bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2023‒03‒19
seventeen papers selected by
Sofia Costa

  1. Biomed Chromatogr. 2023 Mar 15. e5625
      Liver cirrhosis is currently the twelfth leading cause of death globally and the sixth leading cause of death in China. Its treatment is expensive. Changes in the composition of the serum bile acid pool are sensitive indicators of the severity of liver cirrhosis. In this study, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and used to simultaneously determine 15 bile acids in human serum in patients with decompensated cirrhosis. Sample preparation involved spiking with isotope internal standards (ISs) followed by protein precipitation and the analytical run time was 5 min. The LC-MS/MS method was fully validated according to CLSI C62A and the Consensus of method development and validation of liquid chromatography-tandem mass spectrometry in clinical laboratories. The method achieved an acceptable coefficient of variation for precision (0.83-14.80%) and accuracy (89.39-107.62%). Finally, as proof of applicability, the method was applied to patients with decompensated cirrhosis in routine clinical sample analysis. The degree of variation of different bile acids was clearly shown. These results indicated that abnormal metabolic pathways might play important roles in decompensated cirrhosis.
    Keywords:  LC-MS/MS; bile acids; decompensated cirrhosis; human serum
  2. bioRxiv. 2023 Mar 02. pii: 2023.03.01.530642. [Epub ahead of print]
      Nuclear Magnetic Resonance is a powerful platform that reveals the metabolomics profiles within biofluids or tissues and contributes to personalized treatments in medical practice. However, data volume and complexity hinder the exploration of NMR spectra. Besides, the lack of fast and accurate computational tools that can handle the automatic identification and quantification of essential metabolites from NMR spectra also slows the wide application of these techniques in clinical. We present NMRQNet, a deep-learning-based pipeline for automatic identification and quantification of dominant metabolite candidates within human plasma samples. The estimated relative concentrations could be further applied in statistical analysis to extract the potential biomarkers. We evaluate our method on multiple plasma samples, including species from mice to humans, curated using three anticoagulants, covering healthy and patient conditions in neurological disorder disease, greatly expanding the metabolomics analytical space in plasma. NMRQNet accurately reconstructed the original spectra and obtained significantly better quantification results than the earlier computational methods. Besides, NMRQNet also proposed relevant metabolites biomarkers that could potentially explain the risk factors associated with the condition. NMRQNet, with improved prediction performance, highlights the limitations in the existing approaches and has shown strong application potential for future metabolomics disease studies using plasma samples.
  3. J Mass Spectrom. 2023 Mar;58(3): e4911
      The field of mass spectrometry imaging (MSI) is constantly evolving to analyze a diverse array of biological systems. A common goal is the need to resolve cellular and subcellular heterogeneity with high spatial resolution. As the field continues to progress towards high spatial resolution, other parameters must be considered when developing a practical method. Here, we discuss the impacts of high spatial resolution on the time of acquisition and the associated implications they have on an MSI analysis (e.g., area of the region of interest). This work presents a brief tutorial serving to evaluate high spatial resolution MSI relative to time of acquisition and data file size.
    Keywords:  data file; mass spectrometry imaging; memory requirement; spatial resolution; time of acquisition
  4. Methods Mol Biol. 2023 ;2629 247-269
      In this chapter, we review the cutting-edge statistical and machine learning methods for missing value imputation, normalization, and downstream analyses in mass spectrometry metabolomics studies, with illustration by example datasets. The missing peak recovery includes simple imputation by zero or limit of detection, regression-based or distribution-based imputation, and prediction by random forest. The batch effect can be removed by data-driven methods, internal standard-based, and quality control sample-based normalization. We also summarize different types of statistical analysis for metabolomics and clinical outcomes, such as inference on metabolic biomarkers, clustering of metabolomic profiles, metabolite module building, and integrative analysis with transcriptome.
    Keywords:  Imputation; Integrative analysis; Mass spectrometry; Metabolomics; Normalization; Statistical and machine learning
  5. Anal Methods. 2023 Mar 15.
      An analytical method for the determination of glyphosate (GLY) and aminomethylphosphoric acid (AMPA) in biological fluid samples (serum and urine) from poisoning patients using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is established. After the sample pretreatment, including protein precipitation and a modified liquid-liquid extraction method, the chromatographic separation was conducted on a trifunctional modified hydrophilic column. The mobile phases in the gradient program were 2.5% formic acid aqueous solution and acetonitrile. The multiple reaction monitoring (MRM) models and the isotope-labeled internal standards were used in the acquisition process. Good linearities and satisfying recovery rates were obtained in two sample matrices with good RSDs. The detection limits of GLY and AMPA were <2 μg L-1, which were close to those obtained in our previous research. The established method was applied to biological samples from five patients with glyphosate intoxication. The analysis of the trend for the concentration of GLY and AMPA in two biological samples was investigated, and the difference in the downward trend of AMPA in urine was found in patients with a relatively higher concentration of GLY in serum.
  6. J Pharm Biomed Anal. 2023 Mar 05. pii: S0731-7085(23)00090-0. [Epub ahead of print]228 115321
      BACKGROUND: Exogenous progestins are an effective tool for hormonal contraception and family planning. Progestins may be delivered as oral pills, intramuscular or subcutaneous injections, vaginal rings, or intrauterine devices. Drug concentrations may vary based on the route and duration of delivery. Measurement of synthetic steroids in blood plasma can aid in determination of product adherence, evaluation of drug-drug interactions, and investigation of unintended pregnancies.METHODS: Drug-free K2EDTA plasma was spiked with the synthetic steroids etonogestrel (ETO), levonorgestrel (LNG), medroxyprogesterone acetate (MPA), and norethisterone (NET). Plasma was combined with isotopically labeled internal standards, and drugs were extracted via liquid-liquid extraction. Samples were then subjected to liquid chromatographic-tandem mass spectrometric (LC-MS/MS) analysis. The method was validated in accordance with regulatory recommendations. The assay was evaluated in a cohort of remnant plasma samples in individuals using one of the aforementioned progestins.
    RESULTS: The analytical measuring range for ETO, MPA, and NET was 20-10,000 pg/mL; the primary linearity for LNG was 20-20,000 pg/mL. The method showed acceptable precision and accuracy for all progestins. Stability was established for 72 h with room temperature storage and through 3 freeze-thaw cycles. All analytes were stable in whole blood incubated at room temperature for 25 h, and at 40°C and 100% humidity for 2 h. Ion suppression was observed for all analytes spiked in plasma; average ion suppression was 31.6%, 66.6%, 32.1% and 41.2% for ETO, LNG, MPA, and NET, respectively. However, internal standards showed comparable ion suppression, and relative matrix effects were minimal. ETO, LNG, MPA, and NET could also be quantified accurately in K3EDTA plasma and serum. Progestins were successfully measured in remnant samples from individuals using hormonal contraceptives.
    CONCLUSIONS: A multiplexed LC-MS/MS assay for the quantification of ETO, LNG, MPA, and NET has been developed and validated. The assay met acceptable performance characteristics and may be used in downstream studies to evaluate progestin pharmacology.
    Keywords:  Etonogestrel; Hormonal contraceptives; LC-MS; Levonorgestrel; Mass spectrometry; Medroxyprogesterone acetate; Norethisterone; Progestins
  7. J Pharm Anal. 2023 Feb;13(2): 216-222
      The direct coupling of solid-phase microextraction (SPME) to mass spectrometry (MS) (SPME-MS) has proven to be an effective method for the fast screening and quantitative analysis of compounds in complex matrices such as blood and plasma. In recent years, our lab has developed three novel SPME-MS techniques: SPME-microfluidic open interface-MS (SPME-MOI-MS), coated blade spray-MS (CBS-MS), and SPME-probe electrospray ionization-MS (SPME-PESI-MS). The fast and high-throughput nature of these SPME-MS technologies makes them attractive options for point-of-care analysis and anti-doping testing. However, all these three techniques utilize different SPME geometries and were tested with different MS instruments. Lack of comparative data makes it difficult to determine which of these methodologies is the best option for any given application. This work fills this gap by making a comprehensive comparison of these three technologies with different SPME devices including SPME fibers, CBS blades, and SPME-PESI probes and SPME-liquid chromatography-MS (SPME-LC-MS) for the analysis of drugs of abuse using the same MS instrument. Furthermore, for the first time, we developed different desorption chambers for MOI-MS for coupling with SPME fibers, CBS blades, and SPME-PESI probes, thus illustrating the universality of this approach. In total, eight analytical methods were developed, with the experimental data showing that all the SPME-based methods provided good analytical performance with R 2 of linearities larger than 0.9925, accuracies between 81% and 118%, and good precision with an RSD% ≤ 13%.
    Keywords:  Coated blade spray; Drug of abuse; Mass spectrometry; Microfluidic open interface; Probe electrospray ionization; Solid-phase microextraction
  8. J Chromatogr A. 2023 Feb 24. pii: S0021-9673(23)00125-5. [Epub ahead of print]1694 463898
      Nowadays, ion-pairing reversed-phase liquid chromatography (IP-RPLC) is the dominating generic method for the analysis of nucleic acid related compounds, such as antisense-oligonucleotides (ASO), small-interfering ribonucleic acid (siRNA) or other DNA or RNA type molecules and their conjugates. Despite of its effective performance, the usage of a high concentration of ion-pairing reagent in the eluent in IP-RPLC is unfavorable for the hyphenation with mass spectrometry (MS) which is required for a detailed structural characterization of the analytes and their structurally related impurities. In this work, we tested a polybutylene terephthalate (PBT)-bonded silica-based stationary phase for the separation of generically synthesized Patisiran as siRNA (antisense and sense single strands as well as their annealed double strand) giving some unexpected selectivity without any presence of ion-pairing reagents. Important chromatographic conditions affecting the separation have been investigated and evaluated. Furthermore, MS and tandem MS (MS/MS) characterization was possible without contamination of the MS system with ion-pair agent and related problems.
    Keywords:  Impurity profiling; LC-MS; Oligonucleotide; Patisiran; Small interfering RNA (siRNA)
  9. J Proteome Res. 2023 Mar 17.
      Intracellular purine- and pyrimidine-related derivatives are vital molecules for preserving genetic information and are essential for cellular bioenergetics and signal transduction. This study developed a practical liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying intracellular purine- and pyrimidine-related derivatives. To solve the distorted peak shape related to di- and triphosphate nucleotides, in-sample addition of medronic acid and ammonium phosphate was performed. Using the BEH-amide column, the results showed that adding 0.5 mM medronic acid to the sample significantly improved the peak shape without causing an obvious ion suppressive effect. Method validation confirmed that the coefficients of determination (R2) values for linearity evaluation were above 0.94 for all analytes. The intraday and interday accuracies ranged from 85.1 to 128.4%, with the precision below 16.6%. The validated method was successfully applied in characterizing the alterations of purine- and pyrimidine-related derivatives in the A549 cell line with perturbed mitochondrial fission or blockade of the electron transport chain. Collectively, this study demonstrates that the strategy of in-sample medronic acid addition is effective in improving the quantification of intracellular purine- and pyrimidine-related derivatives. We believe that our proposed platform can facilitate the development of novel drugs targeting purine and pyrimidine metabolism in the future.
    Keywords:  lung adenocarcinoma A549 cell; mass spectrometry; medronic acid; mitochondrial dysfunction; purine- and pyrimidine-related derivatives
  10. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Mar 05. pii: S1570-0232(23)00049-1. [Epub ahead of print]1220 123639
      Dried blood spot (DBS) has been used as an alternative matrix in drug testing. In forensic testing it offers enhanced stability of analytes and ease of storage that requires minimal space. This is compatible with long term archiving of large numbers of samples for future investigation. We employed liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantify alprazolam, α-hydroxyalprazolam, and hydrocodone in a DBS sample that has been stored for 17 years. We achieved linear dynamic ranges (0.1-50 ng/mL) that capture wide ranges of concentration of the analytes below and above their reported reference ranges, and limits of detection (0.05 ng/mL) of 40-100X lower than the lower limit of the analyte's reference ranges. The method was validated according to FDA and CLSI guidelines and successfully confirmed and quantified alprazolam and α-hydroxyalprazolam in a forensic DBS sample.
    Keywords:  Alprazolam in DBS; Dried blood spot analysis; Hydrocodone in DBS; LC-MS/MS
  11. J Pharm Biomed Anal. 2023 Mar 05. pii: S0731-7085(23)00093-6. [Epub ahead of print]228 115324
      Cannabidiol (CBD) is the most abundant non-psychotropic phytocannabinoid isolated from Cannabis sativa. To support preclinical studies of ocular pharmacology of CBD, a bioanalytical method was developed and validated for quantification of CBD in aqueous humor using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Aqueous humor samples were subjected to protein precipitation by acetonitrile, followed by chromatographic separation using reversed phase LC on a Raptor ARC-18 column with mobile phase A: 0.1 % (v/v) formic acid in water (B) 0.1 % formic acid in acetonitrile (B) as eluents. Detection was carried out with a triple quadrupole mass spectrometer with electrospray ionization operated in positive ion mode. Stable-isotope labeled CBD (CBD-d3) was used as internal standard. The total run time was 8 min. Quantification was accomplished within the validated concentration range of 0.5-500 ng/mL for CBD using a 5 µL sample. The lower limit of quantitation was 0.5 ng/mL. Inter- and intra-day precision is 4.737-7.620 % and 3.426-5.830 %, respectively. Inter- and intra-day accuracy ranged between 99.01 % and 100.2 % and 99.85-101.4 % respectively. The extraction recoveries were found to be 66.06 ± 5.146 %. The established method was successfully applied to investigate ocular pharmacokinetics of CBD in mice. Following intraperitoneal (i.p.) administration of 50 mg/kg CBD, its concentration reaches a Cmax of 71.55 ± 36.64 ng/mL in aqueous humor, with a Tmax of 2 h and a half-life of 1.046 h. The AUC was 183.4 ± 49.17 ng * h/mL. The development and validation of this LC-MS/MS method is an important step toward the goal of assessing the aqueous humor concentrations of CBD and correlating the concentrations of this phytocannabinoid with its ocular pharmacologic effects.
    Keywords:  Aqueous humor; Cannabidiol; Liquid chromatography; Tandem mass spectrometry
  12. Metabolomics. 2023 Mar 15. 19(3): 18
      INTRODUCTION: Molecular networking (MN) has emerged as a key strategy to organize and annotate untargeted tandem mass spectrometry (MS/MS) data generated using either data independent- or dependent acquisition (DIA or DDA). The latter presents a time-efficient approach where full scan (MS1) and MS2 spectra are obtained with shorter cycle times. However, there are limitations related to DDA parameters, some of which are (i) intensity threshold and (ii) collision energy. The former determines ion prioritization for fragmentation, and the latter defines the fragmentation of selected ions. These DDA parameters inevitably determine the coverage and quality of spectral data, which would affect the outputs of MN methods.OBJECTIVES: This study assessed the extent to which the quality of the tandem spectral data relates to MN topology and subsequent implications in the annotation of metabolites and chemical classification relative to the different DDA parameters employed.
    METHODS: Herein, characterising the metabolome of Momordica cardiospermoides plants, we employ classical MN performance indicators to investigate the effects of collision energies and intensity thresholds on the topology of generated MN and propagated annotations.
    RESULTS: We demonstrated that the lowest predefined intensity thresholds and collision energies result in comprehensive molecular networks. Comparatively, higher intensity thresholds and collision energies resulted in fewer MS2 spectra acquisition, subsequently fewer nodes, and a limited exploration of the metabolome through MN.
    CONCLUSION: Contributing to ongoing efforts and conversations on improving DDA strategies, this study proposes a framework in which multiple DDA parameters are utilized to increase the coverage of ions acquired and improve the global coverage of MN, propagated annotations, and the chemical classification performed.
    Keywords:  Data-dependent acquisition; MS parameter optimisation; MS/MS spectra; Molecular networking; Momordica cardiospermoides; Natural products
  13. Clin Lab. 2023 Mar 01. 69(3):
      BACKGROUND: D4-androstenedione (D4ASD) is an intermediate hormone of androgen biosynthesis by the gonads and the adrenal glands. The interest in D4ASD concentration assessment resides in diagnostics of androgenic hyperproduction pathologies. Currently, many D4ASD quantification methods are available on the market including immunological methods that remain problematic due to the possible cross-reactivity with endogenous or exogenous steroids.METHODS: Recently Roche® launched a new fully automated instrument for the measurement of D4ASD concentration. In this paper, the criteria of analytical performance (repeatability and intermediate precision) of the D4ASD Roche® assay were assessed and compared with 2 different methods including a radioimmunoassay (RIA) as well as a liquid chromatography tandem mass spectrometry (LC-MS/MS) method.
    RESULTS: Repeatability and intermediate precision of the D4ASD Roche® were acceptable according to the prede-fined RICOS standard (CV ≤ 7.9%) and the assay showed a good correlation with other assays considering the 95% CI obtained for the slope and the y-intercept.
    CONCLUSIONS: This method demonstrates acceptable criteria of analytical performance with an intermediate imprecision and a trueness within the fixed acceptance limits.
  14. J Am Soc Mass Spectrom. 2023 Mar 13.
      Over 2.5 million neonatal dried blood spots (DBS) are stored at the Danish National Biobank. These samples offer extraordinary possibilities for metabolomics research, including prediction of disease and understanding of underlying molecular mechanisms of disease development. Nevertheless, Danish neonatal DBS have been little explored in metabolomics studies. One question that remains underinvestigated is the long-term stability of the large number of metabolites typically assessed in untargeted metabolomics over long time periods of storage. Here, we investigate temporal trends of metabolites measured in 200 neonatal DBS collected over a time course of 10 years, using an untargeted liquid chromatography tandem mass spectrometry (LC-MS/MS) based metabolomics protocol. We found that a majority (71%) of the metabolome was stable during 10 years of storage at -20 °C. However, we found decreasing trends for lipid-related metabolites, such as glycerophosphocholines and acylcarnitines. A few metabolites, including glutathione and methionine, may be strongly influenced by storage, with changes in metabolite levels up to 0.1-0.2 standard deviation units per year. Our findings indicate that untargeted metabolomics of DBS samples, with long-term storage in biobanks, is suitable for retrospective epidemiological studies. We identify metabolites whose stability in DBS should be closely monitored in future studies of DBS samples with long-term storage.
    Keywords:  Neonatal metabolome; biobank; dried blood spots; mass spectrometry; metabolomics
  15. Curr Protoc. 2023 Mar;3(3): e701
      Mucopolysaccharidoses (MPSs) are complex lysosomal storage disorders that result in the accumulation of glycosaminoglycans (GAGs) in urine, blood, and tissues. Lysosomal enzymes responsible for GAG degradation are defective in MPSs. GAGs including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), and keratan sulfate (KS) are disease-specific biomarkers for MPSs. This article describes a stable isotope dilution-tandem mass spectrometric method for quantifying CS, DS, and HS in urine samples. The GAGs are methanolyzed to uronic or iduronic acid-N-acetylhexosamine or iduronic acid-N-sulfo-glucosamine dimers and mixed with internal standards derived from deuteriomethanolysis of GAG standards. Specific dimers derived from HS, DS, and CS are separated by ultra-performance liquid chromatography (UPLC) and analyzed by electrospray ionization tandem mass spectrometry (MS/MS) using selected reaction monitoring for each targeted GAG product and its corresponding internal standard. This UPLC-MS/MS GAG assay is useful for identifying patients with MPS types I, II, III, VI, and VII. © 2023 Wiley Periodicals LLC. Basic Protocol: Urinary GAG analysis by ESI-MS/MS Support Protocol 1: Prepare calibration samples Support Protocol 2: Preparation of stable isotope-labeled internal standards Support Protocol 3: Preparation of quality controls for GAG analysis in urine Support Protocol 4: Optimization of the methanolysis time Support Protocol 5: Measurement of the concentration of methanolic HCl.
    Keywords:  LC-ESI-MS/MS; dermatan sulfate; glycosaminoglycans; heparan sulfate; isotope dilution; mucopolysaccharidosis
  16. Anal Chim Acta. 2023 Apr 22. pii: S0003-2670(23)00248-9. [Epub ahead of print]1251 341027
      An analytical method was developed and validated for the analysis of four priority perfluoroalkyl substances (PFAS), namely, perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorohexanesulfonic acid (PFHxS), and perfluorooctanesulfonate (PFOS) in food products using nanoscale liquid chromatography (nano-LC) coupled with nanoscale electrospray ionization (nano-ESI) and Orbitrap mass spectrometry (Orbitrap-MS) detection. The efficiency of two different nano-LC setups for chromatographic separation of selected PFAS was evaluated. The optimal LC separation of analytes was achieved using a reversed phase C18 (RP-C18) nano bore column with an integrated emitter. The effect of matrix concentration factor on signal suppression/enhancement was evaluated for different matrices. The method validation indicated analyte recoveries in the range 83-118% and within-laboratory reproducibility from 7 to 18%, while reanalysis of the materials from proficiency tests (PTs) showed that the accuracy of the obtained concentrations ranged from 85 to 124% of the provided consensus values. The method limits of quantification (m-LOQs) were set as first validation levels ranging from 0.001 to 0.3 ng g-1 sample depending on the type of the food group. The observed method performance characteristics met the criteria stated in Commission Regulation (EU) 2022/1428, Commission Recommendation (EU) 2022/1431, as well as Guidance Document on Analytical Parameters for the Determination of Per- and Polyfluoroalkyl Substances (PFAS) in Food and Feed with regards to the compliance testing of PFAS maximum levels (MLs) and monitoring purposes. The elaborated method was applied for the analysis of selected priority PFAS in different food groups collected from the Latvian retail market.
    Keywords:  Food products; Nanoscale liquid chromatography; Orbitrap mass spectrometry; Per- and polyfluoroalkyl substances (PFAS); Tolerable weekly intake
  17. J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jan 31. pii: S1570-0232(23)00025-9. [Epub ahead of print]1220 123615
      The measurement of dehydroepiandrosterone-sulphate (DHEAs) is an important second-line test to aid in the diagnosis of premature adrenarche, peripubertal gynaecomastia in males and in identifying the source of elevated androgens in females. Historically, DHEAs has been measured by immunoassay platforms which are prone to poor sensitivity and more importantly poor specificity. The aim was to develop an LC-MSMS method for the measurement of DHEAs in human plasma and serum, develop an in-house paediatric (<6 year old) reference limit and compare the performance against the Abbott Alinity DHEAs immunoassay method. Following pre-treatment with an internal standard, samples were loaded onto EVOLUTE® EXPRESS ABN plate. Analytes were separated with reverse-phase chromatography using ACQUITY® UPLC® HSS T3 2.1 mm × 50 mm, 1.8 μm column. Mass spectrometry detection was performed using a Waters® Xevo TQ-XS in electrospray negative mode. For the paediatric reference range, samples were collected from an inpatient setting (age ≤ 6 years old) with no evidence of adrenal dysfunction or history of/current steroid use. The method comparison was performed using samples from this cohort aged between 0 and 52 weeks. The assay demonstrated linearity up to 15 µmol/L (r2 > 0.99) with a functional sensitivity of 0.1 µmol/L. Accuracy results revealed a mean bias of 0.7% (-14% to 15%) when compared against the NEQAS EQA LC-MSMS consensus mean (n = 48). The paediatric reference limit was calculated as ≤ 2.3 µmol/L (95% C.I. 1.4 to 3.8 µmol/L) for ≤ 6 year olds (n = 38). Comparison of neonatal (<52 weeks) DHEAs with the Abbott Alinity revealed that the immunoassay ran at a 166% positive bias (n = 24) which appeared to lessen with increasing age. Described is a robust LC-MSMS method for the measurement of plasma or serum DHEAs validated against internationally recognised protocols. Comparison of paediatric samples of <52 weeks against an immunoassay platform demonstrated that in the immediate new-born period results generated from the LC-MSMS method offer superior specificity than an immunoassay platform.
    Keywords:  Dehydroepiandrosterone-sulphate; Immunoassay; LC-MSMS; Paediatric; Reference limit