bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2022‒02‒20
twenty-four papers selected by
Sofia Costa
Icahn School of Medicine at Mount Sinai

  1. Anal Chem. 2022 Feb 18.
      Untargeted metabolomics and lipidomics LC-MS experiments produce complex datasets, usually containing tens of thousands of features from thousands of metabolites whose annotation requires additional MS/MS experiments and expert knowledge. All-ion fragmentation (AIF) LC-MS/MS acquisition provides fragmentation data at no additional experimental time cost. However, analysis of such datasets requires reconstruction of parent-fragment relationships and annotation of the resulting pseudo-MS/MS spectra. Here, we propose a novel approach for automated annotation of isotopologues, adducts, and in-source fragments from AIF LC-MS datasets by combining correlation-based parent-fragment linking with molecular fragment matching. Our workflow focuses on a subset of features rather than trying to annotate the full dataset, saving time and simplifying the process. We demonstrate the workflow in three human serum datasets containing 599 features manually annotated by experts. Precision and recall values of 82-92% and 82-85%, respectively, were obtained for features found in the highest-rank scores (1-5). These results equal or outperform those obtained using MS-DIAL software, the current state of the art for AIF data annotation. Further validation for other biological matrices and different instrument types showed variable precision (60-89%) and recall (10-88%) particularly for datasets dominated by nonlipid metabolites. The workflow is freely available as an open-source R package, MetaboAnnotatoR, together with the fragment libraries from Github (
  2. Anal Chim Acta. 2022 Mar 08. pii: S0003-2670(22)00063-0. [Epub ahead of print]1197 339492
      A general and deterministic model is derived from the fundamentals of liquid chromatography to calculate retention time, peak width, peak capacity, and density of peak capacity in gradient liquid chromatography. The calculation of these chromatographic properties accounts for 1) the presence of initial (separation of the earliest eluters) and final (column wash) isocratic steps before and after the linear gradient, respectively, 2) the pre- (flow through needle and preheater tubes) and post-column (outlet and emitter tubes before MS detection) dispersion, 3) the compression of the chromatographic band, and 4) the retention of the organic modifier onto the RPLC column. The multiple and variable method parameters may include the column dimensions, particle size, flow rate, temperature, initial and final isocratic hold times, gradient time, gradient steepness, column conditioning/sample load time, and the pre- and post-column tube dimensions. The model enables the users to perform robust multi-dimensional optimization of UHPLC-MS methods and offers the possibility to predict the expected MS feature density for increased method performance. Method optimization can be further improved by matching the observed MS feature density (number of metabolites detected as function of time) to the predicted density of peak capacity. It is directly applied to the optimization of high-throughput RPLC separation methods specifically designed for large-scale urinary metabolic phenotyping.
    Keywords:  Density of peak capacity; LC-MS method optimization; Non-targeted metabolomics; Retention time and peak width prediction; Sample feature density; Urine metabolites
  3. J Chromatogr A. 2022 Feb 02. pii: S0021-9673(22)00068-1. [Epub ahead of print]1667 462870
      The determination of amino acids in biological samples is central to the diagnosis of inherited metabolic disorders and also gives significant information about the metabolisms in the cells and living body. The development of analytical method for reliable quantification of amino acids in biological samples is still challenging because of the polar nature of amino acids and complex nature of biological samples causing a high degree of interferences during analysis. In the present study, a pre-column derivatization method using 2-naphtoyl chloride combined with liquid chromatography-tandem mass spectrometry method was developed for the determination of 17 amino acids in human serum and urine matrices. Low detection limits were obtained in the range of 0.015 - 0.266 µmol kg-1 and acceptable recovery results were obtained in human serum and urine samples. Isotopically labelled (15N labelled) amino acids were spiked to standards and samples before derivatization to compensate for the analytical errors in the whole procedure. The combination of quadrupole isotope dilution strategy with the derivatization based reversed phase chromatography allowed to improve method accuracy and precision.
    Keywords:  Amino acids; Derivatization; Human serum; Human urine; Isotope dilution
  4. Anal Chem. 2022 Feb 15.
      The use of stable isotopically labeled tracers is a long-proven way of specifically detecting and tracking derived metabolites through a metabolic network of interest. While the recently developed stable isotope-assisted methods and associated, supporting data analysis tools have greatly improved untargeted metabolomics approaches, no software tool is currently available that allows us to automatically and flexibly search liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) chromatograms for user-definable isotopolog patterns expected for the metabolism of labeled tracer substances. Here, we present Custom Pattern Extract (CPExtract), a versatile software tool that allows for the first time the high-throughput search for user-defined isotopolog patterns in LC-HRMS data. The patterns can be specified via a set of rules including the presence or absence of certain isotopologs, their relative intensity ratios as well as chromatographic coelution. Each isotopolog pattern satisfying the respective rules is verified on an MS scan level and also in the chromatographic domain. The CPExtract algorithm allows the use of both labeled tracer compounds in nonlabeled biological samples as well as a reversed tracer approach, employing nonlabeled tracer compounds along with globally labeled biological samples. In a proof-of-concept study, we searched for metabolites specifically arising from the malonate pathway of the filamentous fungi Fusarium graminearum and Trichoderma reesei. 1,2,3-13C3-malonic acid diethyl ester and native malonic acid monomethyl ester were used as tracers. We were able to reliably detect expected fatty acids and known polyketides. In addition, up to 46 and 270 further, unknown metabolites presumably including novel polyketides were detected in the F. graminearum and T. reesei culture samples, respectively, all of which exhibited the user-predicted isotopolog patterns originating from the malonate tracer incorporation. The software can be used for every conceivable tracer approach. Furthermore, the rule sets can be easily adapted or extended if necessary. CPExtract is available free of charge for noncommercial use at
  5. J Exp Bot. 2022 Feb 17. pii: erac062. [Epub ahead of print]
      Assessing central carbon metabolism in plants can be challenging due to the dynamic range in pool sizes, with low levels of important phosphorylated sugars relative to more abundant sugars and organic acids. Here, we report a sensitive liquid chromatography-mass spectrometry (LC-MS) method for analyzing central metabolites on a hybrid column, where both anion-exchange and hydrophilic interaction chromatography (HILIC) ligands are embedded in the stationary phase. The LC method was developed for enhanced selectivity of 27 central metabolites in a single run with sensitivity at femtomole levels observed for most phosphorylated sugars. The method resolved phosphorylated hexose, pentose, and triose isomers that are otherwise challenging. Compared to a standard HILIC approach, these metabolites had improved peak areas using our approach due to ion-enhancement or low ion-suppression in the biological sample matrix. The approach was applied to investigate metabolism in high lipid-producing tobacco leaves that exhibited increased levels of the acetyl-CoA, a precursor for oil biosynthesis. The application of the method to isotopologue detection and quantification was considered through evaluating 13C-labeled seeds from Camelina sativa. The method provides a means to analyze intermediates more comprehensively in central metabolism of plant tissues.
    Keywords:  Central metabolism; ion suppression and enhancement; isomer separation; isotopic labeling; liquid chromatography-mass spectrometry; metabolite quantification; mixed-mode column chromatography; oilseeds
  6. Anal Chem. 2022 Feb 16.
      Monosaccharides play important roles in biological processes. Sensitive and accurate analyses of monosaccharides remain challenging because of their high hydrophilicities and poor ionization efficiencies. Here, we developed a paired derivatization approach with H/D-labeled hydroxylamines for simultaneous quantification of 12 monosaccharides by liquid chromatography tandem mass spectrometry (LC-MS/MS). O-(4-Methoxybenzyl)hydroxylamine hydrochloride (4-MOBHA·HCl) showed higher derivatization efficiency for monosaccharides compared to six other hydroxylamine analogues. The derivatization of monosaccharides was readily achieved in an aqueous solution. Furthermore, the deuterium-labeled isotope reagent, d3-4-MOBHA·HCl, was newly synthesized to stably label monosaccharides to improve its accuracy and precision in complex matrix analysis. As a result, 12 monosaccharides were rapidly detected by LC-MS/MS within 16 min with significant improvements in chromatographic separation and retention time. The detection sensitivity increased by 83 to 1600-fold with limits of quantitation ranging from 0.25 to 3.00 fmol. With the paired derivatization strategy, the monosaccharides could be accurately quantified with good linearity (R2 > 0.99) and satisfactory accuracy (recoveries: 85-110%). Using this method, we achieved sensitive and accurate quantification of the monosaccharide composition of herbal polysaccharides and the change in monosaccharide levels in human cell lines under physiopathological conditions. More importantly, the developed method was able to differentiate between the levels of the monosaccharides in fecal samples of human ulcerative colitis (UC) patients and UC mice compared to their respective controls. The differential monosaccharides determined in human feces provided a good diagnostic performance in distinguishing the UC patients from healthy individuals, showing potential for clinical application.
  7. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Feb 11. pii: S1570-0232(22)00039-3. [Epub ahead of print]1193 123135
      Vitamin B12 (VB12) deficiency may lead to hyperhomocysteinemia and methylmalonic acidemia development which are risk factors of cardiovascular disease and nervous system impairment, respectively. However, few analytical methods are available to simultaneously quantify total homocysteine (tHcy) and methylmalonic acid (MMA) due to complex analytical requirements, such as sensitivity at nanomolar concentration, separation performance for succinic acid (SA), an endogenous isomer of MMA, and retention properties for polar compounds. Therefore, we developed and validated a simple and accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of tHcy and MMA with the efficient separation of SA in human serum and urine. The clinical performance of the assay was validated according to CLSI C62-A guidelines. The recovery for serum tHcy was 95.2-105.8%, urine tHcy was 98.1-111.5%, serum MMA was 94.6-99.4%, and urine MMA was 101.6-105.6%. In addition, the LC-MS/MS method was found to be reliable based on the value of inter-assay imprecision and total imprecision coefficient variation (CV), matrix effect, and carryover. Standards and samples were stable in -20 °C for at least 2 months. The limits of quantifications (LOQs) were 0.074 nmol/mL for tHcy and 0.040 nmol/mL for MMA, which are suitable for detecting tHcy and MMA concentrations in human serum and urine. The concentration of tHcy and MMA in samples collected from 148 subjects were measured using this method. The results suggested that the concentrations of serum tHcy and MMA considerably differed between VB12 sufficient and deficient groups. Serum tHcy and serum MMA concentrations were inversely correlated with VB12 status. Our method represents a rapid technique for estimating tHcy and MMA concentrations in serum and urine samples without the need for derivatization and may be used to assess VB12 status in clinical applications.
    Keywords:  Liquid chromatography-tandem mass spectrometry; Methylmalonic acid; Total homocysteine; vitamin B12 deficiency
  8. J AOAC Int. 2022 Feb 17. pii: qsac019. [Epub ahead of print]
      BACKGROUND: Residues of polar pesticides cannot be determined by QuEChERS-based multiresidue extractions because of their non-amenability to reverse-phase chromatographic separation and poor recoveries. On the other hand, single residue methods pose limitations because of the various requirements of sample preparation and LC-MS/MS conditions. A new multiresidue method is thus warranted for rapid and simultaneous analysis of polar pesticides.OBJECTIVE: The study developed a multiresidue method for the simultaneous analysis of glyphosate and its metabolite (aminomethylphosphonic acid, AMPA), glufosinate and its metabolites (3-methylphosphinicopropionic acid and N-acetyl-glufosinate), ethephon, fosetyl-aluminum and its metabolite (phosphonic acid), and trimesium in grape and pomegranate by LC-MS/MS.
    METHODS: The homogenized samples (10 g) were extracted with acidified methanol (20 mL). An aliquot of the extract was diluted with acetonitrile (1 + 1) and measured by LC-MS/MS using a Torus DEA column. The performance of a hydrophilic interaction liquid chromatography (HILIC) column and an "anionic polar pesticides" (APP) column was also evaluated.
    RESULTS: The method performance on the Torus DEA column was satisfactory for all compounds (recoveries = 77-104%, repeatability-RSD, <11%) at LOQ (0.01 mg/kg), and higher levels in grape and pomegranate. The only exception was AMPA, which had an LOQ of 0.05 mg/kg. In the APP column, AMPA could be determined with an LOQ of 0.01 mg/kg. Trimesium, which had poor retention in Torus DEA, performed better in an XBridge HILIC column (retention time= 4.2 min, LOQ = 0.01 mg/kg). The inter-laboratory validation experiment yielded comparable results with high accuracy and precision.
    CONCLUSIONS: The method could screen the residues of all compounds on a Torus DEA column. For AMPA and trimesium, the APP and XBridge HILIC columns provided superior method performances. Since isotopically-labelled internal standards were not required, the method appeared cost-effective. Considering its compliance with the SANTE/12682/2019 validation guidelines and EU-MRLs, the method can be recommended for regulatory testing purposes.
    HIGHLIGHTS: A high-throughput residue analysis method targeting nine polar and ionic compounds in grape and pomegranate involved a single multiresidue extraction, followed by direct analysis using LC-MS/MS. A satisfactory method performance was achieved through intra- and inter-laboratory validation. The method sensitivity met the EU-MRLs and the SANTE/12682/2019 analytical quality control criteria.
  9. Molecules. 2022 Feb 01. pii: 998. [Epub ahead of print]27(3):
      A fast, accurate and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for simultaneous quantification of ivermectin (IVER), doramectin (DORA), and moxidectin (MOXI) in bovine plasma. A priority for sample preparation was the eradication of possible infectious diseases to avoid travel restrictions. The sample preparation was based on protein precipitation using 1% formic acid in acetonitrile, followed by Ostro® 96-well plate pass-through sample clean-up. The simple and straightforward procedure, along with the short analysis time, makes the current method unique and suitable for a large set of sample analyses per day for PK studies. Chromatographic separation was performed using an Acquity UPLC HSS-T3 column, with 0.01% acetic acid in water and methanol, on an Acquity H-Class ultra-high performance liquid chromatograph (UHPLC) system. The MS/MS instrument was a Xevo TQ-S® mass spectrometer, operating in the positive electrospray ionization mode and two multiple reaction monitoring (MRM) transitions were monitored per component. The MRM transitions of m/z 897.50 > 753.4 for IVER, m/z 921.70 > 777.40 for DORA and m/z 640.40 > 123.10 for MOXI were used for quantification. The method validation was performed using matrix-matched calibration curves in a concentration range of 1 to 500 ng/mL. Calibration curves fitted a quadratic regression model with 1/x2 weighting (r ≥ 0.998 and GoF ≤ 4.85%). Limits of quantification (LOQ) values of 1 ng/mL were obtained for all the analytes, while the limits of detection (LOD) were 0.02 ng/mL for IVER, 0.03 ng/mL for DORA, and 0.58 ng/mL for MOXI. The results of within-day (RSD < 6.50%) and between-day (RSD < 8.10%) precision and accuracies fell within acceptance ranges. No carry-over and no peak were detected in the UHPLC-MS/MS chromatogram of blank samples showing good specificity of the method. The applicability of the developed method was proved by an analysis of the field PK samples.
    Keywords:  UHPLC-MS/MS; bioanalysis; bovine; macrocyclic lactones; method development; plasma
  10. Curr Opin Biotechnol. 2022 Feb 10. pii: S0958-1669(22)00013-1. [Epub ahead of print]75 102693
      Single-cell metabolomics with mass spectrometry enables a large variety of metabolites to be simultaneously detected from individual cells, without any preselection or labelling, to map phenotypes on the single cell level. Although the field is relatively young, it is steadily progressing with an increasing number of active research groups, techniques for cell sampling and ionization, tools for data analysis, and applications to answer important biomedical and environmental questions. In addition, the community shows great creativity in overcoming challenges associated with low sample volumes, a wide range of metabolite species, and large datasets. Here, we briefly discuss publications since 2019 and aim to provide the unfamiliar reader with an insight into the field and the expert reader with an update on the current status of the field.
  11. Anal Chim Acta. 2022 Mar 01. pii: S0003-2670(21)01231-9. [Epub ahead of print]1196 339405
      Metabolomics (both targeted and untargeted) has become the gold standard in biomarker discovery. Whereas targeted approaches only provide information for the selected markers, thus hampering the determination of out-of-the-box markers, the common bottleneck of untargeted metabolomics is the identification of detected biomarkers. In this study, we developed a strategy based on derivatization and LC-MS/MS detection in a precursor ion scan for the untargeted determination of a specific part of the metabolome (carbonyl-containing metabolites). The usefulness of this guided metabolomics approach has been demonstrated by elucidating carbonyl-containing biomarkers of COVID-19 severity. First, the LC-MS/MS behavior of 63 model compounds after O-benzylhydroxylamine derivatization was studied. A precursor ion scan of m/z 91 was selected as a suitable approach for the untargeted detection of carbonyl-containing metabolites. The method was able to detect ≈300 potential carbonyl-containing molecules in plasma, including mono-/di-/tricarbonylic compounds with satisfactory intra-day and inter-day repeatability and RSDs commonly <15%. Additionally, the semiquantitative nature of the precursor ion scan method was confirmed by comparison with a fully validated targeted method. The application of the guided metabolomics method to COVID-19 plasma samples revealed the presence of four potential COVID-19 severity biomarkers. Based on their LC-MS/MS behavior, these biomarkers were elucidated as 2-hydroxybutyrate, 2,3-dihydroxybutyrate, 2-oxobutyrate and 2-hydroxy-3-methylbutyrate. Their structures were confirmed by comparison with reference materials. The alterations of these biomarkers with COVID-19 severity were confirmed by a target analysis of a larger set of samples. Our results confirm that guided metabolomics is an alternative approach for the untargeted detection of selected families of metabolites; this approach can accelerate their elucidation and provide new perspectives for the establishment of health/disease biomarkers.
    Keywords:  Biomarker discovery; COVID-19; Carbonyl metabolome; Liquid chromatography-tandem mass spectrometry; Precursor ion scan; Untargeted metabolomics
  12. Int J Mol Sci. 2022 Jan 20. pii: 1136. [Epub ahead of print]23(3):
      Clinical data indicate that low circulating l-homoarginine (HArg) concentrations are associated with cardiovascular (CV) disease, CV mortality, and all-cause mortality. A high number of LC-based analytical methods for the quantification of HArg, in combination with the l-arginine (Arg)-related pathway metabolites, have been reported. However, these methods usually consider a limited panel of analytes. Thus, in order to achieve a comprehensive picture of the Arg metabolism, we described an improved targeted metabolomic approach based on a multiple reaction monitoring (MRM) mass spectrometry method for the simultaneous quantification of the Arg/nitric oxide (NO) pathway metabolites. This methodology was then employed to quantify the plasma concentrations of these analytes in a cohort of individuals with different grades/types of coronary artery disease (CAD) in order to increase knowledge about the role of HArg and its associated metabolites in the CV field. Our results showed that the MRM method here implemented is suitable for the simultaneous assessment of a wide panel of amino acids involved in the Arg/NO metabolic pathway in plasma samples from patients with CV disease. Further, our findings highlighted an impairment of the Arg/NO metabolic pathway, and suggest a sex-dependent regulation of this metabolic route.
    Keywords:  cardiovascular diseases; endothelial dysfunction; l-arginine/nitric oxide metabolic pathway; l-homoarginine; mass spectrometry; targeted metabolomics
  13. Anal Chem. 2022 Feb 14.
      Chromatography is often used as a method for reducing sample complexity prior to analysis by mass spectrometry, and the use of retention time (RT) is becoming increasingly popular to add valuable supporting information in lipid identification. The RT of lipids with the same headgroup in reversed-phase separation can be predicted using the equivalent carbon number (ECN) model. This model describes the effects of acyl chain length and degree of saturation on lipid RT. For the first time, we have found a robust correlation in the chromatographic separation of lipids with different headgroups that share the same fatty acid motive. This relationship can be exploited to perform interclass RT conversion (IC-RTC) by building a model from RT measurements from lipid standards that allows the prediction of RT of one lipid subclass based on another. Here, we utilize ECN modeling and IC-RTC to build a glycerophospholipid RT library with 517 entries based on 136 tandem mass spectrometry-characterized lipid RTs from NIST SRM-1950 plasma and lipid standards. The library was tested on a patient cohort undergoing coronary artery bypass grafting surgery (n = 37). A total of 156 unique circulating glycerophospholipids were identified, of which 52 (1 LPG, 24 PE, 5 PG, 18 PI, and 9 PS) were detected with IC-RTC, thereby demonstrating the utility of this technique for the identification of lipid species not found in commercial standards.
  14. Bioanalysis. 2022 Feb 17.
      Background: This article describes the development and validation of a bioanalytical assay to quantify CPI-613 and its major metabolites, CPI-2850 and CPI-1810, in human plasma matrix using LC-MS/MS. Methodology: Sample extraction procedure following protein precipitation with acetonitrile was optimized to extract all three analytes from plasma with maximum recovery. The final extracted supernatants were diluted with water and injected onto an Xbridge C18 (50 × 2.1 mm; 5 μm) column for analysis. The analytes were separated by a gradient elution, and detection was performed on a triple quadrupole mass spectrometer (Sciex API 5000) operating in the negative ion mode. Results: The assay was linear over a range of 50-50,000 ng/ml for CPI-613, 250-250,000 ng/ml for CPI-2850 and 10-10,000 ng/ml for CPI-1810. Benchtop stability was established for 24 h, and four freeze-thaw cycles were evaluated for CPI-613 and its metabolites. Long-term freezer (-60 to -80°C) stability for about 127 days was established in this validation. Mean matrix recovery was more than 80% for all analytes. Conclusion: A robust LC-MS/MS method was developed for the quantification of CPI-613 and its major metabolites. The current assay will be used to support ongoing and future CPI-613 clinical trials.
    Keywords:  CPI-613 (devimistat); LC-MS/MS; clinical trials; human plasma; investigational new drug; protein precipitation
  15. Anal Chem. 2022 Feb 15.
      Nanospray desorption electrospray ionization mass spectrometry, a powerful ambient sampling and imaging technique, is herein coupled as an isolated source with 21 Tesla (21T) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Absorption-mode data, enabled by an external data acquisition system, is applied for improved mass resolution, accuracy, and dynamic range without compromising spectral acquisition rates. Isotopic fine structure (IFS) information is obtained from the ambient sampling of living Bacillus and Fusarium species, allowing for high confidence in molecular annotations with a resolution >830 k (at m/z 825). Tandem mass spectrometry experiments for biological samples are shown to retain the IFS in addition to gained fragmentation information, providing a further degree of annotation confidence from ambient analyses. Rat brain was imaged by nanospray desorption electrospray ionization (nano-DESI) 21T FTICR MS in ∼5 h using 768 ms transients, producing over 800 molecular annotations using the METASPACE platform and low-parts-per-billion mass accuracy at a spatial resolution of ∼25 × 180 μm. Finally, nano-DESI 21T FTICR MS imaging is demonstrated to reveal images corresponding to the IFS, as well as hundreds of additional molecular features (including demonstrated differences as low as 8.96 mDa) that are otherwise undetected by a more conventional imaging methodology.
  16. Methods Mol Biol. 2022 ;2462 125-133
      Phytohormones plays crucial physiological functions in plants, where they are involved in plant development, reproduction, defense, and many other functions. Phytohormones production has been found to be regulated in response to abiotic and biotic factors affecting the plant metabolism, and therefore, biosynthesis of primary and secondary metabolites. Thus, the detection and quantification of phytohormones in different plant tissues are essential to be determined unraveling the various plant metabolic pathways and behavior. Yet phytohormones analysis is always problematic, since they are found in extremely low concentrations and have a wide range of chemical and physicochemical properties. As a result, the ideal method should start with an appropriate extraction procedure followed by quantification by highly sensitive instrumental techniques providing precise and robust results. The current chapter presents an improved extraction method based on liquid-liquid extraction from a 50-mg aliquot of plant tissue for analysis of the major classes of phytohormones. Then, mass spectrometry (MS) analysis is conducted using quadrupole/linear ion trap (QLIT) mass analyzer equipped with electrospray ionization (ESI) source after a liquid chromatographic separation step. The developed method demonstrates an appropriate feasibility addressing biological questions related to phytohormones production and regulation.
    Keywords:  Extraction; High resolution mass spectrometry; Liquid chromatography; Plant hormones; Quantitative analysis
  17. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Mar 01. pii: S1570-0232(22)00008-3. [Epub ahead of print]1192 123104
      Measurement of drug concentration in biological matrices (such as serum, plasma, blood, urine, and saliva) is important to determine Bioavailability (BA) and/or Bioequivalence (BE) of a drug product which are required during the drug product development and approval process to support applications for new active substances (INDs, NDAs) and generic (ANDAs) drug products to make critical decisions on safety and efficacy. Because of their vital role, bioanalytical methods should be well-characterized, fully validated and documented to yield reliable results. In present work, a simple, specific, high throughput, accurate and sensitive UHPLC-MS/MS method has been developed and validated for quantification of Minoxidil in human plasma. The analyte and the internal standard were extracted from plasma by Liquid-Liquid Extraction using ethyl acetate. The chromatographic separation was achieved on Thermo Hypersil Gold column (4.6x50mm, 5μm) using acetonitrile-0.1% formic acid in water (60:40, v/v) at a flow rate of 0.400 ml/min. Detection by turbospray positive ionization mass spectrometry in the multiple reaction monitoring mode with a mass transition ion-pair of m/z 210.152 → 163.965 (Minoxidil) and m/z 220.267 → 169.089 (Internal Standard-Minoxidil D10) was found to be linear over the concentration range of 1.280 to 151.075 ng/ml. The method was fully validated as per USFDA guidelines and the results were within regulatory limits. The inter and intra-day precision ranged from 5.42 to 9.27% and 2.55-9.42% respectively. The inter and intra-day accuracy ranged from 89.2 to 98.9% and 102-105% respectively. The method was successfully applied to a BE study involving human volunteers.
    Keywords:  BA/BE studies; Bioanalysis; LC-MS/MS; LLE; Minoxidil
  18. Molecules. 2022 Jan 20. pii: 682. [Epub ahead of print]27(3):
      The routine techniques currently applied for the determination of nicotine and its major metabolites, cotinine, and trans-3'-hydroxycotinine, in biological fluids, include spectrophotometric, immunoassays, and chromatographic techniques. The aim of this study was to develop, and compare two new chromatographic methods high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS), and RP-HPLC enriched with chaotropic additives, which would allow reliable confirmation of tobacco smoke exposure in toxicological and epidemiological studies. The concentrations of analytes were determined in human plasma as the sample matrix. The methods were compared in terms of the linearity, accuracy, repeatability, detection and quantification limits (LOD and LOQ), and recovery. The obtained validation parameters met the ICH requirements for both proposed procedures. However, the limits of detection (LOD) were much better for HPLC-QQQ-MS/MS (0.07 ng mL-1 for trans-3'-hydroxcotinine; 0.02 ng mL-1 for cotinine; 0.04 ng mL-1 for nicotine) in comparison to the RP-HPLC-DAD enriched with chaotropic additives (1.47 ng mL-1 for trans-3'-hydroxcotinine; 1.59 ng mL-1 for cotinine; 1.50 ng mL-1 for nicotine). The extraction efficiency (%) was concentration-dependent and ranged between 96.66% and 99.39% for RP-HPLC-DAD and 76.8% to 96.4% for HPLC-QQQ-MS/MS. The usefulness of the elaborated analytical methods was checked on the example of the analysis of a blood sample taken from a tobacco smoker. The nicotine, cotinine, and trans-3'-hydroxycotinine contents in the smoker's plasma quantified by the RP-HPLC-DAD method differed from the values measured by the HPLC-QQQ-MS/MS. However, the relative errors of measurements were smaller than 10% (6.80%, 6.72%, 2.04% respectively).
    Keywords:  HPLC-QQQ-MS/MS chaotropic effect; RP-HPLC-DAD; cotinine; nicotine; trans-3′-hydroxycotinine
  19. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Feb 09. pii: S1570-0232(22)00069-1. [Epub ahead of print]1193 123165
      Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has become a mainstay analytical technique in pharmaceutical research and development and clinical diagnosis due to several advantages including excellent selectivity, specificity, and high sensitivity. LC-MS/MS has become the method of choice for steroids analysis due to its fast analytical time and improved specificity yet has a challenge in the separation and measurement of isomers with the same product ions. Here we describe a high-sensitivity LC/LC-MS/MS method that combines chiral chromatography and reverse-phase chromatography (LC/LC) along with MS/MS to rapidly separate and quantify steroid isomers of 11ß-methyl-19-nortestosterone (11ß-MNT) and endogenous testosterone in serum.
    Keywords:  11β-methyl-19-nortestosterone; Chiral separation; LC/LC-MS/MS; Reverse phase chromatography; Steroid isomers; Testosterone
  20. J Cheminform. 2022 Feb 16. 14(1): 6
      Unknown features in untargeted metabolomics and non-targeted analysis (NTA) are identified using fragment ions from MS/MS spectra to predict the structures of the unknown compounds. The precursor ion selected for fragmentation is commonly performed using data dependent acquisition (DDA) strategies or following statistical analysis using targeted MS/MS approaches. However, the selected precursor ions from DDA only cover a biased subset of the peaks or features found in full scan data. In addition, different statistical analysis can select different precursor ions for MS/MS analysis, which make the post-hoc validation of ions selected following a secondary analysis impossible for precursor ions selected by the original statistical method. Here we propose an automated, exhaustive, statistical model-free workflow: paired mass distance-dependent analysis (PMDDA), for reproducible untargeted mass spectrometry MS2 fragment ion collection of unknown compounds found in MS1 full scan. Our workflow first removes redundant peaks from MS1 data and then exports a list of precursor ions for pseudo-targeted MS/MS analysis on independent peaks. This workflow provides comprehensive coverage of MS2 collection on unknown compounds found in full scan analysis using a "one peak for one compound" workflow without a priori redundant peak information. We compared pseudo-spectra formation and the number of MS2 spectra linked to MS1 data using the PMDDA workflow to that obtained using CAMERA and RAMclustR algorithms. More annotated compounds, molecular networks, and unique MS/MS spectra were found using PMDDA compared with CAMERA and RAMClustR. In addition, PMDDA can generate a preferred ion list for iterative DDA to enhance coverage of compounds when instruments support such functions. Finally, compounds with signals in both positive and negative modes can be identified by the PMDDA workflow, to further reduce redundancies. The whole workflow is fully reproducible as a docker image xcmsrocker with both the original data and the data processing template.
    Keywords:  Data analysis; High-resolution mass spectrometry; Metabolomics; Open science; Reproducible research; Workflow
  21. J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Feb 12. pii: S1570-0232(22)00073-3. [Epub ahead of print]1193 123169
      Illegal drug residues in animal derived foods are closely related to human's life and health. Studies on illegal drug residues and the metabolism, such as β2-agonists in animals have attracted more and more attention. In most cases, β2-agonists are suppliedand used astheracemate. The metabolic process and distribution of the two enantiomers in animal tissues are different. Therefore, it is very necessary to develop a simple and fast method for chiral resolution of these drugs in animal tissues. In this paper, a reliable resolution and determination method was presented using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for fourteen enantiomers of seven β2-agonist racemates, clenbuterol (CLE), salbutamol (SAL), cimaterol (CIM), terbutaline (TER), clorprenaline (CLO), tulobuterol (TUL), penbuterol (PEN) in pork, beef, and lamb muscle samples. The samples were added the internal standard solution (IS) and extracted in the alkaline medium with acetonitrile. The further sample purification was accomplished through MCX solid phase extraction cartridge. Chromatographic chiral separation was carried out on a VancoShell chiral column (100 mm × 4.6 mm, 2.7 μm) with an isocratic mobile phase consisting of methanol and 10 mmol mL-1 ammonium formate aqueous solution (85:15, v/v). Under the optimized conditions, the resolution (R) of CIM was 2.0, CLE and PEN were 1.5, the others were all greater than 1.0. Enantiomeric determination was performed in the positive electrospray ionization mode using multiple reaction monitoring (MRM). The correlation coefficient (r) in the range of 0.2-25.0 μg L-1 was above 0.993. The average recoveries at the three spiking levels ranged from 95.3% to 117.7% with the relative standard deviation (RSD) lower than 15%. The limit of detection (LOD) and the limit of quantification (LOQ) of β2-agonist enantiomers was 0.2 μg kg-1 and 0.5 μg kg-1 respectively. The method was successfully applied in the analysis and evaluation of β2-agonist enantiomers in positive food animal muscle samples, CLE, SAL, TEB and CIM enantiomers were detected. The concentrations of the corresponding enantiomers were in the range of 1.06-17.3 μg kg-1, the lowest enantiomer fraction (EF) value was 0.42, and the highest value was 0.69. The work is expected to provide a method for chiral separation and enantiomeric determination of the further study of pharmacology, toxicity and residue elimination of β2-agonist enantiomers.
    Keywords:  Enantiomer fraction; Enantiomeric determination; Enantioseparation; HPLC-MS/MS; Multi-residues; muscle tissues; β(2)-agonists
  22. J Am Soc Mass Spectrom. 2022 Feb 17.
      Neutral loss (NL) spectral data presents a mirror of MS2 data and is a valuable yet largely untapped resource for molecular discovery and similarity analysis. Tandem mass spectrometry (MS2) data is effective for the identification of known molecules and the putative identification of novel, previously uncharacterized molecules (unknowns). Yet, MS2 data alone is limited in characterizing structurally related molecules. To facilitate unknown identification and complement the METLIN-MS2 fragment ion database for characterizing structurally related molecules, we have created a MS2 to NL converter as a part of the METLIN platform. The converter has been used to transform METLIN's MS2 data into a neutral loss database (METLIN-NL) on over 860 000 individual molecular standards. The platform includes both the MS2 to NL converter and a graphical user interface enabling comparative analyses between MS2 and NL data. Examples of NL spectral data are shown with oxylipin analogues and two structurally related statin molecules to demonstrate NL spectra and their ability to help characterize structural similarity. Mirroring MS2 data to generate NL spectral data offers a unique dimension for chemical and metabolite structure characterization.
  23. Nat Protoc. 2022 Feb 18.
      Organoid culture systems are self-renewing, three-dimensional (3D) models derived from pluripotent stem cells, adult derived stem cells or cancer cells that recapitulate key molecular and structural characteristics of their tissue of origin. They generally form into hollow structures with apical-basolateral polarization. Mass spectrometry imaging (MSI) is a powerful analytical method for detecting a wide variety of molecules in a single experiment while retaining their spatiotemporal distribution. Here we describe a protocol for preparing organoids for MSI that (1) preserves the 3D morphological structure of hollow organoids, (2) retains the spatiotemporal distribution of a vast array of molecules (3) and enables accurate molecular identification based on tandem mass spectrometry. The protocol specifically focuses on the collection and embedding of the organoids in gelatin, and gives recommendations for MSI-specific sample preparation, data acquisition and molecular identification by tandem mass spectrometry. This method is applicable to a wide range of organoids from different origins, and takes 1 d from organoid collection to MSI data acquisition.
  24. Molecules. 2022 Feb 04. pii: 1056. [Epub ahead of print]27(3):
      A simple, fast and reliable analytical method was developed for 20 free amino acids (FAAs) determination in honey samples through a dilute-and-shoot strategy and hydrophilic interaction liquid chromatography tandem mass spectrometry. Compared with previous reports, direct dilution by water has significantly reduced the matrix effect and facilitated full extraction of FAAs. Further, a 5 min determination method was established with an acetonitrile-water mobile phase system with 0.1% formic acid addition. The established method was validated and demonstrated several advantages including short detection time, wide linear range over 3-4 orders of magnitude, high sensitivity down to 0.1 ng/mL and negligible matrix effect. Twenty FAAs were determined in 10 honey samples from different botanical origins by this method, and 19 FAAs were found. This general applicable method was also promising for fast determination of FAAs in other practical samples.
    Keywords:  dilute-and-shoot; free amino acid; honey sample; hydrophilic interaction liquid chromatography