrenal hyperplasia (CAH) is a group of autosomal-recessive disorders due to deficiency of 11- or 21-hydroxylase. The analysis of cortisol, androstenedione, 17-hydroxyprogesterone (OHPG), dehydroepiandrosterone (DHEA), 11-deoxycortisol, and testosterone is generally performed in the diagnosis and/or follow-up of CAH. Analysis of specific steroids is also performed in other disorders such as evaluation of hirsutism or infertility in females and hypogonadism in males. Cortisol is generally analyzed by immunoassays, whereas other hormones are preferably assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). A multiple reaction monitoring, positive mode atmospheric pressure chemical ionization, LC-MS/MS method is described for the simultaneous quantification of androstenedione, 17-hydroxyprogesterone, DHEA, 11-deoxycortisol and testosterone. The method involves addition of labeled internal standards to serum samples and extraction of steroids in methyl tert-butyl ether. The extract is evaporated under stream of nitrogen, and the residue is reconstituted in methanol and analyzed by LC-MS/MS.
    Keywords:  11-Deoxycortisol; 17-Hydroxyprogesterone and testosterone; Androstenedione; Dehydroepiandrosterone; Tandem mass spectrometry (LC-MS/MS)
  15. J Am Soc Mass Spectrom. 2022 Sep 22.
      In 2017, the United States Department of Health and Human Services declared the widespread misuse and abuse of prescription and illicit opioids an epidemic. However, this epidemic dates back to the 1990s when opioids were extensively prescribed for pain management. Currently, opioids are still recommended for pain management, and given their abuse potential, rapid screening is imperative for patient treatment. Of particular importance is assessing pain management patient compliance, where evaluating drug use is crucial for preventing opioid abuse and potential overdoses. In this work, we utilized drift tube ion mobility spectrometry coupled with mass spectrometry (DTIMS-MS) to develop a rapid screening method for 33 target opioids and opioid urinary metabolites. Collision cross section values were determined for all target molecules using a flow-injection DTIMS-MS method, and clear differentiation of 27 out of the 33 opioids without prior chromatographic separation was observed when utilizing a high resolution demultiplexing screening approach. An automated solid phase extraction (SPE) platform was then coupled to DTIMS-MS for 10 s sample-to-sample analyses. This SPE-IMS-MS approach enabled the rapid screening of urine samples for opioids and presents a major improvement in sample throughput compared to traditional chromatographic analyses coupled with MS, which routinely take several minutes per sample. Overall, this vast reduction in analysis time facilitates a faster turn-around for patient samples, providing great benefits to clinical applications.
  16. Methods Mol Biol. 2022 ;2546 119-128
      Monitoring urinary free cortisol (UFC) excretion helps assess adrenal function and is used to screen for endogenous Cushing's syndrome caused by an adrenal or pituitary tumor. While serum cortisol levels fluctuate in response to time of day, stress, and concentrations of cortisol-binding globulin (CBG), a 24-h urine collection measures the cortisol produced over the entire day and does not suffer from as much variability as a serum measurement.We describe here a method of measurement of urinary free cortisol (UFC) and cortisone using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine samples, combined with stable isotope-labeled internal standards, are extracted by liquid-liquid extraction using ethyl acetate and hexane. An API 5500 mass spectrometer operated in positive atmospheric pressure chemical ionization (APCI) mode is used for detection.
    Keywords:  APCI; Cortisone; Cushing’s syndrome; LC-MS/MS; Urinary free cortisol
  17. Methods Mol Biol. 2022 ;2546 175-183
      Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are catecholamine metabolites used in the diagnostic workup of neuroendocrine tumors. Here we describe a simple dilute-and-shoot method for simultaneously quantitating HVA and VMA in human urine specimens. The method employs analyte separation on a reverse-phase liquid chromatography column followed by detection using electrospray ionization triple quadrupole mass spectrometry (ESI-MS/MS), wherein qualifier and quantifier ion transitions are monitored. This is a simple and fast analytical method with an injection-to-injection time of 4 min.
    Keywords:  Adrenal medulla; Homovanillic acid; Neuroblastoma; Pheochromocytoma; Vanillylmandelic acid
  18. Methods Mol Biol. 2022 ;2546 509-521
      Peroxisomal disorders are a heterogeneous group of genetic disorders caused by impaired peroxisomal biogenesis or by defects in single peroxisomal proteins. The most common peroxisomal disorders are Zellweger spectrum disorders (ZSDs), due to pathogenic variants in one of the 13 PEX genes, and X-linked adrenoleukodystrophy/adrenomyeloneuropathy (X-ALD/AMN), due to pathogenic variants in ATP-binding cassette transporter type D1 (ABCD1) gene. Peroxisomes perform multiple essential cellular functions, including β-oxidation of very-long-chain fatty acids (VLCFAs), pristanic acid and some bile acid intermediates, and α-oxidation of phytanic acid. In most patients, abnormal levels of VLCFAs and/or branched-chain fatty acids (BCFAs, e.g., phytanic and pristanic acids) are present; hence, measuring these analytes is critical when suspecting a peroxisomal disorder. This chapter describes a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify VLCFAs and BCFAs in plasma or serum for the diagnosis of peroxisomal disorders. The method consists of an acid hydrolysis step to release the fatty acids from their coenzyme A esters followed by derivatization using oxalyl chloride, dimethylaminoethanol, and then methyl iodide. The trimethyl-amino-ethyl (TMAE) iodide ester derivatives are analyzed using UPLC-MS/MS in positive electrospray ionization and multiple reaction-monitoring (MRM) mode. Quantitation is performed using a five-point calibration curve after normalizing with deuterated internal standards.
    Keywords:  Branched-chain fatty acids; Liquid chromatography-tandem mass spectrometry; Peroxisomal disorders; Very-long-chain fatty acids
  19. Methods Mol Biol. 2023 ;2571 95-103
      Capillary electrophoresis-mass spectrometry (CE-MS) is gaining interest for metabolomics studies because of its high separation efficiency, selectivity, and versatility. The ability to inject nanoliters from only a few microliters of sample in the injection vial makes this approach very suited for volume-limited applications. However, the low injection volumes could compromise the detection sensitivity of CE-MS, thereby potentially limiting its scope in metabolomics. To overcome this issue, online sample preconcentration methods have been developed to increase sample-loading volumes without hampering the intrinsic high separation efficiency of CE. In this protocol, online preconcentration with sample stacking based on pH junction was assessed for the direct profiling of endogenous metabolites in rat brain microdialysates. Sample stacking was realized by a pre-injection of ammonium hydroxide, followed by a large sample injection (i.e., about 17% of the total capillary volume). It is shown that this relatively simple and fast preconcentration procedure is fully compatible with the high-salt concentration in microdialysates and significantly improves the detection sensitivity of the CE-MS method.
    Keywords:  Brain microdialysate; Capillary electrophoresis; Direct analysis; Mass spectrometry; Metabolic profiling
  20. Methods Mol Biol. 2022 ;2546 149-163
      Quantitation of long-chain fatty acids in serum/plasma and red blood cells is a useful diagnostic tool in the evaluation of nutritional status and assessment of risk for essential fatty acid deficiency (EFAD). Serum/plasma has been the traditional sample type for this method, yet it requires prolonged fasting which is not compatible with some patient populations. More recently, red blood cells have become an important sample type due to less intraindividual variability and obviating the need for fasting. Here we present a method for the quantitation of 22 fatty acids in serum/plasma or red blood cells. Fatty acids are hydrolyzed and extracted from the biological matrix, followed by derivatization with pentafluorobenzyl bromide and subsequent analysis by gas chromatography-negative chemical ionization-mass spectrometry (GC-NCI-MS).
    Keywords:  Chemical ionization; Essential fatty acid deficiency; Fatty acids; Gas chromatography; Mass spectrometry; Plasma; Red blood cells; Selected ion monitoring; Serum
  21. Methods Mol Biol. 2023 ;2576 49-66
      The adequate quantification of endocannabinoids and related N-acylethanolamines can be complex due to their low endogenous levels, structural diversity, and metabolism. Therefore, advanced analytical approaches, involving LC-MS, are required to quantify these molecules in plasma, tissues, and other matrices. It has been shown that endocannabinoid congeners synthesized from n-3 poly-unsaturated fatty acids (n-3 PUFAs), such as docosahexaenoylethanolamide (DHEA) and eicosapentaenoylethanolamide (EPEA), have interesting immunomodulatory and tumor-inhibiting properties. Recent work has shown that DHEA and EPEA can be further enzymatically metabolized by cyclo-oxygenase 2 (COX-2), forming oxygenated metabolites. Here, an LC-MS-based method for the quantification of the n-3 PUFA-derived endocannabinoid congeners DHEA and EPEA is described, which is also suited to measure a wider spectrum of endocannabinoids. The chapter contains a step-by-step protocol for the analysis of (n-3) endocannabinoids in plasma, including sample collection and solid phase extraction, LC-MS analysis, and data processing. In addition, protocol modifications are provided to allow quantification of n-3 PUFA-derived endocannabinoids and their COX-2 metabolites in tissues and cell culture media. Finally, conditions that alter endocannabinoid concentrations are briefly discussed.
    Keywords:  Docosahexaenoylethanolamide; Eicosapentaenoylethanolamide; Endocannabinoids; LC–MS; Solid phase extraction; n-3 fatty acid
  22. Methods Mol Biol. 2022 ;2546 195-204
      Serotonin (5-hydroxytryptamine) is a neurotransmitter produced in excess by carcinoid tumors, which develop from enterochromaffin cells. 5-Hydroxyindoleacetic acid (5-HIAA) is the primary urinary metabolite of serotonin, making measurement of 5-HIAA useful in the diagnosis and management of carcinoid tumors. Here we describe a simple, inexpensive, and fast method for the detection and quantification of 5-HIAA in urine. Samples are prepared by simple 1:1 dilution. The instrumental analysis is performed by chromatographic separation on a reverse-phase analytical column followed by detection using a triple quadrupole mass spectrometer with electrospray ionization in positive ion mode. Data are acquired by multiple reaction monitoring (MRM).
    Keywords:  5-Hydroxyindolacetic acid; Carcinoid tumor; Tandem mass spectrometry; Ultra-performance liquid chromatography
  23. STAR Protoc. 2022 Sep 22. pii: S2666-1667(22)00595-0. [Epub ahead of print]3(4): 101715
      Differences in metabolic profiles can link to functional changes of immune cells in disease conditions. Here, we detail a protocol for the detection and quantitation of 19 metabolites in one analytical run. We provide the parameters for chromatographic separation and mass spectrometric analysis of isotopically labeled and unlabeled metabolites. We include steps for incubation and sample preparation of PBMCs and monocytes. This protocol overcomes the chromatographic challenges caused by the chelating properties of some metabolites.
    Keywords:  Chemistry; Immunology; Mass spectrometry; Metabolism; Metabolomics
  24. Methods Mol Biol. 2022 ;2546 523-537
      Liquid-chromatography tandem mass spectrometry (LC-MS/MS) has been shown to be an effective approach in the clinical analysis of 25-OH-vitamin D in patient serum. Test volumes vary among laboratories and different levels of throughput are required for different settings. LC-MS/MS assays with multiple LC channels can be beneficial for labs with the demand of large sample volume (e.g., 300 or more samples) to control costs and fulfill a reasonable turnaround time. We hereby present an assay that employs 4 LC channels (4-plex), which are coupled to the TSQ Endura triple-quadrupole (QqQ) MS instrument, for a high-throughput solution. Briefly, the pre- and postelution segments of the LC gradient are diverted to waste via solenoid valve controls, reserving the data acquisition for only the elution segment per injection per channel at a time. The multiplexing affords a manifold increase in throughput and the optimization of the duty cycle, without compromise in assay performance and precision.
    Keywords:  25-OH-vitamin D; LC-to-LC channel precision; Multiple-reaction-monitoring (MRM); atmospheric pressure chemical ionization (APCI); duty cycle; high-throughput LC-MS/MS; multiplex HPLC
  25. J Chromatogr A. 2022 Sep 13. pii: S0021-9673(22)00687-2. [Epub ahead of print]1682 463495
      The application of proton transfer ionization reaction mass spectrometry (PTR MS) combined with microscale supercritical fluid extraction (SFE) and supercritical fluid chromatography (SFC) aiming to quantitate single-cell fatty acid analysis levels was investigated. Using a microscale extraction vessel, the obtained low limits of quantitation (LLOQs) of arachidonic acid and arachidic acid were 1.2 and 2.7 fmol, respectively, by using less than 1 µL of sample on stainless steel frit. A series of phthalate, vitamin K1, and α-tocopherol were also tested, and the LLOQ was less than one femtomole for phthalate and 35 and 13 fmol for vitamin K1 and α-tocopherol, respectively. A microliter portion of SFE extracts was introduced into the SFC column by split injection, improving the reproducibility of the chromatography and separation efficiency. The method in the present study has great potential to quantitate lipophilic molecules on the nanogram scale of a sample without complex preparation procedures.
    Keywords:  Proton-transfer-reaction; Supercritical fluid chromatography; Supercritical fluid extraction; Time-of-flight mass spectrometer; Trace-level fatty acid analysis
  26. Anal Sci. 2022 Sep 20.
      In this work, dispersive liquid-liquid microextraction (DLLME) based on high-density extraction solvent was applied as a simple, fast and sensitive method for extraction and preconcentration of methamphetamine from human plasma and urine samples. The efficiency of positive corona discharge ionization ion mobility spectrometry was investigated for direct analysis of the extracted analyte. Effective parameters on the extraction efficiency, such as type and volume of the extraction and disperser solvents, centrifugation time, and sample solution pH were optimized. Trichloromethane and isopropanol were selected as the extracting and disperser solvents, respectively. Under the optimized conditions, the linear dynamic range (R2 = 0.9969) was found to be 0.5-18 µg/L, and 0.15 µg/L was calculated as the limit of detection. The relative standard deviations of intra- and inter-day were obtained 4 and 10%, respectively, and finally, in the analysis of human plasma and urine samples, the extraction recovery was obtained 104%.
    Keywords:  Biological samples; DLLME; Ion mobility spectrometry; Methamphetamine; Sample extraction method
  27. Molecules. 2022 Sep 07. pii: 5796. [Epub ahead of print]27(18):
      Solid-phase analytical derivatization (SPAD) is a promising hybrid sample preparation technique combining the clean-up and preconcentration of the sample in a single step. In this work, a novel SPAD method based on the preparation of trimethylsilyl (TMS) derivatives of steroid hormones (testosterone, estrone, DHT, estriol, estradiol, and progesterone) in Phenomenex Strata C18-E (100 mg, 1 mL) cartridges has been developed and applied for their GC-MS/MS determination in human urine samples. The proposed procedure allows the detection and quantification of steroids with limits of 1.0-2.5 and 2.5-5 ng/mL, respectively. These characteristics are comparable with those obtained with a conventional liquid-liquid extraction, while the recovery of analytes in the proposed SPAD procedure is higher. The major advantages of SPAD are a short derivatization time, high efficiency, and the possibility to automatize the procedure. However, its cost-effectiveness in routine practice is still questionable.
    Keywords:  GC-MS/MS; LLE; SPAD; androgens; estrogens; sample preparation; steroid hormones
  28. J Cheminform. 2022 Sep 22. 14(1): 64
      The majority of primary and secondary metabolites in nature have yet to be identified, representing a major challenge for metabolomics studies that currently require reference libraries from analyses of authentic compounds. Using currently available analytical methods, complete chemical characterization of metabolomes is infeasible for both technical and economic reasons. For example, unambiguous identification of metabolites is limited by the availability of authentic chemical standards, which, for the majority of molecules, do not exist. Computationally predicted or calculated data are a viable solution to expand the currently limited metabolite reference libraries, if such methods are shown to be sufficiently accurate. For example, determining nuclear magnetic resonance (NMR) spectroscopy spectra in silico has shown promise in the identification and delineation of metabolite structures. Many researchers have been taking advantage of density functional theory (DFT), a computationally inexpensive yet reputable method for the prediction of carbon and proton NMR spectra of metabolites. However, such methods are expected to have some error in predicted 13C and 1H NMR spectra with respect to experimentally measured values. This leads us to the question-what accuracy is required in predicted 13C and 1H NMR chemical shifts for confident metabolite identification? Using the set of 11,716 small molecules found in the Human Metabolome Database (HMDB), we simulated both experimental and theoretical NMR chemical shift databases. We investigated the level of accuracy required for identification of metabolites in simulated pure and impure samples by matching predicted chemical shifts to experimental data. We found 90% or more of molecules in simulated pure samples can be successfully identified when errors of 1H and 13C chemical shifts in water are below 0.6 and 7.1 ppm, respectively, and below 0.5 and 4.6 ppm in chloroform solvation, respectively. In simulated complex mixtures, as the complexity of the mixture increased, greater accuracy of the calculated chemical shifts was required, as expected. However, if the number of molecules in the mixture is known, e.g., when NMR is combined with MS and sample complexity is low, the likelihood of confident molecular identification increased by 90%.
    Keywords:  DFT; Metabolomics; NMR; Quantum chemistry; Small molecules
  29. Methods Mol Biol. 2022 ;2546 45-54
      Accurate determination of serum and plasma aldosterone is essential for screening, diagnosis, and subtype classification of primary aldosteronism (PA). Its measurement is also used in the investigation of adrenal incidentaloma, adrenal carcinoma, Addison's disease, congenital adrenal hyperplasia, renal artery stenosis, and renal tubular channelopathies. We describe a simple and robust method for the accurate and precise measurement of aldosterone in serum or plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). After addition of internal standard, aldosterone is extracted from serum samples using supported liquid extraction (SLE) with methyl tert-butyl ether (MtBE). The MtBE is evaporated to dryness, and the sample is reconstituted with mobile phase before injection onto the LC-MS/MS and quantitation using an eight-point calibration curve. The assay calibration range is approximately 50-6500 pM (0.16-234 ng/dL) with total imprecision between 6.8% and 4.1% for concentrations between about 50 and 1000 pM, respectively.
    Keywords:  Aldosterone; Hypokalemia; Mass spectrometry; Mineralocorticoid hypertension; Plasma renin activity; Primary aldosteronism; Secondary hypertension
  30. Methods Mol Biol. 2022 ;2546 459-472
      We describe an LC-MS/MS method for serum testosterone using a novel extraction media, AC Extraction Plate™ (AC Plate,Tecan Schweiz). The AC Plate principle is essentially that of a liquid-liquid extraction (LLE) but employs a stationary nonpolar phase coated on the wells of 96-well plates instead of a nonmiscible organic solvent for partitioning testosterone out of serum, leaving interfering substances behind. This low complexity sample preparation protocol has been validated for and used in production in our laboratory with both manual and automated liquid handling. The primary advantage of this method is the highly reproducible nature of an extraction method that does not require LC-MS/MS expertise or specialized extraction equipment. We modified the existing vendor application and validated the method for matrix effect, recovery, precision, trueness [accuracy relative to certified reference material (CRM)], specificity, reportable range, sample stability, various sample containers, and correlation with other methods.Method performance is excellent, with a reportable range of 4-750 ng/dL, between-day quality control coefficient of variation (CV) over 12 months of <8%, mean accuracy of <4.0% bias against CRM, no interference from hemolysis, icterus, lipemia, serum separator tube gel, or common steroids/metabolites, and mean bias of 1.3% versus 4 other LC-MS/MS testosterone methods. An investigation of calibration stability and robustness supports sparse (3 versus 6 calibrators) and/or historical calibration for routine use.
    Keywords:  Automation; Ease-of-use; Extraction; LC-MS/MS; Reproducibility; Testosterone
  31. Methods Mol Biol. 2023 ;2571 115-122
      The circulating metabolome of human peripheral blood provides valuable information to investigate the molecular mechanisms underlying the development of diseases and to discover candidate biomarkers. In particular, erythrocytes have been proposed as potential systemic indicators of the metabolic and redox status of the organism. To accomplish wide-coverage metabolomics analysis, the combination of complementary analytical techniques is necessary to manage the physicochemical complexity of the human metabolome. Herein, we describe an untargeted metabolomics method to capture the plasmatic and erythroid metabolomes based on ultrahigh-performance liquid chromatography coupled to high-resolution mass spectrometry, combining reversed-phase liquid chromatography and hydrophilic interaction liquid chromatography. The method provides comprehensive metabolomics fingerprinting of plasma and erythrocyte samples, thereby enabling the elucidation of the distinctive metabolic disturbances behind childhood obesity and associated comorbidities, such as insulin resistance.
    Keywords:  Childhood obesity; Erythrocyte; Liquid chromatography; Mass spectrometry; Metabolomics; Plasma
  32. Metabolites. 2022 Aug 28. pii: 807. [Epub ahead of print]12(9):
      Pre-column fluorescent derivatization has been used for the fast quantification of amino acids using high-performance liquid chromatography (HPLC) systems. However, it generally requires an offline in-vial derivatization process with multiple derivatization reagents. The offline derivatization requires the same number of reaction vials as the number of sample vials for use as a reaction chamber for the derivatization reaction in an autosampler. Therefore, the number of samples analyzed per batch using the pre-column derivatization method is halved. To benefit from the pre-column derivatization method, we transformed the derivatization process from an offline chamber process to an online in-needle process (in-needle Pre-column Derivatization for Amino acids Quantification; iPDAQ). Fluorescent derivatization in the injection needle obviated the need for vacant vials as reaction chambers. Consequently, the throughput per batch improved up to two times, and the consumption of derivatization reagents was reduced to less than one-tenth of that in the conventional vial method. We demonstrated to separate and quantify the amino acids in various biological samples. Herein, we presented a novel HPLC-based amino acid quantification method that enables the continuous analysis of a large number of samples. The iPDAQ facilitates accurate amino acid quantification due to the automation of derivatization and achieves improvement in the throughput and reduction of analysis labor.
    Keywords:  HPLC; SRM1950; amino acids quantification; pre-column derivatization
  33. Methods Mol Biol. 2023 ;2571 157-168
      Imaging mass spectrometry (IMS) allows for visualization of the spatial distribution of proteins, lipids, and other metabolites in a targeted or untargeted approach. The identification of compounds through mass spectrometry combined with the mapping of compound distribution in the sample establishes IMS as a powerful tool for metabolomics. IMS analysis for serotonin will allow researchers to pinpoint areas of deficiencies or accumulations associated with ocular disorders such as serotonin selective reuptake inhibitor optic neuropathy. Furthermore, L-DOPA has shown great promise as a therapeutic approach for disorders such as age-related macular degeneration, and IMS allows for localization, and relative magnitudes, of L-DOPA in the eye. We describe here an end-to-end approach of IMS from sample preparation to data analysis for serotonin and L-DOPA analysis.
    Keywords:  High-resolution mass spectrometry; Imaging mass spectrometry; L-DOPA; Localization; MALDI; Mass spectrometry; Metabolite localization; Metabolite visualization; Metabolomics; Serotonin
  34. Methods Mol Biol. 2022 ;2546 13-25
      Quality assurance (QA) activities enable continuous improvement through ongoing post-implementation monitoring to identify, evaluate, and correct problems. QA for clinical liquid chromatography tandem mass spectrometry (LC-MS/MS) assays should include specific components that address the unique aspects of these methods. This chapter briefly describes approaches for clinical LC-MS/MS system performance monitoring using batch and peak review metrics, largely following CLSI-C62A guidance. Though routine checks ensure the quality of results reported for each run, there is also a need to evaluate metrics between runs over time. Post-implementation performance monitoring of LC-MS/MS methods is typically focused on calibration curves, retention times, peak intensities, and ion ratios.
    Keywords:  Data analytics; LC-MS/MS; Mass spectrometry; Quality assurance
  35. Methods Mol Biol. 2022 ;2546 65-81
      Branched-chain amino acids (BCAA), including valine, alloisoleucine, isoleucine, and leucine, play significant roles in a number of metabolic pathways in the body. Deficiency in branched-chain ketoacid dehydrogenase complex, an enzyme required for metabolism of those amino acids, will lead to elevation and accumulation of BCAA and ketoacids in bodily fluids. This results in maple syrup urine disease (MSUD), a condition estimated to affect 1 in 100,000-300,000 births. If MSUD is not diagnosed in the first few days of life, progression of this disease can lead to intellectual disability, coma, irreversible brain damage, seizures, or even death. If diagnosed early, MSUD can be managed by monitoring the blood concentrations of BCAA and adjusting the patient's dietary intake accordingly. Therefore, it is critical to have a rapid, accurate, and reliable BCAA assay for confirmation of MSUD in newborns as well as routine monitoring of MSUD patients. Here, we describe a high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for BCAA measurement which requires only 20 μL of plasma. The sample preparation does not require derivatization and only involves protein precipitation with LC/MS-grade methanol, which contains leucine(13C6;15N), isoleucine(13C6;15N), and valine(13C5;15N) as the internal standards. The final sample extracts do not require dry-down and reconstitution and are readily compatible with the liquid chromatography (LC) method. BCAA are separated using the isocratic gradient method on a mixed-mode Intrada column. Multiple-reaction monitoring (MRM) mode is used for MS/MS detection to monitor the parent-to-daughter transitions m/z 132.2 to 86.4 for leucine, isoleucine, and alloisoleucine; m/z 118.2 to 72.4 for valine; m/z 139.2 to 92.4 for leucine(13C6;15N) and isoleucine(13C6;15N); and m/z 124.2 to 77.4 for valine(13C5;15N).
    Keywords:  Branched-chain amino acid; Liquid chromatography; Maple syrup urine disease; Mass spectrometry; Plasma; Quantification
  36. J Am Soc Mass Spectrom. 2022 Sep 21.
      Ion signals in electrospray ionization (ESI) mass spectrometry (MS) are affected by addition of acid or base. Acids or bases are typically added to samples to enhance detection of analytes in positive- or negative-ion mode, respectively. To carry out simultaneous monitoring of analytes with different ionogenic moieties by ESI-MS, a rapid acid/base switching system was developed. The system was further coupled with flow injection analysis (FIA) and liquid chromatography (LC) MS. The two variants enable detection of separated analytes immediately after alternating addition of acid and base. The methods were tested using a set of phospholipids (PLs) as analytes. The rapid acid/base switching enhanced signals of some of the PL analytes in both ion modes of MS. Both FIA-MS and LC-MS with acid/base switching show signal enhancements (∼1.3-23.2 times) of some analyte signals when compared with analysis conducted without acid/base switching. The proposed methods are suitable for simultaneous analysis of cationic and anionic analytes. The FIA-MS and LC-MS methods with acid/base switching were also applied in analysis of lipid extract from real samples (sausage and porcine liver). However, the FIA-MS results were affected by ionization competition and isobaric interference due to the complexity of the sample matrix and diversity of PL species. In contrast, the LC-MS mode provides adequate selectivity to observe signal enhancement for specific analyte ions. Overall, alternating addition of acid and base immediately before the ESI source can improve analytical performance without the need to carry out separate analyses targeting different types of analytes.
  37. Methods Mol Biol. 2022 ;2546 105-117
      Cortisol is one of the most important glucocorticoids involved in the regulation of human metabolism and physiological stress. Monitoring of levels of cortisol is of immense clinical benefit. In particular, salivary cortisol levels have been shown to correlate well with diurnal changes in cortisol levels in serum and have been used widely for monitoring of cortisol levels for diagnosis and prognosis purposes. We present a sensitive, fast, and robust quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) assay for salivary cortisol in negative mode. This assay employs protein precipitation followed by reversed-phase liquid chromatographic separation, negative-mode electrospray ionization (ESI), and MS/MS detection. This assay has a total run time of 5.8 minutes and a limit of quantification of 0.5 ng/mL with a linear range up to 100 ng/mL. No carryover was observed at 10 μg/mL. This assay also incorporates the routine monitoring of prednisolone, a potential interferent to salivary cortisol.
    Keywords:  Cushing’s syndrome; Interferents; LC-MS/MS; Prednisolone; Quality control; Salivary cortisol
  38. Int J Mol Sci. 2022 Sep 18. pii: 10908. [Epub ahead of print]23(18):
      Metabolomics is a potential approach to paving new avenues for clinical diagnosis, molecular medicine, and therapeutic drug monitoring and development. The conventional metabolomics analysis pipeline depends on the data-independent acquisition (DIA) technique. Although powerful, it still suffers from stochastic, non-reproducible ion selection across samples. Despite the presence of different metabolomics workbenches, metabolite identification remains a tedious and time-consuming task. Consequently, sequential windowed acquisition of all theoretical MS (SWATH) acquisition has attracted much attention to overcome this limitation. This article aims to develop a novel SWATH platform for data analysis with a generation of an accurate mass spectral library for metabolite identification using SWATH acquisition. The workflow was validated using inclusion/exclusion compound lists. The false-positive identification was 3.4% from the non-endogenous drugs with 96.6% specificity. The workflow has proven to overcome background noise despite the complexity of the SWATH sample. From the Human Metabolome Database (HMDB), 1282 compounds were tested in various biological samples to demonstrate the feasibility of the workflow. The current study identified 377 compounds in positive and 303 in negative modes with 392 unique non-redundant metabolites. Finally, a free software tool, SASA, was developed to analyze SWATH-acquired samples using the proposed pipeline.
    Keywords:  MS/MS library; SWATH; data-independent acquisition; high-resolution LC-MS; metabolomics
  39. Methods Mol Biol. 2022 ;2546 485-492
      A method for free thyroxine measurement in human serum using equilibrium dialysis followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) is described. Free thyroxine in serum is first separated from protein-bound thyroxine by equilibrium dialysis and then measured by LC-MS/MS.
    Keywords:  Equilibrium dialysis; Free thyroxine; LC-MS/MS; T4
  40. Metabolites. 2022 Aug 29. pii: 812. [Epub ahead of print]12(9):
      Untargeted metabolomics is a promising tool for identifying novel disease biomarkers and unraveling underlying pathomechanisms. Nuclear magnetic resonance (NMR) spectroscopy is particularly suited for large-scale untargeted metabolomics studies due to its high reproducibility and cost effectiveness. Here, one-dimensional (1D) 1H NMR experiments offer good sensitivity at reasonable measurement times. Their subsequent data analysis requires sophisticated data preprocessing steps, including the extraction of NMR features corresponding to specific metabolites. We developed a novel 1D NMR feature extraction procedure, called Bucket Fuser (BF), which is based on a regularized regression framework with fused group LASSO terms. The performance of the BF procedure was demonstrated using three independent NMR datasets and was benchmarked against existing state-of-the-art NMR feature extraction methods. BF dynamically constructs NMR metabolite features, the widths of which can be adjusted via a regularization parameter. BF consistently improved metabolite signal extraction, as demonstrated by our correlation analyses with absolutely quantified metabolites. It also yielded a higher proportion of statistically significant metabolite features in our differential metabolite analyses. The BF algorithm is computationally efficient and it can deal with small sample sizes. In summary, the Bucket Fuser algorithm, which is available as a supplementary python code, facilitates the fast and dynamic extraction of 1D NMR signals for the improved detection of metabolic biomarkers.
    Keywords:  NMR metabolomics; data preprocessing; feature extraction
  41. J Chromatogr A. 2022 Sep 13. pii: S0021-9673(22)00691-4. [Epub ahead of print]1682 463499
      There are many challenges associated with analysing gas chromatography - mass spectrometry (GC-MS) data. Many of these challenges stem from the fact that electron ionization (EI) can make it difficult to recover molecular information due to the high degree of fragmentation with concomitant loss of molecular ion signal. With GC-MS data there are often many common fragment ions shared among closely-eluting peaks, necessitating sophisticated methods for analysis. Some of these methods are fully automated, but make some assumptions about the data which can introduce artifacts during the analysis. Chemometric methods such as Multivariate Curve Resolution (MCR), or Parallel Factor Analysis (PARAFAC/PARAFAC2) are particularly attractive, since they are flexible and make relatively few assumptions about the data - ideally resulting in fewer artifacts. These methods do require expert user intervention to determine the most relevant regions of interest and an appropriate number of components, k, for each region. Automated region of interest selection is needed to permit automated batch processing of chromatographic data with advanced signal deconvolution. Here, we propose a new method for automated, untargeted region of interest selection that accounts for the multivariate information present in GC-MS data to select regions of interest based on the ratio of the squared first, and second singular values from the Singular Value Decomposition (SVD) of a window that moves across the chromatogram. Assuming that the first singular value accounts largely for signal, and that the second singular value accounts largely for noise, it is possible to interpret the relationship between these two values as a probabilistic distribution of Fisher Ratios. The sensitivity of the algorithm was tested by investigating the concentration at which the algorithm can no longer pick out chromatographic regions known to contain signal. The algorithm achieved detection of features in a GC-MS chromatogram at concentrations below 10 pg on-column. The resultant probabilities can be interpreted as regions that contain features of interest.
    Keywords:  Chemometrics; Fisher ratio analysis; Gas chromatography mass spectrometry; Region of interest selection
  42. Diagnostics (Basel). 2022 Sep 09. pii: 2184. [Epub ahead of print]12(9):
      Profiling bodily fluids is crucial for monitoring and discovering metabolic markers of disease. In this study, a comprehensive analysis approach based on 1D-LC-MS/MS and 2D-LC-MS/MS was applied to profile normal human urine metabolites from 348 children and 315 adults. A total of 2357 metabolites were identified, including 1831 endogenous metabolites and 526 exogenous ones. In total, 1005 metabolites were identified in urine for the first time. The urinary metabolites were mainly involved in amino acid metabolism, small molecule biochemistry, lipid metabolism and cellular compromise. The comparison of adult's and children's urine metabolomes showed adults urine had more metabolites involved in immune response than children's, but the function of binding of melatonin, which belongs to the endocrine system, showed a higher expression in children. The urine metabolites detected by the 1D-LC-MS/MS method were mainly related to amino acid metabolism and lipid metabolism, and the 2D-LC-MS/MS method not only explored metabolites from 1D-LC-MS/MS but also metabolites related to cell signaling, cell function and maintenance, etc. Our analysis comprehensively profiled and functionally annotated the metabolome of normal human urine, which would benefit the application of urinary metabolome to clinical research.
    Keywords:  metabolomics database; urinary metabolites; urinary metabolome
  43. Methods Mol Biol. 2022 ;2546 83-94
      Acylcarnitines are formed when an acyl group is transferred from coenzyme A to a molecule of L-carnitine. In organic acidemias, and in fatty acid oxidation disorders, specific acylcarnitine species accumulate in a pattern that is characteristic for each disease. For this reason, acylcarnitine analysis is widely used for screening and diagnosis of inherited disorders of metabolism. The most common method for acylcarnitine analysis uses flow injection tandem mass spectrometry. Flow injection analysis allows for high throughput, however, does not provide separation of isomeric and isobaric compounds. Among the acylcarnitine species which can be affected by the presence of isomeric/isobaric compounds, C4-carnitine and C5DC-carnitine are probably the ones encountered most often. The method presented here is performed on urine and utilizes butanolic HCL to derivatize acylcarnitines, ultra-performance liquid chromatography to resolve C4- and C5-DC isomers and isobars, and quantitation of these species using multiple-reaction monitoring (MRM).
    Keywords:  Butyrylcarnitine; Fatty acid oxidation disorders; Glutaric acidemia type I; Glutarylcarnitine; Isobutyryl-CoA dehydrogenase deficiency; Isobutyrylcarnitine; Organic acidemia; Short-chain acyl-CoA dehydrogenase (SCAD) deficiency
  44. Methods Mol Biol. 2022 ;2546 205-216
      Serum IgG subclasses (IgGSC) are measured for a number of indications, but the most common are the identification of selective immunodeficiency disease and the diagnosis of IgG4-related disease (IgG4RD). Traditional nephelometric (IN) assays can suffer from two issues impacting the accuracy of the results: (1) hook effect and (2) antibody cross-reactivity between the subclasses. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is not vulnerable to these modes of interference and therefore serves as an excellent and relatively inexpensive means of diagnosing and/or monitoring the relevant clinical conditions.We describe a semiautomated and simple method for the accurate and precise measurement of IgGSC from 20 μL of serum using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method following digestion of serum proteins in 96-well plate format. Due to the high abundance of the target proteins, no specialized sample preparation (such as solid phase extraction) is required. Twenty microliters are injected to the LC-MS/MS system. Quantitation is performed against a five-point duplicate linear calibration curve prepared in blank matrix. The assay calibration range is 0.38-7.74 g/L for IgG1, 0.24-4.46 g/L for IgG2, 0.038-0.752 g/L for IgG3, 0.025-0.435 g/L for IgG4, and 0.62-15.5 g/L for total IgG. Total IgG is used as an internal quality control marker and is compared to the sum of the four subclass results. Total imprecision in clinical production has been observed to be 5.1-10.6% for in-house prepared control materials having IgGSC mean values in the range of 0.38-8.43 g/L for IgG1, 0.22-3.76 g/L for IgG2, 0.0387-0.721 g/L for Ig3, and 0.0279-1.46 g/L for IgG4. Limit of quantitation (LoQ) was determined to be 0.29 g/L for IgG1, 0.22 g/L for IgG2, 0.019 g/L for IgG3, and 0.0067 g/L for IgG4.
    Keywords:  IgG subclasses; IgG4RD; Immunoglobulin G; Mass spectrometry
  45. Methods Mol Biol. 2022 ;2546 539-544
      Vitamin D plays an important role not only in bone health but also in many other body functions. Vitamin D deficiency is very common in the general population. Measurement of blood 25-hydroxyvitamin D is a common practice to evaluate vitamin D deficiency. Immunoassays and liquid chromatography tandem mass spectrometry (LC-MS/MS) are the most commonly used methods for the measurement of 25-hydroxyvitamin D. Immunoassays suffer from specificity issues and do not distinguish between 25-hydroxyvitamin D2 and D3. Therefore, LC-MS/MS is a preferred method for quantification of 25-hydroxyvitamin. We describe an LC-MS/MS method, which involves protein precipitation and analysis of the extract using atmospheric pressure chemical ionization and multiple reaction monitoring. 25-hydroxyvitamin D3-d6 is used as an internal standard. The method is linear from 1-100 ng/mL for both 25-hydroxyvitamin D2 and D3 and has imprecision of <10%.
    Keywords:  25-Hydroxyvitamin D2; 25-Hydroxyvitamin D3; Tandem Mass Spectrometry; Vitamin D
  46. Methods Mol Biol. 2022 ;2546 439-450
      Accurate determination of plasma renin activity (PRA) is essential for the development and maintenance of an effective screening program for primary aldosteronism (PA). PRA measurement can also be useful in the investigation of renal artery stenosis, syndrome of mineralocorticoid excess, Addison's disease, congenital adrenal hyperplasia, Bartters and Gitelman syndromes, and for inherited defects in the renin angiotensin aldosterone system (RAAS). We describe a semiautomated and simple method for the accurate and precise measurement of PRA from 500 μL of plasma (250 μL if blank subtraction is omitted, as discussed) using a liquid chromatography and tandem mass spectrometry (LC-MS/MS) method for angiotensin I (AngI) in 96-well format. After a 3 h AngI generation step at 37 °C in buffering conditions at pH 6, the reaction is quenched with 10% formic acid containing AngI internal standard. Sample preparation then proceeds with offline solid phase extraction, two wash steps, and methanol elution followed by injection into the LC-MS/MS system. Quantitation is performed against a 7-point calibration linear curve prepared in buffer. The assay calibration range is 0.34-30.0 ng/mL, which corresponds to PRA values of 0.11-10.0 ng/mL/h: much wider than was possible using traditional competitive antibody-based methods. Total precision in clinical production has been observed to be 5.8-5.0% for BioRad Hypertension Control materials having nominal PRA values ranging from 1.73 to 12.43 ng/mL/h. At AngI concentrations of 0.06 ng/L (corresponding to a PRA of 0.02 ng/mL/h), signal-to-noise ratio is 50:1, indicating that the limit of quantitation is well below the level required for clinical use.
    Keywords:  Angiotensin I; Hypokalemia; Mass spectrometry; Mineralocorticoid hypertension; Plasma renin activity; Primary aldosteronism; Secondary hypertension
  47. Talanta. 2022 Sep 14. pii: S0039-9140(22)00718-4. [Epub ahead of print]253 123922
      This work highlights the efficient approach to highly sensitive determination of dipeptides that can present in biological liquids at very low and trace quantities. The approach involves preliminary derivatization of peptides with tris(2,4,6-trimethoxyphenyl)-methyl carbenium hexafluoroborate followed by ESI and MALDI high-resolution mass spectrometry. Using model dipeptides with various amino acid compositions and sequences, it was shown that the derivatization reaction proceeded smoothly in mild conditions and gave rise to pink-red colored salt derivatives. Ready cations of interest for the analysis are easily desorbed from the salt-derivatives providing strong signals in ESI and MALDI mass spectra and this ensures high sensitivity of the analysis. Another positive aspect is the removal of the target signal from the region of a matrix noise, since the introduced fragment possesses a large mass increment (359 Da). High resolution mass spectrometry, which provides the determination of accurate weights and elemental compositions of ions, was used to reliably detect model dipeptides added to artificial urine and blood serum. A number of these dipeptides was shown to be present in real blood serum collected from volunteers. Collision induced dissociation of precursor cations composed of derivatizing reagent and dipeptide moieties gives rise to characteristic and simple fragmentation mass spectra. A comparison of limits of detection (LOD) measured for non-modified and derivatized dipeptides showed that the latter derivatives provide the highest sensitivity when LOD is determined by using multiple reaction monitoring (MRM) transitions. The suggested derivatization approach was shown to be useful for unambiguous identification of special dipeptides in artificial media and dietary supplements.
    Keywords:  Charge-fixed reagent; Derivatization; Dipeptide analysis; High resolution; MALDI mass Spectrometry ESI mass Spectrometry
  48. Methods Mol Biol. 2022 ;2546 165-174
      We describe a simple stable isotope dilution method for accurate and precise measurement of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in human cerebrospinal fluid (CSF) as a clinical diagnostic test. Determination of CSF GABA has clinical utility in diagnosing inborn errors of GABA metabolism, specifically for deficiencies of GABA-transaminase and succinic semialdehyde dehydrogenase. Quantitation of CSF GABA is performed utilizing high-performance liquid chromatography coupled with electrospray positive ionization tandem mass spectrometry (HPLC-ESI-MS/MS). Analysis of free and total GABA requires two individual sample preparations and mass spectrometry analyses. Free GABA in CSF is determined by a 1:2 dilution with internal standard (GABA-D2) and injected directly onto the HPLC-ESI-MS/MS system. Quantitation of total GABA in CSF requires additional sample preparation in order to hydrolyze all the conjugated GABA in the sample to free GABA. Complete hydrolysis is performed incubating sample at >100 °C in acidic conditions (hydrochloric acid) for 4 h. The sample is then further diluted 1:10 with a 90% acetonitrile/0.1% formic acid solution and injected into the HPLC-ESI-MS/MS system. Each assay is quantified using a five-point standard curve and is linear from 6 to 1000 nM and 0.63 to 80 μM for free and total GABA, respectively.
    Keywords:  Cerebrospinal fluid; GABA; Mass spectrometry; Seizures
  49. Molecules. 2022 Sep 08. pii: 5827. [Epub ahead of print]27(18):
      Mass spectrometry (MS) is widely used for the identification of chemical compounds by matching the experimentally acquired mass spectrum against a database of reference spectra. However, this approach suffers from a limited coverage of the existing databases causing a failure in the identification of a compound not present in the database. Among the computational approaches for mining metabolite structures based on MS data, one option is to predict molecular fingerprints from the mass spectra by means of chemometric strategies and then use them to screen compound libraries. This can be carried out by calibrating multi-task artificial neural networks from large datasets of mass spectra, used as inputs, and molecular fingerprints as outputs. In this study, we prepared a large LC-MS/MS dataset from an on-line open repository. These data were used to train and evaluate deep-learning-based approaches to predict molecular fingerprints and retrieve the structure of unknown compounds from their LC-MS/MS spectra. Effects of data sparseness and the impact of different strategies of data curing and dimensionality reduction on the output accuracy have been evaluated. Moreover, extensive diagnostics have been carried out to evaluate modelling advantages and drawbacks as a function of the explored chemical space.
    Keywords:  LC-MS/MS; chemometrics; classification; fingerprints; multi-task; neural networks; similarity matching
  50. Methods Mol Biol. 2023 ;2571 149-155
      Aqueous humor (AH) is a transparent fluid that fills the anterior segment of the eye. The composition and level of metabolites in AH are important for understanding its physiology and changes caused by the occurrence of eye disease. A simple method for the preparation and analysis of AH samples was developed using the liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) technique. The analyses were performed using two types of chromatography: reversed-phase liquid chromatography-mass spectrometry (LC-RP-MS) and hydrophilic interaction liquid chromatography-mass spectrometry (LC-HILIC-MS), in the sample prepared by one protocol.
    Keywords:  Aqueous humor; HILIC analysis; LC–MS; Reversed-phase analysis; Untargeted metabolomics
  51. Methods Mol Biol. 2023 ;2576 21-40
      Different mass spectrometric techniques have been used over the past decade to quantify endocannabinoids (eCBs) and related lipids. Even with the level of molecular fingerprinting accuracy of an instrument like the most advanced triple quadrupole mass spectrometer, if one is not getting the most optimized sample to the detector in a way that this improved technology can be of use, then advancements can be stymied. Here, our focus is on review and discussion of sample preparation methodologies used to isolate the eCB anandamide and its close congeners N-acyl ethanolamines and structural congeners (i.e., lipo amino acids, lipoamines, N-acyl amides) in biological fluids. Most of our focus will be on the analysis of these lipids in plasma/serum, but we will also discuss how the same techniques can be used for the analysis of saliva and breast milk.
    Keywords:  Acyl amino acids; Endocannabinoids; HPLC; Lipidomics; Lipoamino acids; Mass spectrometry; Solid phase extraction
  52. Anal Chem. 2022 Sep 19.
      Complex carbohydrates are ubiquitous in nature and represent one of the major classes of biopolymers. They can exhibit highly diverse structures with multiple branched sites as well as a complex regio- and stereochemistry. A common way to analytically address this complexity is liquid chromatography (LC) in combination with mass spectrometry (MS). However, MS-based detection often does not provide sufficient information to distinguish glycan isomers. Ion mobility-mass spectrometry (IM-MS)─a technique that separates ions based on their size, charge, and shape─has recently shown great potential to solve this problem by identifying characteristic isomeric glycan features such as the sialylation and fucosylation pattern. However, while both LC-MS and IM-MS have clearly proven their individual capabilities for glycan analysis, attempts to combine both methods into a consistent workflow are lacking. Here, we close this gap and combine hydrophilic interaction liquid chromatography (HILIC) with IM-MS to analyze the glycan structures released from human alpha-1-acid glycoprotein (hAGP). HILIC separates the crude mixture of highly sialylated multi-antennary glycans, MS provides information on glycan composition, and IMS is used to distinguish and quantify α2,6- and α2,3-linked sialic acid isomers based on characteristic fragments. Further, the technique can support the assignment of antenna fucosylation. This feature mapping can confidently assign glycan isomers with multiple sialic acids within one LC-IM-MS run and is fully compatible with existing workflows for N-glycan analysis.
  53. Anal Bioanal Chem. 2022 Sep 19.
      The chemical composition of wine samples comprises numerous bioactive compounds responsible for unique flavor and health-promoting properties. Thus, it's important to have a complete overview of the metabolic profile of new wine products in order to obtain peculiar information in terms of their phytochemical composition, quality, and traceability. To achieve this aim, in this work, a mass spectrometry-based phytochemical screening was performed on seven new wine products from Villa D'Agri in the Basilicata region (Italy), i.e., Aglianico Bianco, Plavina, Guisana, Giosana, Malvasia ad acino piccolo, Colata Murro and Santa Sofia. Ultra-high-resolution mass spectrometry data were processed into absorption mode FT-ICR mass spectra, in order to remove artifacts and achieve a higher resolution and lower levels of noise. Accurate mass-to-charge ratio (m/z) values were converted into putative elemental formulas. Therefore, 2D van Krevelen diagrams were used as a tool to obtain molecular formula maps useful to perform a rapid and more comprehensive analysis of the wine sample metabolome. The presence of important metabolite classes, i.e., fatty acid derivatives, amino acids and peptides, carbohydrates and phenolic derivatives, was assessed. Moreover, the comparison of obtained metabolomic maps revealed some differences among profiles, suggesting their employment as metabolic fingerprints. This study shed some light on the metabolic composition of seven new Italian wine varieties, improving their value in terms of related bioactive compound content. Moreover, different metabolomic fingerprints were obtained for each of them, suggesting the use of molecular maps as innovative tool to ascertain their unique metabolic profile.
    Keywords:  High-resolution mass spectrometry; Metabolomics; Untargeted; Van Krevelen; Wine
  54. Methods Mol Biol. 2022 ;2544 129-144
      Hepatocytes play an important role in maintaining homeostasis in living organisms by carrying out various metabolic functions. The urea cycle, one of the metabolic pathways taking place in hepatocytes, is an important metabolic pathway that converts toxic ammonia to nontoxic urea. Performing quantitative assessments of individual metabolite levels using a mass spectrometer is useful for assessing the metabolic state of the urea cycle in hepatocytes. In addition, metabolic flux analysis using stable isotopes and a mass spectrometer is a new technique for measuring the metabolic state. It enables conducting specific, objective, and quantitative measurement of the activated state of the target metabolic pathway regardless of external disturbing factors. This section describes the technical background and methodology of performing metabolic flux analysis of the urea cycle by mass spectrometry.
    Keywords:  Hepatocytes;  Metabolic flux analysis;  Urea cycle