bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒03‒13
27 papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

  1. Anal Chem. 2022 Mar 11.
      Sugar phosphates are important metabolic intermediates in organisms and play a vital role in energy and central carbon metabolism. Profiling of sugar phosphates is of great significance but full of challenges due to their high structural similarity and low sensitivities in liquid chromatography (LC)-mass spectrometry (MS). In this study, we developed a novel stable isotope chemical labeling combined with the reversed-phase (RP)LC-MS method for ultrasensitive determination of sugar phosphates at the single-cell level. By chemical derivatization with 2-(diazo-methyl)-N-methyl-N-phenyl-benzamide (2-DMBA) and d5-2-DMBA, sugar phosphate isomers can obtain better separation and identification, and the detection sensitivities of sugar phosphates increased by 3.5-147 folds. The obtained limits of detection of sugar phosphates ranged from 5 to 16 pg/mL. Using this method, we achieved ultrasensitive and accurate quantification of 12 sugar phosphates in different trace biological samples. Benefiting from the improved separation and detection sensitivity, we successfully quantified five sugar phosphates (d-glucose 1-phosphate, d-mannose 6-phosphate, d-fructose 6-phosphate, d-glucose 6-phosphate, and seduheptulose 7-phosphate) in a single protoplast of Arabidopsis thaliana.
  2. Rapid Commun Mass Spectrom. 2022 Mar 11. e9295
      RATIONALE: The ability to perform absolute quantitation and non-targeted analysis on a single mass spectrometry instrument would be advantageous to many researchers studying PFAS. High resolution accurate mass (HRAM) instrumentation (typically deployed for non-targeted work) carries several advantages over traditional triple quadrupole workflows when performing absolute quantitation. Processing this data using a vendor-neutral software would promote collaboration for these environmental studies.METHODS: LC-MS (Orbitrap Exploris 240) was used for absolute quantitation of 45 PFAS using precursor (MS1) peak areas for quantitation while isotope pattern matching and fragmentation (MS2) pattern matching were used for qualitative identification. Additionally, a fluorinated chromatographic column provided superior separation compared to the typical C18 columns typically used in PFAS analyses. This method is validated across 8 different chemical classes using recommended guidelines found in EPA Method 537.1 and Skyline data processing software.
    RESULTS: The validated limits of all 45 compounds are reported as well as metrics or accuracy and reproducibility. Most compounds achieved limits of quantitation in the range of 2-50 ng/L. Also validated were four newly released Chemours-specific compounds (PEPA, PFO3OA, PFO4DA, and PFO5DoA). Aspects of data analysis specific to high resolving power absolute quantitation are reviewed as are the details of processing this data via Skyline.
    CONCLUSIONS: This method demonstrates the feasibility of performing reproducible absolute quantitation of PFAS on an HRAM platform and does so using an open-source vendor-neutral data processing software to facilitate sharing of data across labs and institutions.
  3. J Mass Spectrom. 2022 Apr;57(4): e4817
      The study of short (SCFAs) and branched chain fatty acids (BCFAs) in human stool related to gastrointestinal diseases, gut microbiota, metabolism, and diet has dramatically increased. As a result, a fast, reliable method with minimal pretreatment is needed for quantification of these metabolites (acetic, propionic, isobutyric, butyric, isovaleric, valeric, and caproic acid) in stool. Therefore, a GC-MS method meeting this criterion was developed. A bias sampling study showed no statistical difference (p > 0.05) in analyte means when comparing 100 mg subsamples of homogenized to non-homogenized samples (n = 6, p values 0.153-0.910). Stool samples were homogenized, diluted with 80:20 water:methanol (v/v), and adjusted to a pH of 1.5-2.5. Samples were vortexed, centrifuged, and directly injected into the GC-MS using pulsed splitless injection offering twofold-to-threefold signal enhancement over a 10:1 split injection. DB-FATWAX Ultra Inert Polyethylene Glycol (PEG) Column showed no peak tailing, reduced responses, or retention time shifts after 1,476 stool injections, while other columns failed before 361 injections. Intra- and inter-day accuracy for stool supernatant samples ranged from -10.21% to 8.88% and -13.25% to 9.91%, while intra- and inter-day precision ranged from 0.21% to 1.21% and 0.89% to 2.84% coefficient of variation (CV), respectively. This method demonstrates excellent linearity (0.9999-1.0000) and low limits of quantification (1.50-8.01 μM). Stool samples proved stable stored at -20°C up to 28 days, and recoveries ranged from 85.04% to 106.59%. Matrix effects in stool are non-significant determined by comparing standard and stool supernatant calibration curve slopes (p > 0.05).
    Keywords:  branched chain fatty acids; direct injection; gas chromatography-mass spectrometry; short chain fatty acids; stool
  4. Anal Chim Acta. 2022 Apr 01. pii: S0003-2670(22)00188-X. [Epub ahead of print]1200 339617
      Bile acids are steroid compounds involved in biological mechanisms of neurodegenerative diseases making them potential biomarkers for diagnosis or treatment. These compounds exist as structural and conformational isomers, which hinder distinguishing them in physiological processes. We aimed to develop tandem mass spectrometry-ion mobility spectrometry (MS/MS-IMS) methodologies to explore and understand the behaviour of isomeric steroids in the gas-phase and rapidly separate them. Unlike previously published ion mobility data, various isomers were investigated in mixtures to better mimic complex (pre-) clinical samples. The experimental collision cross sections (CCS)s were compared to the theoretical CCS values for an in-depth analysis of isomeric ions' behaviour in the gas-phase. Based on density-functional theory, we identified the impact of adduct positioning on the 3D conformation of enantiomers, diastereomers and structural isomers. The curling of the large side chains hedged the small differences among the isomers and lowered the CCS values. On the other hand, fragmenting off the identical side branches as well as imposing the bending of the steroid ring resulted in ion mobility differentiation. Careful data evaluation revealed the tendency of isomers to form homo-cluster in the mixture solutions and assist the separation. Our fundamental and experimental findings enable the ion mobility separation of isomeric steroids to be predicted. The introduced rapid and optimal MS/MS-IMS analytical methodology can be applied to distinguish isomeric bile acids both in a solution and potentially in patients' tissue samples, and consequently, reveal their molecular pathways.
    Keywords:  Bile acids; Collision cross section; Density functional theory; Gas-phase molecular ion; Ion mobility; Traveling wave ion mobility
  5. Anal Chim Acta. 2022 Apr 01. pii: S0003-2670(22)00185-4. [Epub ahead of print]1200 339614
      It seems to be well received that nonlinear electrospray ionization (ESI) distorts the signal distribution in mass spectrometry (MS) analysis, thus leading to diminished statistical power for t-test. However, the exact consequence and possible solutions to this quantitative issue have not been systematically explored. In this work, using a serial diluted urine metabolomics dataset, we demonstrated that over 80% of the metabolic features present nonlinear ESI response patterns, causing either left-skewed or right-skewed MS signal distributions. Among them, right-skewed MS distributions cannot be rescued using conventional data transformation (e.g., log transformation, power transformation). Furthermore, using a Monte Carlo simulation, we quantitatively assessed the reduced statistical power for t-test calculated using MS signal data in various sample sizes and effect sizes. In all these comparisons, t-test using MS signal data has consistently lower statistical power than t-test using metabolic concentration data. To address this statistical issue, we proposed a bioinformatic workflow, termed PowerU, to minimize the diminished statistical power caused by both the nonlinear ESI response and the intrinsic non-normal distribution of metabolic concentrations. The PowerU workflow is composed of two steps. The first step is to convert MS signals to quality control (QC) sample injection amounts to solve the skewed MS signal distributions. The second step is to perform a Shapiro-Wilk test to determine data normality and then use the normality results to guide the application of t-test and Mann-Whitney U test for the best statistical outcome. PowerU was tested using a metabolomics study of mouse cecum samples. Results demonstrate that the PowerU workflow can significantly boost statistical power for t-test and facilitate the discovery of significantly altered metabolites for downstream biological interpretation.
    Keywords:  Electrospray ionization; Liquid chromatography-mass spectrometry; Monte Carlo simulation; Nonlinear response; Statistical power; t-test
  6. Rapid Commun Mass Spectrom. 2022 Mar 09. e9292
      RATIONALE: Cardiovascular disease as a multifactorial disease caused by genetics and environment, has emerged as a leading cause of mortality. The discovery of metabolic biomarkers for the clinical diagnosis, early warning, and elucidation of the molecular pathogenesis of cardiovascular disease, using metabolomics, has attracted broad interest. Therefore, this work aimed to develop a sensitive and reliable targeted metabolomics method for the quantification of cardiovascular disease-related biomarkers in plasma.METHODS: The method was developed and validated using ultra performance liquid chromatography augmented with tandem mass spectrometry (UPLC-MS/MS). The LC conditions and MS parameters were optimized, by using multiple reaction monitoring scanning mode to high throughput and sensitively separating, and could detect 20 metabolic biomarkers in a single experiment. And the linearity, selectivity, accuracy, precision, stability, and recovery of the developed method were assessed according to the "Bioanalytical Method Validation" guidelines of the United States (US) Food and Drug Administration (FDA).
    RESULTS: These quantified metabolic biomarkers are involved in pathways such as aromatic amino acid catabolism (e.g., phenylalanine, tryptophan, tyrosine), trimethylamine N-oxide (TMAO) biosynthesis (e.g., TMAO, choline, carnitine, betaine), and histidine metabolism (e.g., histidine), among others. All analytes exhibited excellent linearities with coefficients of determination greater than 0.99. Accuracies deviated by less than 15% for medium- and high-concentration samples and less than 20% for low-concentration samples, with intra- and inter-day precisions of 1.12-14.12% and 0.30-13.74%, respectively. Recoveries and stabilities also met the analysis requirements of biological samples.
    CONCLUSIONS: This targeted metabolomics method was shown to have a powerful ability to accurately analyze metabolic biomarkers, thereby providing valuable information for large-scale biomarker validation and clarifying the potential material basis of cardiovascular disease for clinical diagnosis or early warning.
  7. J Pharm Biomed Anal. 2022 Feb 18. pii: S0731-7085(22)00099-1. [Epub ahead of print]213 114678
      A high-throughput method using microwave-assisted extraction, chemical derivatization and liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) was developed for the simultaneous analysis of illicit drugs and their metabolites, including amphetamine (AMP), methamphetamine (MA), methylenedioxy- amphetamine (MDA), methylenedioxy-methamphetamine (MDMA), morphine (MOR), 6-acetylmorphine (6-AM), ketamine (K), and norketamine (NK) in a micro-segment of a single hair (0.4 mm). In order to elevate the throughput and sensitivities of selected compounds, 3 min microwave-assisted extraction and 10 min derivatization with dansyl chloride (DC) were employed. After derivatization, all compounds except ketamine and norketamine were derivatized and enhanced the sensitivities significantly. Derivatives showed intense fragment ions and low background noise on DC-MOR, DC-6-AM, and four DC-amphetamine-type stimulants. The total sample preparation and analysis time was 50 min. The calibration range was from LOQ to 5000 pg/mg, the coefficient of determination was better than 0.997. Intra-assay precision and accuracy were generally less than 15%. Limits of detection ranged from 15 to 50 pg/mg, limits of quantitation ranged between 45 and 125 pg/mg. The matrix effect was better than 90%. The method was successfully applied to the analysis of actual hair samples collected from multi-drug abusers. This advanced method showed practicality in hair analysis and was suitable for the extremely insufficient sample.
    Keywords:  Dansyl chloride; Drugs of abuse; Hair; LC-MS/MS; Segmental analysis
  8. Anal Chim Acta. 2022 Apr 01. pii: S0003-2670(22)00184-2. [Epub ahead of print]1200 339613
      Collision-induced dissociation (CID) is a common fragmentation strategy in tandem mass spectrometry (MS2) analysis. A conventional understanding is that fragment ions generated in low-energy CID should follow the even-electron rule. As such, (de)protonated ([M+H]+/[M-H]-) or even-electron precursor ions should follow heterolytic cleavages and predominately generate even-electron fragment ions with very few radical fragment ions (RFIs). However, the extent to which RFIs present in MS2 spectra has not been comprehensively investigated. This work uses the annotated high-resolution MS2 spectra from the latest NIST 20 tandem mass spectral library to investigate the occurrence of RFIs in CID MS2 experiments. In particular, RFIs were recognized using integer double bond equivalent (DBE) values calculated from their annotated molecular formulas. Our study shows that 65.4% and 68.8% of MS2 spectra of even-electron precursors contain at least 10% RFIs by ion-count (total number of ions) in positive and negative electrospray ionization modes, respectively. Furthermore, we classified chemicals based on their compound classes and chemical substructures, and calculated the percentages of RFIs in each class. As expected, compounds that can stabilize the radical site via resonance, such as aromatic and conjugated double bond-containing chemicals, are more likely to form RFIs. We also found four possible patterns of change in RFI percentages as a function of CID collision energy. Finally, we demonstrate that the inadequate consideration of RFIs in most conventional bioinformatic tools might be problematic during in silico fragmentation and de novo annotation of MS2 spectra. This work provides a further understanding of CID MS2 mechanisms, and the unexpectedly large percentage of RFIs suggests that the even-electron rule seems to be challenged in numerous cases where it is disobeyed.
    Keywords:  Collision-induced dissociation; Fragmentation mechanism; MS(2) interpretation; Radical fragment ions; Tandem MS
  9. Anal Chim Acta. 2022 Apr 01. pii: S0003-2670(22)00181-7. [Epub ahead of print]1200 339610
      Phthalate esters are a group of synthetic industrial chemicals that are widely used in plastics. Urinary phthalate metabolites are short-term exposure markers frequently used to represent exposure levels in environmental epidemiology studies. Human hair is an alternative matrix for recording long-term exposure, but there are still analytical challenges that need to be addressed. In this study, an analytical method was established for simultaneously measuring nine major phthalate metabolites in human hair and successfully applied to measure phthalate metabolites in 30 hair samples collected from 30 individual human volunteers without known occupational exposure to phthalates. Two portions of 25 mg of hair samples were extracted by acidified methanol and water in 240 min of ultrasonication and then analyzed using a liquid chromatography-tandem mass spectrometry system. The limit of quantification ranged from 0.72 to 10.7 ng/g hair for nine phthalate metabolites. All nine phthalate metabolites were detected in more than 70% of the 30 individual human hair samples. The measured levels of hair phthalate metabolites were (in descending order): MEHP > MMP ≫ MEP > MBP (MnBP + MiBP) > MiNP > MEHHP ≈ MEOHP ≈ MECPP. The primary metabolite, MEHP (692 ± 582 ng/g), is the major DEHP metabolite in hair. This result is consistent with the findings in blood but not in urine, in which the secondary metabolites are the major DEHP metabolites. This method is easy to foresee with a clinical application and applies to human biomonitoring studies to assess long-term environmental phthalate exposure.
    Keywords:  Biomarker; Hair; Long-term exposure; Phthalate
  10. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Feb 28. pii: S1570-0232(22)00104-0. [Epub ahead of print]1195 123200
      Untargeted lipidomics using liquid chromatography high-resolution mass spectrometry (LC-HRMS) was performed using polarity switching, and in positive and negative polarity separately on the same set of serum samples, and the performances of the methods were evaluated. Polarity switching causes an increase in the cycle time of the HRMS measurements (1.18 s/cycle vs 0.27 s/cycle), resulting in fewer data points across chromatographic peaks. The coefficient of variation (CV) was on average lower for the added isotopically labelled standards in pooled samples (QC) and patient samples using separate polarities (QC = 5.6%, samples = 12.5%) compared to polarity switching (QC = 8.5%, samples = 13.4%), but the difference was not statistically significant. For the endogenous features measured in the QCs polarity switching resulted in on average significantly higher CVs (3.80 (p = 4.25e-30) and 3.3 percentage points (p = 6.84e-40), for positive and negative modes, respectively) however still acceptable for an untargeted method (mean CVs of 17.9% and 12.2% in positive and negative modes respectively). A slightly larger number of endogenous features were detected using the separate polarities, but the large majority of features (>95%) were detected with both methodologies. The overlap of features detected in both positive and negative polarities was low (4.1%) demonstrating the importance of using both polarities for untargeted lipidomics. When investigating the effects of a treatment on multiple sclerosis patients it was found that both methodologies gave highly similar biological results, further confirming the applicability of polarity switching.
    Keywords:  Lipidomics; Polarity switching; Positive/negative switching; Untargeted mass spectrometry
  11. J Sep Sci. 2022 Mar 05.
      Meropenem, a representative β-lactam antibiotic, is widely used to treat complicated and serious infections. Therefore, it is a great significance to monitor the plasma drug concentration for individualized antimicrobial therapy. This study first describes the development and validation of high performance liquid chromatography-tandem mass spectrometry cubed method for monitoring meropenem in human plasma. Protein precipitation with methanol and a chromatographic analysis time of 7 min make this method is simple and high-throughput. Meropenem was extracted from human plasma with recoveries greater than 94.1%. Calibration curves were linear (R2 >0.995) in the concentration range of 0.5-50 μg/mL. Overall accuracy and precision did not exceed 8.0% as well as no significant matrix effect was observed. The novelty of this method is that the triple-stage MS technology improves the selectivity and sensitivity. A comparison of the presented method and traditional liquid chromatography-tandem mass spectrometry method was assessed in 44 patients treated with meropenem and Passing-Bablok regression coefficients and Bland-Altman plots showed that no significant difference between the two methods. So the triple-stage MS method developed in this study is appropriate and practical for the monitor of meropenem in the daily clinical laboratory practice. This article is protected by copyright. All rights reserved.
    Keywords:  high performance liquid chromatography-tandem mass spectrometry; meropenem; quantitation strategy; triple-stage MS
  12. Anal Chim Acta. 2022 Apr 01. pii: S0003-2670(22)00182-9. [Epub ahead of print]1200 339611
      A simple flame-induced atmospheric pressure chemical ionization (FAPCI) source was developed to couple a gas chromatograph (GC) with a mass spectrometer (MS). The interface consisted of a heated transfer line and a high voltage-free ambient FAPCI source. Nitrogen gas flowing through the heated transfer line was utilized to deliver the analytes eluted from a GC column to the ionization region. A micro oxyacetylene flame was positioned under the exit of heated transfer line, which generated primary charged species in the ionization region. Since the temperature at the ionization region was below 200 °C, the analytes were not thermally decomposed. Protonated analytes were formed by reacting the analytes with flame-induced charged species through ion-molecule reactions (IMRs). The simple GC-FAPCI/MS was used to characterize a series of fatty acid methyl esters (FAMEs) and long-chain normal alkanes, which showed protonated FAME and oxidized n-alkane ions on the mass spectra. The limits of detection (LODs) for C15:0 to C25:0 FAMEs were 1-2.5 pg. A calibration curve ranging from 2.5 to 500 pg, with a R2 value of 0.9821, was obtained.
    Keywords:  Fatty acid methyl ester; Flame-induced atmospheric pressure chemical ionization; GC-MS interface; Micro-flame
  13. J Pharm Biomed Anal. 2022 Mar 01. pii: S0731-7085(22)00118-2. [Epub ahead of print]213 114697
      Catecholamines and their metabolites act as neurotransmitters in the brain and are important for nervous system function. In the current work, a highly selective and sensitive UPLC-MS/MS assay was developed for quantitation of six catecholamines and their metabolites, including dopamine, norepinephrine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindolacetic acid from rat and mouse striatum as pharmacodynamic biomarkers to support neuroscience and pharmaceutical research. A fit-for-purpose strategy for method development, assay qualification and study support were adopted for this assay. A surrogate matrix (brain homogenizing solution absent of targeted analytes) was used for preparation of calibration samples and certain levels of quality control samples to avoid interference from endogenous baselines. Homogenized rodent striatum was derivatized by dansyl chloride to enhance the sensitivity, followed by liquid-liquid extraction with ethyl acetate in 96-well plate format. The lower limit of quantitation (LLOQ) was 0.2 ng/mL in tissue homogenate, equivalent to 3.2 pg/mg in brain tissue, which could be further reduced to ten times lower by changing the re-dissolving and injecting volume in the last sample purification step. Acceptable accuracy, precision, linearity, specificity, recovery, and matrix effect was obtained. Bench-top stability (2 h), freeze-thaw stability (3 cycles at -20 °C), and - 80 °C storage stability (up to 51 days) in both tissue homogenate and surrogate matrix along with autosampler stability (60 h at 4°C) all met acceptance criteria. This assay was successfully applied to measure the six analytes in striatum from mice treated with the neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), an animal model of Parkinsonism for which dosing protocols can vary widely, and further confirmed the metabolic pathway of neurotoxicity by the quantification of catecholamine metabolites. Our study is the first detailed the step-by-step recovery and pointed out the key factors for the assay to simultaneously quantify these six neurotransmitters in rodent striatum with superior sensitivity.
    Keywords:  Catecholamines; Derivatization; LC-MS; MPTP; Mouse striatum; Rat striatum
  14. Biomed Chromatogr. 2022 Mar 08. e5360
      A method to determine 8 polychlorinated biphenyls (PCBs), 23 organochlorine pesticides (OCPs) and 16 polycyclic aromatic hydrocarbons (PAHs) was described using dispersive liquid-liquid microextraction (DLLME) of a small amount of plasma or serum sample and gas chromatography-tandem mass spectrometry (GC-MS/MS). The appropriate selection of the extraction solvent and dispersing solvent contributes to a high extraction yield and a clean extract. To verify the developed method, the interference, linearity of the calibration curve, detection limit, precision and accuracy were evaluated. The calibration curves were linear by 2-3 orders of magnitude with correlation coefficients above 0.997 in all cases. The LODs of PCBs, OCPs and PAHs were measured in the ranges of 0.0006-0.0029, 0.001-0.029 and 0.0002-0.012 ng/ml. The intraday precision achieved by this method was 2.19-10.3% (PCBs), 1.65-14.3% (OCPs) and 0.91-12.8% (PAHs), and the intraday accuracy 1.56-7.37% (PCBs), 2.34-19.6% (OCPs) and 1.49-15.7% (PAHs). The advantage of this method is that the analysis of PCBs, OCPs, and PAHs can be performed in a single chromatographic run, and the low detection limit enables monitoring of target substances in low exposure general public samples, and the analysis procedure is relatively simple and fast.
    Keywords:  Polychlorinated biphenyls; biomonitoring; dispersive liquid-liquid microextraction; gas chromatography-tandem mass spectrometry; organochlorine pesticides; polycyclic aromatic hydrocarbons; serum
  15. J Chromatogr A. 2022 Feb 16. pii: S0021-9673(22)00105-4. [Epub ahead of print]1668 462907
      In this study, non-targeted gas chromatography-Orbitrap-mass spectrometry (GC-Orbitrap-MS) analysis of semi-volatile organic compounds (SVOCs) in indoor environmental dust samples is proposed. High-resolution mass spectrometry (HRMS) provides massive amounts of information-rich mass data which presents storage and processing challenges. Thus, a combination of the regions of interest (ROI) data filtering and mass compression method, together with the multivariate curve resolution-alternating least squares (MCR-ALS) data resolution method (which is called the ROIMCR procedure), is applied to solve huge data analysis challenges. The ROI method assures a significant reduction of the computer storage requirements of mass spectrometry data without any significant loss of spectral resolution nor of accuracy on m/z measures. On the other side, the MCR-ALS method allows the total resolution of the elution and spectral profiles of the different constituents present in the analyzed samples, not requiring their chromatographic peak alignment nor their chromatographic peak shape modelling using natural constraints like non-negativity. Since all the possible species are investigated by the ROIMCR method, it is a powerful tool for non-targeted analysis. In order to check that the sample constituents are correctly recovered and identified by the proposed ROIMCR procedure when is applied to non-targeted GC-Orbitrap-MS analysis, a set of lab-emulated dust samples at different concentration levels were qualitatively and quantitatively analyzed in detail. Then, to evaluate the performance of the proposed ROIMCR procedure, this method was applied to the same type of non-targeted GC-Orbitrap-MS analysis data of two real dust samples with unknown compositions. Many chemical compounds present in the lab-emulated dust samples were correctly identified and quantified in these dust samples. An additional number of extra chemical compounds were resolved in these real dust samples, whose identification as putative constituents of these samples is proposed. The ROIMCR procedure proposed in this work facilitates the simultaneous data processing of complex analytical samples and allows the detection and identification of possible extra sample constituents. As a final conclusion of this work, the combination of the GC-Orbitrap-MS and ROIMCR methods, is shown to be a reliable tool for the non-targeted qualitative and quantitative analysis of complex analytical and environmental samples.
    Keywords:  Chemometrics; Dust; GC-Orbitrap-MS; MCR-ALS; Non-targeted analysis; ROI; ROIMCR
  16. Anal Chem. 2022 Mar 07.
      Novichok A-series compounds, novel nerve agents, pose an increasing threat to citizens worldwide; however, no analytical methods have been reported for detecting their hydrolysis products. Herein, a screening method was developed to detect and identify Novichok A-series degradation products (hydrolysates of A230, A232, A234, A262, and one related compound) and alkyl methylphosphonic acids (RMPAs, conventional nerve agent hydrolysates) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified a suitable derivatization reagent, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), and optimized the reaction conditions. The derivatized esters of Novichok A-series degradation products were stable and easily detected. We used this derivatization to achieve the first analytical method for Novichok hydrolysis products in urine (0.40-4.0 ng/mL). The detection limits of the RMPAs (0.1-0.4 ng/mL) were comparable to those presented in previous reports involving pentafluorobenzylation or direct LC-MS/MS. The applicability of the newly developed method was evaluated by analyzing urine samples from the OPCW Fifth Biomedical Proficiency Test.
  17. Biomed Chromatogr. 2022 Mar 10. e5366
      Schizophrenia is a debilitating psychiatric disorder affecting approximately 1% of the population worldwide. Disturbances of the homocysteine metabolism are important factors in the pathophysiology of schizophrenia. In this research, a novel validated LC-MS/MS quantification procedure was developed to investigate three significant compounds of homocysteine metabolism, homocysteine, cysteine and methionine in patients with schizophrenia and healthy controls. Sample preparation involved a reduction with dithiothreitol followed by protein precipitation, and the chromatographic runtime was 2 min. The LC-MS/MS method was validated according to CLSI C62-A and Chinese Guidance for Liquid Chromatography and Mass Spectrometry Clinical Application. The performance of the method was excellent with a coefficient of variation for precision in the range 0.5%-6.9%, an accuracy from 90.4% to 101.6%. In addition, the practical applicability of the method was demonstrated by applying it in the routine sample analysis for the schizophrenic patient. Increased homocysteine levels and decreased cysteine levels were observed in the patient with schizophrenia. These results indicate that the activity of transsulfuration pathway may play a key role in the pathogenesis of schizophrenia.
    Keywords:  Cysteine; Homocysteine; LC-MS/MS; Methionine; Schizophrenia
  18. Molecules. 2022 Mar 07. pii: 1726. [Epub ahead of print]27(5):
      Untargeted metabolomics approaches are emerging as powerful tools for the quality evaluation and authenticity of food and beverages and have been applied to wine science. However, most fail to report the method validation, quality assurance and/or quality control applied, as well as the assessment through the metabolomics-methodology pipeline. Knowledge of Mexican viticulture, enology and wine science remains scarce, thus untargeted metabolomics approaches arise as a suitable tool. The aim of this study is to validate an untargeted HS-SPME-GC-qTOF/MS method, with attention to data processing to characterize Cabernet Sauvignon wines from two vineyards and two vintages. Validation parameters for targeted methods are applied in conjunction with the development of a recursive analysis of data. The combination of some parameters for targeted studies (repeatability and reproducibility < 20% RSD; linearity > 0.99; retention-time reproducibility < 0.5% RSD; match-identification factor < 2.0% RSD) with recursive analysis of data (101 entities detected) warrants that both chromatographic and spectrometry-processing data were under control and provided high-quality results, which in turn differentiate wine samples according to site and vintage. It also shows potential biomarkers that can be identified. This is a step forward in the pursuit of Mexican wine characterization that could be used as an authentication tool.
    Keywords:  HS-SPME GC-QTOFMS; Mexican wine; metabolomics; untargeted method validation; wine
  19. J Appl Lab Med. 2022 Mar 08. pii: jfac009. [Epub ahead of print]
      BACKGROUND: Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard for the measurement of fentanyl and norfentanyl (NF) in urine and is favored over immunoassays due to its superior specificity. NF is the principal metabolite of fentanyl found in the urine and is typically present in higher abundance than fentanyl. Thus, the sensitivity and specificity of LC-MS/MS relies largely on the ability to identify and quantitate NF.METHODS: We analyzed urine specimens from women who had received bupivacaine and fentanyl for epidural analgesia during labor. We analyzed the contents of the epidural bag itself and purified bupivacaine metabolite N-desbutyl bupivacaine [or N-(2,6-dimethylphenyl)piperidine-2-carboxamide (NDB)] by LC-MS/MS.
    RESULTS: NDB interferes with the LC-MS/MS assay for NF. NDB passes through the Q1 mass selection filter because it is isobaric with the NF precursor ion (233 m/z). Further, it shares product ions with NF (84 m/z and 150 m/z), used as quantifier and qualifier ions, respectively, in our urine NF detection method. Baseline resolution of NDB and NF using these quantifier and qualifier ions could not be achieved. A unique product ion of NF (177 m/z) was useful for distinguishing NDB from NF.
    CONCLUSION: Bupivacaine is a commonly used drug. Recognition of this interference by laboratories is critical for preventing the misidentification of NF, which can have profound effects on patient care.
    Keywords:   N-(2,6-dimethylphenyl)piperidine-2-carboxamide; N-desbutyl bupivacaine; N-despropyl (R)-ropivacaine; (−)-2ʹ,6ʹ-pipecoloxylidide; 2-piperidinecarboxamide; 2ʹ,6ʹ-pipecoloxylidide, (−)−; LC–MS/MS; bupivacaine, fentanyl; desbutylbupivacaine, (−)−; epidural; mass spectrometry; norfentanyl; urine
  20. J Pharm Biomed Anal. 2022 Mar 02. pii: S0731-7085(22)00119-4. [Epub ahead of print]213 114698
      The antiretroviral agents rilpivirine (RPV) and cabotegravir (CAB) are approved as a combined treatment regimen against human immunodeficiency virus (HIV). To fully understand the biodistribution of these agents and determine their concentration levels in various parts of the body, a simple, selective and sensitive bioanalytical method is essential. In the present study, a high performance liquid chromatography method with mass spectrometry detection (HPLC-MS) was developed for simultaneous detection and quantification of RPV and CAB in various biological matrices. These included plasma, skin, lymph nodes, vaginal tissue, liver, kidneys and spleen, harvested from female Sprague Dawley rats. The suitability of the developed method for each matrix was validated based on the guidelines of the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) on bioanalytical method validation. Analytes were extracted from biological samples employing a simple one-step protein precipitation method using acetonitrile. Samples were analysed using an Apex Scientific Inertsil ODS-3 column (4.6 mm × 250 mm, 5 µm particle size), maintained at 40 °C, on a HPLC system coupled with a single quadrupole MS detector. RPV was detected at a mass-to-charge ratio (m/z) of 367.4 and CAB at 406.3. Separation was achieved using isocratic elution at 0.3 mL/min with a mixture of acetonitrile and 0.1% (v/v) trifluoroacetic acid in water (81:19, v/v) as the mobile phase. The run time was set at 13 min. The presented method was selective, sensitive, accurate and precise for detection and quantification of RPV and CAB in all matrices. The developed and validated bioanalytical method was successfully employed for in vivo samples with both drugs simultaneously.
    Keywords:  Biodistribution; Cabotegravir; HPLC; MS; Pharmacokinetic; Rilpivirine
  21. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Feb 25. pii: S1570-0232(22)00054-X. [Epub ahead of print]1195 123150
      Insulin is a peptide hormone that is secreted by the β cells of the pancreas and is essential to the metabolism of carbohydrates, fats, and proteins in the body. The marmoset insulin peptide is not homologous with human insulin and therefore commonly available assays do not work for this species. Due to the increasing popularity of marmoset research, a simple, specific assay for the quantitation of marmoset insulin is needed. This study aimed to develop and validate a bottom-up proteomic workflow with trypsin digestion and analysis using LC coupled with triple quadrupole mass spectrometry (LC-MS/MS). Marmoset serum proteins were subjected to denaturation, reduction, and enzymatic cleavage to extract a unique, 7 amino acid peptide for quantitation of marmoset insulin. Resolution of the peptide was achieved by LC-MS/MS using electrospray ionization operating in positive mode. Calibration was achieved by aliquot dilution of fully synthetic marmoset insulin tryptic peptide into macaque serum. A stable-isotope labeled (13C, 15N) synthetic marmoset insulin tryptic peptide was used as internal standard. The assay was fully validated according to bioanalytical method validation guidelines for linearity, precision, and dilution linearity using purified marmoset insulin. The limit of detection was 15.49 pmol/L and the limit of quantitation was 140.78 pmol/L. Biological validation was achieved by comparison of samples previously run by radioimmunoassay and measurement of the marmoset insulin response to glucose via an oral glucose tolerance test (OGTT). The physiological range of marmoset insulin was shown to be 84.5 to 1222 pmol/L. In summary, this paper presents a simple, reproducible method to measure marmoset insulin in serum using LC-MS/MS.
    Keywords:  Insulin; LC-MS/MS; Marmosets; Metabolic syndrome; Nonhuman primate model
  22. J Pharm Biomed Anal. 2022 Feb 26. pii: S0731-7085(22)00112-1. [Epub ahead of print]213 114691
      Depression is a mental health disorder characterized by chronic negative mood, and depression has become a major threat to human health and quality of life. Anyupeibo capsule, a fifth-class new Chinese medicine, was prepared with extracts of Piper laetispicum C.DC. (Piperaceae), and the alkaloid K6 (5'-methoxy-3',4'-methyl-enedioxycinnamic-acidisob-utylamide-isobutylamide) was found to be the main active component. Using high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (LC-MS/MS), we developed a method to quantify the concentration of K6 in serum samples from patients with depression. Pretreatment of samples was completed based on solid-phase extraction, and the mobile phase for subsequent LC analysis consisted of aqueous ammonium acetate (0.1 mmol/L, phase A) and acetonitrile (phase B) with isocratic elution at 60% B. Chromatographic separation of K6 was achieved within 3 min with an Agilent ZORBAX SB-C18 column (2.1 × 150 mm, 3.5 µm) at a flow rate of 0.3 mL/min. A linear regression equation for K6 yielded correlation coefficients of r2 > 0.99 within a linear range 0.0503-100.5000 ng/mL. Extraction recovery ranged from 85.33% to 101.18%, and the matrix effect ranged from 87.15% to 100.28%. The inter-day and intra-day precision values-expressed as relative standard deviation-were less than 15%, and the corresponding accuracy values were within ±15%. All validation results for stability, specificity, and carry-over met the requirements of Pharmacopoeia. The LC-MS/MS method was applied to determine the K6 concentration in serum samples from patients with depression in a phase III clinical trial of Anyupeibo capsule.
    Keywords:  Depressed patients; Exposure determination; Human serum; K6; LC-MS/MS
  23. Bioanalysis. 2022 Mar 07.
    Keywords:  IMS; MSI; bioanalysis; mass spectrometry imaging
  24. Shokuhin Eiseigaku Zasshi. 2022 ;63(1): 12-19
      In this study, we simultaneously determined three antioxidants, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ), using HPLC equipped both with a photodiode array detector and a fluorescence detector in 25 minutes per sample. Due to the combined use of the two detectors, we could achieve improved target selectivity. Further, quantification at the specific wavelengths for each target substance particularly increased BHT detection sensitivity. This approach enabled us to avoid repeated measurements during daily inspections. Furthermore, detections were performed using LC-MS/MS instead of GC-MS to overcome the problem of helium gas shortage.In addition, we investigated antioxidant stability in standard solutions during storage. Although TBHQ was stable in methanol with ascorbic acid at -20℃, ascorbic acid storage has possibility to lead to decrease in BHT and BHA concentrations. We recognized that the mixture of BHT and BHA dissolved in methanol at 4℃ and that of BHT, BHA and TBHQ dissolved in methanol with ascorbic acid at -20℃ were suitable for about one year.
    Keywords:  HPLC; LC-MS/MS; antioxidant; butylated hydroxyanisole (BHA); butylated hydroxytoluene (BHT); fluorescence detection; l-ascorbic acid (AsA); stability; tertiary butylhydroquinone (TBHQ)
  25. Rapid Commun Mass Spectrom. 2022 Mar 09. e9291
      RATIONALE: Electrospray mass spectrometry (ESI-MS) is one of the most popular techniques for obtaining structural information, which is commonly used in bioanalysis and clinical diagnostics. However, for the detection of complicated samples with high reactivities (such as reactive sulfur species, RSS), traditional ESI-MS usually suffers from overlapped and inaccurate signals. Herein, based on the multiphase flow of extractive electrospray ionization (MF-EESI), an ambient MS technique of online derivatization was proposed to detect thiols without any other sample pretreatment.METHODS: RSS molecules and the derivatization reagent of 4-Chloro-7-nitro-1,2,3-benzoxadiazole (NBD-Cl) were introduced into the internal and innermost capillary of the MF-EESI system, respectively. By a high-velocity nebulizing stream of N2 gas through an external capillary, both flows of innermost biothiols and internal NBD-Cl were electrosprayed and mixed for online reactions. Therefore, the fast derivatization of thiols was employed to generate stable ionized derivatives for MS detection.
    RESULTS: By evaluating the changes of MS signals before and after the derivatization, the ions of RSS were identified simply and correctly. Without any sample pretreatment, the fast detection of cysteine, homocysteine, and glutathione has been achieved in the complicated samples.
    CONCLUSIONS: The present online derivatization-based MF-EESI was successfully used for fast, simple, and accurate detection of biothiols. This presented a potential pathway for the fast identification of thiols in complicated samples.
  26. Molecules. 2022 Mar 01. pii: 1620. [Epub ahead of print]27(5):
      Reactive oxygen species (ROS) are continuously produced in living cells due to metabolic and biochemical reactions and due to exposure to physical, chemical and biological agents. Excessive ROS cause oxidative stress and lead to oxidative DNA damage. Within ROS-mediated DNA lesions, 8-oxoguanine (8-oxoG) and its nucleotide 8-oxo-2'-deoxyguanosine (8-oxodG)-the guanine and deoxyguanosine oxidation products, respectively, are regarded as the most significant biomarkers for oxidative DNA damage. The quantification of 8-oxoG and 8-oxodG in urine, blood, tissue and saliva is essential, being employed to determine the overall effects of oxidative stress and to assess the risk, diagnose, and evaluate the treatment of autoimmune, inflammatory, neurodegenerative and cardiovascular diseases, diabetes, cancer and other age-related diseases. High-performance liquid chromatography with electrochemical detection (HPLC-ECD) is largely employed for 8-oxoG and 8-oxodG determination in biological samples due to its high selectivity and sensitivity, down to the femtomolar range. This review seeks to provide an exhaustive analysis of the most recent reports on the HPLC-ECD determination of 8-oxoG and 8-oxodG in cellular DNA and body fluids, which is relevant for health research.
    Keywords:  8-oxo-2′-deoxyguanosine (8-oxodG); 8-oxoguanine (8-oxoG); HPLC–ECD; oxidative DNA damage; oxidative DNA damage biomarker
  27. Rapid Commun Mass Spectrom. 2022 Mar 09. e9293
      RATIONALE: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has exhibited great advantages in rapid analysis of metabolites. However, the influence of lipid fragments generated by in-source fragmentation (ISD) and/or post-source fragmentation (PSD) on the accurate qualification and quantification of metabolites has not been fully demonstrated.METHODS: Phospholipid standards and serum extract were analyzed by MALDI MS with both TiO2 nanoparticle (TiO2 NP) and 2,5-DHB matrices to illustrate the structures of lipid fragments and their influence on the qualitative and quantitative analysis of metabolites in biological samples. Monophasic and biphasic extraction methods were also compared for their efficiency of removing potential interferents.
    RESULTS: The fragment ions derived from the phosphocholine head group of phosphatidylcholines (PC) interfere with peaks of low molecular weight (LMW) metabolites in both MS and MS2 levels. The biphasic extraction system with methanol-chloroform could high efficiently remove the interference from PC fragments, and the metabolites choline and carnitine in serum were directly and accurately quantified by MALDI MS by using this biphasic extraction.
    CONCLUSIONS: The phospholipids could produce fragment ions through ISD and PSD in MALDI MS with both nanoparticle and organic matrices. The fragments have great influence on the qualification and qualification of metabolites in serum. By choosing the proper extraction method, the interference of lipid fragments could be efficiently alleviated.