bims-metlip 2021-09-05 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–09–05
23 papers selected by
Sofia Costa, Cold Spring Harbor Laboratory

CRB-SWATH: A Method for Enhancing Untargeted Precursor Ion Extraction and Automatically Constructing Their Tandem Mass Spectra from SWATH Datasets by Chromatographic Retention Behaviors.
EVA: Evaluation of Metabolic Feature Fidelity Using a Deep Learning Model Trained With Over 25000 Extracted Ion Chromatograms.
Parallel Derivatization Strategy for Comprehensive Profiling of Unconjugated and Glycine-conjugated Bile Acids Using: Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry.
Determination of Saccharides in Honey Using Supercritical Fluid Chromatography Coupled with Single Quadrupole Mass Spectrometry.
A versatile method to separate complex lipid mixtures using 1-butanol as eluent in a reverse-phase UHPLC-ESI-MS system.
Streamlined three step total vitamin C analysis by HILIC-UV for laboratory testing.
Development of a novel analytical method for determining trazodone in human plasma by LC-MS coupled with automatic 2-dimensional liquid chromatograph-mass spectrometer coupler 9500 and its application to therapeutic drug monitoring.
Comparison of High-Resolution Fourier Transform Mass Spectrometry Platforms for Putative Metabolite Annotation.
ADAP-KDB: A Spectral Knowledgebase for Tracking and Prioritizing Unknown GC-MS Spectra in the NIH's Metabolomics Data Repository.
A Workflow to Perform Targeted Metabolomics at the Untargeted Scale on a Triple Quadrupole Mass Spectrometer.
Development of a sensitive liquid chromatography-tandem mass spectrometry method for quantification of human plasma arginine vasopressin.
Profiling of sphingolipids in Caenorhabditis elegans by two-dimensional multiple heart-cut liquid chromatography - mass spectrometry.
Nontargeted screening method for veterinary drugs and their metabolites based on fragmentation characteristics from ultrahigh-performance liquid chromatography-high-resolution mass spectrometry.
Simultaneous quantification of oxcarbazepine and its active metabolite in spiked human plasma using ultra performance liquid chromatography-MS/MS.
[Determination of Albendazole Metabolite in Livestock Products Using LC-MS/MS].
A new strategy for dynamic metabolic flux estimation by integrating transient metabolome data into genome-scale metabolic models.
Development and Validation of a Liquid Chromatography Mass Spectrometry Method for Simultaneous Measurement of 25(OH)D3, epi-25(OH)D3, 25(OH)D2, Vitamin A, α-Tocopherol, and γ-Tocopherol.
Gas chromatography-mass spectrometry-based metabolite profiling and sensory profile of Indonesian fermented food (tempe) from various legumes.
Investigation of stationary phases performance for eicosanoids profiling in RP-HPLC.
Surface molecularly imprinted solid-phase extraction for the determination of vancomycin and norvancomycin in milk by liquid chromatography coupled to tandem mass spectrometry.
mspack: efficient lossless and lossy mass spectrometry data compression.
An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in E. coli.
Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry.

Anal Chem. 2021 Aug 30.

CRB-SWATH: A Method for Enhancing Untargeted Precursor Ion Extraction and Automatically Constructing Their Tandem Mass Spectra from SWATH Datasets by Chromatographic Retention Behaviors.

Ren-Qi Wang, Jun Ding, Ye Geng, Yuan-Zheng Li, Ying-Wu Mei, Kai Bao, Huai-Dong Yu, Yu-Qi Feng.

Sequential window acquisition of all theoretical spectra (SWATH) as a typical data-independent acquisition (DIA) strategy is favorable for untargeted metabolomics. It could theoretically acquire product ions of all precursor ions, including precursor ions showing chromatographic peaks of rather poor qualities. However, existing data processing methods present limited capabilities in capturing poor-quality peaks of precursor ions. Thus, although their product ions could be acquired, their precursor ions are absent. Here, we present a new strategy, chromatographic retention behavior-SWATH (CRB-SWATH), that could unbiasedly capture poor-quality peaks and provide high resolutions of multiplexed mass spectroscopy (MS/MS) spectra in SWATH datasets. CRB-SWATH monitors CRBs of SWATH-MS signals under a series of altered elution gradients. As signals of compounds differ from noise by showing CRBs, both the precursor and fragment ions are captured, while ignoring their peak qualities. Moreover, CRB-SWATH offers good chances to resolve highly multiplexed MS/MS spectra in SWATH datasets because precursor ions coeluted in a single elution gradient often present different CRBs. In the untargeted metabolic analysis of Hela cell extracts, CRB-SWATH showed the advantage in exclusively capturing 2645 ions of poor-quality peaks (i.e., tiny peaks, discontinuous ion traces, tailing peaks, zigzag peaks, etc.), accounting for 34.4% of all the untargeted precursor ions extracted. Therein, it is noteworthy that among 2116 negative ions detected in hydrophilic interaction liquid chromatography (HILIC) mode, 1284 poor-quality ion peaks (>60%) were exclusively captured by CRB-SWATH. As CRB-SWATH automatically captures a large sum of true ion peaks of poor qualities, extracts MS/MS spectra of high purities, and provides chromatographic retention behaviors of untargeted metabolites for identification and classification, it could be a useful metabolomics tool for understanding biological phenomena better.

DOI: https://doi.org/10.1021/acs.analchem.1c01841
Anal Chem. 2021 Aug 28.

EVA: Evaluation of Metabolic Feature Fidelity Using a Deep Learning Model Trained With Over 25000 Extracted Ion Chromatograms.

Jian Guo, Sam Shen, Shipei Xing, Ying Chen, Frank Chen, Elizabeth M Porter, Huaxu Yu, Tao Huan.

Extracting metabolic features from liquid chromatography-mass spectrometry (LC-MS) data relies on the recognition of extracted ion chromatogram (EIC) peak shapes using peak picking algorithms. Unfortunately, all peak picking algorithms present a significant drawback of generating a problematic number of false positives. In this work, we take advantage of deep learning technology to develop a convolutional neural network (CNN)-based program that can automatically recognize metabolic features with poor EIC shapes, which are of low feature fidelity and more likely to be false. Our CNN model was trained using 25095 EIC plots collected from 22 LC-MS-based metabolomics projects of various sample types, LC and MS conditions. Notably, we manually inspected all the EIC plots to assign good or poor EIC quality for accurate model training. The trained CNN model is embedded into a C#-based program, named EVA (short for evaluation). The EVA Windows Application is a versatile platform that can process metabolic features generated by LC-MS systems of various vendors and processed using various data processing software. Our comprehensive evaluation of EVA indicates that it achieves over 90% classification accuracy. EVA can be readily used in LC-MS-based metabolomics projects and is freely available on the Microsoft Store by searching "EVA Metabolomics".

DOI: https://doi.org/10.1021/acs.analchem.1c01309
J Steroid Biochem Mol Biol. 2021 Aug 25. pii: S0960-0760(21)00179-5. [Epub ahead of print] 105986

Parallel Derivatization Strategy for Comprehensive Profiling of Unconjugated and Glycine-conjugated Bile Acids Using: Ultra-high Performance Liquid Chromatography-Tandem Mass Spectrometry.

Ting Hu, Han Li, Benshan Xu, Ping Du, Lihong Liu, Zhuoling An.

  Bile acids (BAs) are steroidal compounds that play important roles in the occurrence and development of liver injury. However, comprehensive characterization of BAs was rarely reported due to the limitations of both standards access and detection sensitivity. In this study, a parallel derivatization strategy was established for the sensitive and comprehensive profiling of unconjugated and glycine-conjugated BAs by using ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Two structural analogues 2-hydrazinyl-4,6-dimethylpyrimidine (DMP) and 2-hydrazinylpyrimidine (DP) were used as the parallel derivatization reagents for BAs labeling, facilitating the improvements of both detection sensitivities and chromatographic performances. The derivatization reactions can be completed in 20 min at room temperature, with derivatization efficacy higher than 99%. Through derivatization, the sensitivity of BAs increased dozens or hundreds of times compared to their non-derivatized forms. Due to the structural similarities of derivatized BAs, general MS parameters can be forged for the analysis of DMP and DP labeled BAs. In addition, the DP labeled BAs were incorporated into the DMP derivatized biological samples for both the discovery and comprehensive characterization of BAs. Retention time shift (RTS) and peak area ratio (PAR) induced by the parallel DMP and DP labeled BAs were used for the rapid identification of BAs from complex biological samples. More than 200 BAs were profiled in rat serum using this parallel derivatization strategy. Further, the new strategy was successfully implemented in BAs profiling of serum samples from tripterysium glycosides-induced liver injury rat model. The disturbance of the BA metabolism network was further interpreted.

Keywords:  UHPLC-MS/MS; bile acid; derivatization; drug-induced liver injury; tripterysium glycosides

DOI:  https://doi.org/10.1016/j.jsbmb.2021.105986
J Chromatogr Sci. 2021 Sep 01. pii: bmab104. [Epub ahead of print]

Determination of Saccharides in Honey Using Supercritical Fluid Chromatography Coupled with Single Quadrupole Mass Spectrometry.

Yang Huang, Zhengjin Jiang.

Saccharides are one of the most important biomoleculars in the world, which are responsible for various roles in living organisms. In this work, a combined use of mass spectrometry (MS) together with supercritical fluid chromatography (SFC) was applied for the separation of eight saccharides. A satisfactory separation was achieved within 15 min on a Unitary Diol column using 10 mM ammonium acetate in methanol as mobile phase additive at a temperature of 60°C. Compared to liquid chromatography-MS (LC-MS), the SFC-MS presented faster running time and better selectivity. The SFC-MS was validated and applied to the analysis of three saccharides (fructose, glucose and sucrose) present in different honey samples. The limits of detection (LODs) and limits of quantification (LOQs) of SFC-MS method for fructose, glucose and sucrose were 5.2, 4.5, 7.4 μg/mL and 17.2, 14.9, 24.1 μg/mL, respectively. The relative standard deviations for intrabatch and interbatch precision were <2.5% and the recoveries ranged from 97.6% to 103.1% for three concentration levels. The values of LODs and LOQs using SFC-MS method were lower (1.5-2.4-fold) than using LC-MS method. The results demonstrated the potential of SFC-MS for fast and sensitive determination of saccharides in honey.

DOI: https://doi.org/10.1093/chromsci/bmab104
Chem Phys Lipids. 2021 Aug 25. pii: S0009-3084(21)00078-5. [Epub ahead of print]240 105125

A versatile method to separate complex lipid mixtures using 1-butanol as eluent in a reverse-phase UHPLC-ESI-MS system.

Niels A W de Kok, Marten Exterkate, Ruben L H Andringa, Adriaan J Minnaard, Arnold J M Driessen.

  Simple, robust and versatile LC-MS based methods add to the rapid assessment of the lipidome of biological cells. Here we present a versatile RP-UHPLC-MS method using 1-butanol as the eluent, specifically designed to separate different highly hydrophobic lipids. This method is capable of separating different lipid classes of glycerophospholipid standards, in addition to phospholipids of the same class with a different acyl chain composition. The versatility of this method was demonstrated through analysis of lipid extracts of the bacterium Escherichia coli and the archaeon Sulfolobus acidocaldarius. In contrast to 2-propanol-based methods, the 1-butanol-based mobile phase is capable of eluting highly hydrophobic analytes such as cardiolipins, tetraether lipids and mycolic acids during the gradient instead of the isocratic purge phase, resulting in an enhanced separation of cardiolipins and extending the analytical range for RPLC.

Keywords:  Cardiolipin; Electrospray ionization mass spectrometry (ESI-MS); Lipidomics; Phospholipid; Reversed-phase liquid chromatography (RPLC); Tetraether lipid

DOI:  https://doi.org/10.1016/j.chemphyslip.2021.105125
Clin Chem Lab Med. 2021 Sep 03.

Streamlined three step total vitamin C analysis by HILIC-UV for laboratory testing.

Michael Fitzpatrick, Paul Bonnitcha, Van Long Nguyen.

   OBJECTIVES: In the clinical setting, the analysis and quantification of vitamin C (ascorbic acid) poses several challenges including analyte instability and poor retention by reverse phase HPLC systems. In this article we describe a rapid hydrophilic interaction chromatography ultraviolet method for the measurement of total vitamin C in plasma which overcomes these issues.
METHODS: Ascorbic acid and the internal standard were separated under isocratic conditions using a Waters BEH-Amide column and a mobile phase containing 0.005 M potassium phosphate in 80% acetonitrile.
RESULTS: The proposed method was validated and showed good precision (coefficient of variation <5%), accuracy (>99%), and analyte stability after extraction (>24 h).
CONCLUSIONS: The simple sample preparation allows full automation and rapid analytical run times of the assay and is therefore suitable for a high-throughput clinical chromatography laboratory.

Keywords:  ascorbic acid; high performance liquid chromato-graphy-diode array detection (HPLC-DAD); high performance liquid chromatography-ultraviolet detection (HPLC-UV); hydrophilic interaction chromatography (HILIC); plasma; vitamin C

DOI:  https://doi.org/10.1515/cclm-2021-0614
Ther Drug Monit. 2021 Aug 27.

Development of a novel analytical method for determining trazodone in human plasma by LC-MS coupled with automatic 2-dimensional liquid chromatograph-mass spectrometer coupler 9500 and its application to therapeutic drug monitoring.

Jing Ding, Yan Zhang, Xiaohua Cui, Caiping Zhao, Suo Zhang.

BACKGROUND: Trazodone (TZD) is a tetracyclic serotonin antagonist and reuptake inhibitor that is used as a second-generation phenylpiperazine antidepressant. However, the plasma concentrations of TZD have shown individual variations in clinical practice. Quantification of TZD plasma concentrations may be an effective and valuable method to balance the clinical efficacy and adverse reactions. This study aimed to establish a novel LC-MS assay for measuring TZD concentrations in human plasma for therapeutic drug monitoring (TDM).
METHODS: After protein precipitation with acetonitrile, LC-MS quantification of TZD was performed in the multiple reaction monitoring mode with chromatographic separation using a mobile phase of MeOH and 0.1% formic acid in water. This method validation intends to investigate specificity, sensitivity, linearity, precision, accuracy, recovery, matrix effect, and stability according to USFDA guidelines.
RESULTS: This method showed good selectivity because no interfering peaks were observed in the plasma samples during the 2-min run time. The range of the calibration curve was 1-3000 ng/mL. The detection and quantification limits were 0.3 and 1 ng/mL, respectively. The intra- and inter-day accuracies were 96.5-103.4%, with precision RSD% values of <5%, except for the limit of quality. The mean TZD recovery from human plasma was 95.4-104.5%. Finally, this method was successfully applied to TDM in 20 patients. The TZD plasma concentrations of the patients ranged between 21.5 and 2267.3 ng/mL.
CONCLUSIONS: A novel analytical method was established to measure TZD by LC-MS coupled with an automatic 2-dimensional liquid chromatograph mass spectrometer coupler 9500 (LC-MS/MS-Mate 9500), which is superior to the ordinary LC-MS system in terms of separation, transport, anti-interference, sensitivity, and quantitative analysis stability.

DOI: https://doi.org/10.1097/FTD.0000000000000921
Anal Chem. 2021 Aug 30.

Comparison of High-Resolution Fourier Transform Mass Spectrometry Platforms for Putative Metabolite Annotation.

Danning Huang, Marcos Bouza, David A Gaul, Franklin E Leach, I Jonathan Amster, Frank C Schroeder, Arthur S Edison, Facundo M Fernández.

Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap mass spectrometry (MS) are among the highest-performing analytical platforms used in metabolomics. Non-targeted metabolomics experiments, however, yield extremely complex datasets that make metabolite annotation very challenging and sometimes impossible. The high-resolution accurate mass measurements of the leading MS platforms greatly facilitate this process by reducing mass errors and spectral overlaps. When high resolution is combined with relative isotopic abundance (RIA) measurements, heuristic rules, and constraints during searches, the number of candidate elemental formula(s) can be significantly reduced. Here, we evaluate the performance of Orbitrap ID-X and 12T solariX FT-ICR mass spectrometers in terms of mass accuracy and RIA measurements and how these factors affect the assignment of the correct elemental formulas in the metabolite annotation pipeline. Quality of the mass measurements was evaluated under various experimental conditions (resolution: 120, 240, 500 K; automatic gain control: 5 × 104, 1 × 105, 5 × 105) for the Orbitrap MS platform. High average mass accuracy (<1 ppm for UPLC-Orbitrap MS and <0.2 ppm for direct infusion FT-ICR MS) was achieved and allowed the assignment of correct elemental formulas for over 90% (m/z 75-466) of the 104 investigated metabolites. 13C1 and 18O1 RIA measurements further improved annotation certainty by reducing the number of candidates. Overall, our study provides a systematic evaluation for two leading Fourier transform (FT)-based MS platforms utilized in metabolite annotation and provides the basis for applying these, individually or in combination, to metabolomics studies of biological systems.

DOI: https://doi.org/10.1021/acs.analchem.1c02224
Anal Chem. 2021 Aug 29.

ADAP-KDB: A Spectral Knowledgebase for Tracking and Prioritizing Unknown GC-MS Spectra in the NIH's Metabolomics Data Repository.

Aleksandr Smirnov, Yunfei Liao, Eoin Fahy, Shankar Subramaniam, Xiuxia Du.

We report the development of a spectral knowledgebase named ADAP-KDB for tracking and prioritizing unknown gas chromatography-mass spectrometry (GC-MS) spectra in the NIH's Metabolomics Data Repository-a national and international repository for metabolomics data. ADAP-KDB consists of two parts. One part is a computational workflow that preprocesses raw mass spectrometry data and derives consensus mass spectra. The other part is a web portal for users to browse the consensus spectra and match query spectra against them. For each consensus spectrum, the Gini-Simpson diversity index and the p-value from χ2 goodness-of-fit test are calculated to measure its statistical significance, which enables prioritization of unknown mass spectra for subsequent costly compound identification.

DOI: https://doi.org/10.1021/acs.analchem.1c00355
ACS Meas Sci Au. 2021 Aug 18. 1(1): 35-45

A Workflow to Perform Targeted Metabolomics at the Untargeted Scale on a Triple Quadrupole Mass Spectrometer.

Michaela Schwaiger-Haber, Ethan Stancliffe, Valerie Arends, Bharat Thyagarajan, Miriam Sindelar, Gary J Patti.

The thousands of features commonly observed when performing untargeted metabolomics with quadrupole time-of-flight (QTOF) and Orbitrap mass spectrometers often correspond to only a few hundred unique metabolites of biological origin, which is in the range of what can be assayed in a single targeted metabolomics experiment by using a triple quadrupole (QqQ) mass spectrometer. A major benefit of performing targeted metabolomics with QqQ mass spectrometry is the affordability of the instruments relative to high-resolution QTOF and Orbitrap platforms. Optimizing targeted methods to profile hundreds of metabolites on a QqQ mass spectrometer, however, has historically been limited by the availability of authentic standards, particularly for "unknowns" that have yet to be structurally identified. Here, we report a strategy to develop multiple reaction monitoring (MRM) methods for QqQ instruments on the basis of high-resolution spectra, thereby enabling us to use data from untargeted metabolomics to design targeted experiments without the need for authentic standards. We demonstrate that using high-resolution fragmentation data alone to design MRM methods results in the same quantitative performance as when methods are optimized by measuring authentic standards on QqQ instruments, as is conventionally done. The approach was validated by showing that Orbitrap ID-X data can be used to establish MRM methods on a Thermo TSQ Altis and two Agilent QqQs for hundreds of metabolites, including unknowns, without a dependence on standards. Finally, we highlight an application where metabolite profiling was performed on an ID-X and a QqQ by using the strategy introduced here, with both data sets yielding the same result. The described approach therefore allows us to use QqQ instruments, which are often associated with targeted metabolomics, to profile knowns and unknowns at a comprehensive scale that is typical of untargeted metabolomics.

DOI: https://doi.org/10.1021/acsmeasuresciau.1c00007
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Aug 23. pii: S1570-0232(21)00384-6. [Epub ahead of print]1181 122903

Development of a sensitive liquid chromatography-tandem mass spectrometry method for quantification of human plasma arginine vasopressin.

Masaki Takiwaki, Fumio Nomura, Mamoru Satoh, Sachio Tsuchida, Kazuo Otake, Junko Takagi.

   BACKGROUND AND AIMS: Direct measurement of arginine vasopressin (AVP) via immunoassays is not widely conducted, mainly because of technical constraints. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) has been widely used as the gold standard in clinical chemistry. Here, we aimed to develop an MS-based assay to determine human plasma AVP and compare the results with those obtained using a conventional immunoassay.
MATERIALS AND METHODS: We developed a protocol using triple quadrupole MS coupled with LC for the measurement of human plasma AVP. Analytical evaluations of the method were performed, and the results obtained using LC/MS/MS and radioimmunoassay (RIA) were compared.
RESULTS: The lower limit of quantification (LLOQ) for plasma AVP obtained using LC/MS/MS and RIA were 0.2 and 0.4 pg/mL, respectively. Although there was a weak overall correlation between the results obtained using the two different methods, the RIA results did not agree with the LC/MS/MS results, particularly at low concentrations.
CONCLUSIONS: AVP detection through RIA is not satisfactory compared with that using LC/MS/MS. Diagnostic values of direct AVP measurements must be evaluated based on the results obtained via sensitive and accurate MS-based methods rather than those obtained through RIA.

Keywords:  Arginine vasopressin; Liquid chromatography-tandem mass spectrometry; Radioimmunoassay

DOI:  https://doi.org/10.1016/j.jchromb.2021.122903
J Chromatogr A. 2021 Aug 18. pii: S0021-9673(21)00605-1. [Epub ahead of print]1655 462481

Profiling of sphingolipids in Caenorhabditis elegans by two-dimensional multiple heart-cut liquid chromatography - mass spectrometry.

Johannes Scholz, Patrick O Helmer, Merle M Nicolai, Julia Bornhorst, Heiko Hayen.

  Sphingolipids exert important functions in cells, ranging from stabilising the cell membrane to bioactive signalling in signal transduction pathways. Changed concentrations of sphingolipids are associated with, among others, neurodegenerative and cardiovascular diseases. In this work, we present a novel two-dimensional liquid chromatography method (2D-LC) coupled to tandem mass spectrometry (MS/MS) for the identification of ceramides, hexosylceramides and sphingomyelins in the model organism Caenorhabditis elegans (C. elegans). The method utilises a multiple heart-cut approach with a hydrophilic interaction liquid chromatography (HILIC) separation in the first dimension. The fractions of the sphingolipid classes were cut out and thereby separated from the abundant glycerolipids, which offers a simplified sample preparation and a high degree of automation as it compensates the alkaline depletion step usually conducted prior to the chromatographic analysis. The fractions were stored in a sample loop and transferred onto the second column with the combination of two six port valves. A reversed phase liquid chromatography was performed as the second dimension and allowed for a separation of the species within a sphingolipid class and according to the fatty acid moiety of the sphingolipid. The segregation of the abundant glycerolipids and the reduced matrix effects allowed for better identification of low abundant species, especially dihydro-sphingolipids with a saturated sphingoid base. In addition, the separation of the three fractions was carried out parallel to the separation and equilibration in the first dimension, which leads to no extension of the analysis time for the 2D-LC compared to the one-dimensional HILIC method. In total 45 sphingolipids were detected in the C. elegans lipid extract and identified via accurate mass and MS/MS fragments.

Keywords:  Caenorhabditis elegans; Ceramides; Multiple heart-cut; Online 2D-LC; Sphingolipids; Sphingomyelins

DOI:  https://doi.org/10.1016/j.chroma.2021.462481
Food Chem. 2021 Aug 21. pii: S0308-8146(21)01934-8. [Epub ahead of print]369 130928

Nontargeted screening method for veterinary drugs and their metabolites based on fragmentation characteristics from ultrahigh-performance liquid chromatography-high-resolution mass spectrometry.

Wenying Liang, Fujian Zheng, Tiantian Chen, Xiuqiong Zhang, Yueyi Xia, Zaifang Li, Xin Lu, Chunxia Zhao, Guowang Xu.

  Nontargeted screening of both veterinary drugs and their metabolites is important for comprehensive safety evaluation of animal-derived foods. In this study, a novel nontargeted screening strategy was developed for veterinary drugs and their metabolites based on fragmentation characteristics from ultrahigh-performance liquid chromatography-high-resolution mass spectrometry. First, an in-house database of mass spectra including 3,710 veterinary drugs and their metabolites was constructed. Second, fragmentation characteristics of parent drugs and their metabolites in mass spectrometry were investigated and summarized. Then, a nontargeted screening procedure was established based on fragmentation characteristics to screen unknown parent drugs and their metabolites. Finally, the strategy was applied to 33 egg samples, and four veterinary drugs and three drug metabolites were determined and identified. These results showed that the developed strategy can realize suspect and nontargeted screening of veterinary drugs and their metabolites, and can also be applied to other animal-derived foods.

Keywords:  Drug metabolites; Fragmentation characteristics; Liquid chromatography-high-resolution mass spectrometry; Nontargeted screening; Suspect screening; Veterinary drugs

DOI:  https://doi.org/10.1016/j.foodchem.2021.130928
Bioanalysis. 2021 Aug;13(16): 1259-1273

Simultaneous quantification of oxcarbazepine and its active metabolite in spiked human plasma using ultra performance liquid chromatography-MS/MS.

Karthik Rajendran, Bhadram Kalyan Chekraverthy, Rajendran Sankham Devendran, R Jasmin Sajini, Krishnaveni Nagappan.

  Aim: Clinical monitoring of oxcarbazepine (OXC) and its metabolite licarbazepine (MHD) in biological matrix requires a sensitive and validated analytical method. The aim of this study is to develop and validate an optimized ultra performance liquid chromatography-MS/MS based bioanalytical method for the simultaneous estimation of OXC and its metabolite MHD in human plasma, using deuterated internal standard method. Materials & methods: A reverse phase ultra performance liquid chromatography analysis and mass spectrometric detection was performed using electrospray ionization in positive ion mode as interface, multiple reaction monitoring as mode of acquisition. Results & conclusion: The linearity range was 10-4011 ng/ml for OXC and 40-16061 ng/ml for MHD. The kinetic parameters were calculated and compared for bioequivalence. This method fulfilled the validation guidelines, could be employed for determining bioavailability and in new formulation development studies.

Keywords:  anti-epileptic drugs; bioanalytical method; bioequivalence; metabolite; oxcarbazepine; plasma

DOI:  https://doi.org/10.4155/bio-2021-0125
Shokuhin Eiseigaku Zasshi. 2021 ;62(4): 113-118

[Determination of Albendazole Metabolite in Livestock Products Using LC-MS/MS].

Ryo Okabe, Akiko Kubota, Satoru Nemoto, Mitsutoshi Aoyagi.

  A method for determining albendazole metabolite (metabolite I) in livestock products using LC-MS/MS was proposed. Livestock samples were hydrolyzed with 6 mol/L HCl at 110℃ for an hour and defatted with ethyl acetate and n-hexane (1 : 1, v/v) mixture. Metabolite I was extracted with acetonitrile from the sample, and the extracts were salted out under basic conditions, allowing the acetonitrile layer to separate. The acetonitrile solution was cleaned up using a cartridge column packed with divinylbenzene-N-vinylpyrolidone copolymer bearing sulfo groups. The HPLC separation was conducted on an Inertsil ODS-4 column with a gradient formed from water containing 0.05% (v/v) formic acid and acetonitrile containing 0.05% (v/v) formic acid. To detect metabolite I, tandem mass spectrometry with positive ion electrospray ionization was used. Truenesses (n=5) of metabolite I from cattle meat, fat, liver, and milk spiked at the maximum residue limits or the 0.01 mg/kg were in the range from 83.6 to 97.9%, and the relative standard deviations were from 1.6 to 6.1%.

Keywords:  LC-MS/MS; albendazole; livestock products; metabolite

DOI:  https://doi.org/10.3358/shokueishi.62.113
Bioprocess Biosyst Eng. 2021 Aug 30.

A new strategy for dynamic metabolic flux estimation by integrating transient metabolome data into genome-scale metabolic models.

Peng Liu, Ye Hua, Wei Zhang, Tingting Xie, Yingping Zhuang, Jianye Xia, Henk Noorman.

  Metabolic flux analysis (MFA) is a powerful tool for studying microbial cell physiology. Isotope-based MFA is the accepted standard for studying metabolic fluxes under steady-state conditions. However, its application under dynamic extracellular conditions is limited due to lack of proper techniques, such as rapid sampling and quenching, high cost and laborious execution. Here, we propose a new strategy to tackle this through incorporating dynamic metabolite abundance data into genome-scale metabolic models (GEM). First, a dummy extracellular pool concept is proposed for each dynamically changing metabolite, which represents a "sink" or "source", with corresponding dummy reactions coded into the GEM model. The dynamic model (expressed as differential equations) is then transformed into a quasi-steady-state model (expressed as linear equations), which can be easily solved by constraining the GEM model with the dynamic metabolite quantification data. For this, common linear-programming optimization algorithms were utilized to estimate the dynamic fluxes. Dynamic high-accuracy metabolite abundance data were obtained through the Isotope Dilution Mass Spectrometry (IDMS) method and high-speed sampling-quenching, and it was demonstrated that the newly proposed strategy could be successfully applied to obtain intracellular dynamic fluxes of Aspergillus niger under regimes of single and periodic extracellular glucose pulses. The applicability of the new method was also tested on dynamic fluxes estimation in a glucose pulse-response study of Saccharomyces cerevisiae. The proposed method provides a powerful tool to investigate cell physiology under dynamic conditions, especially relevant for bioprocess scale-up to industrial-scale bioreactors.

Keywords:  Dynamic metabolic flux; Fermentation scale-up; Genome-scale metabolic model; Glucose pulse; Metabolic flux analysis

DOI:  https://doi.org/10.1007/s00449-021-02626-3
Am J Clin Pathol. 2021 Aug 31. pii: aqab088. [Epub ahead of print]

Development and Validation of a Liquid Chromatography Mass Spectrometry Method for Simultaneous Measurement of 25(OH)D3, epi-25(OH)D3, 25(OH)D2, Vitamin A, α-Tocopherol, and γ-Tocopherol.

Yi Xiao, Edward Ki Yun Leung.

   OBJECTIVES: Fat-soluble vitamins are measured to identify deficiencies that may lead to rickets, osteomalacia, night blindness, and reversible motor and sensory neuropathies. We present a rapid liquid chromatography-mass spectrometry (LC-MS/MS) method that simultaneously measures 25-hydroxyvitamin D3 (25[OH]D3), epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol.
METHODS: We mixed 100 µL serum with internal standard and extracted it by using supported liquid extraction plates. Reconstituted specimens were analyzed by LC-MS/MS with a 10-minute gradient.
RESULTS: The method was linear, covering physiological levels with r2 > 0.99, and the total precision was less than 15% at all quality control levels. The lower limit of the measuring intervals for 25(OH)D3, epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol were 4 ng/mL, 4 ng/mL, 4 ng/mL, 1 µg/dL, 0.2 µg/mL, and 0.2 µg/mL, respectively, with coefficient of variation of 20% or less. Recoveries were between 92% and 111% for National Institute of Standards and Technology reference materials and 81% and 122% for spike-recovery studies. Comparison studies for vitamin D total, vitamin A, and α-tocopherol demonstrated slopes between 1.04 and 1.11 and r2 between 0.94 and 0.96. Minimal matrix effect was observed for all analytes.
CONCLUSIONS: We developed and validated a rapid LC-MS/MS method for the simultaneous measurement of 25(OH)D3, epi-25(OH)D3, 25(OH)D2, vitamin A, α-tocopherol, and γ-tocopherol.

Keywords:  Fat-soluble vitamins; LC-MS/MS; Vitamin A; Vitamin D; Vitamin E; epi-25(OH)D3

DOI:  https://doi.org/10.1093/ajcp/aqab088
J Biosci Bioeng. 2021 Aug 28. pii: S1389-1723(21)00180-8. [Epub ahead of print]

Gas chromatography-mass spectrometry-based metabolite profiling and sensory profile of Indonesian fermented food (tempe) from various legumes.

Della Rahmawati, Made Astawan, Sastia Prama Putri, Eiichiro Fukusaki.

  Tempe is an Indonesian fermented food prepared from soybean or other non-soy legumes. Non-soy legumes based tempes have been reported to have a nutritional profile different from that of soybean-based tempe. However, a comprehensive study of the metabolite and sensory profiles of legume tempes has not been conducted. This study investigated the metabolite profile of legumes before and after fermentation using a metabolomic approach and its relation to the sensory profile. Four different legumes, namely red kidney bean, soybean, cowpea bean, and jack bean, and four kinds of tempe prepared from the abovementioned legumes were subjected to gas chromatography-mass spectrometry (GC-MS) analysis. These tempe samples were fermented in two different production places, Japan and Indonesia. In total, 83 metabolites were annotated from all samples. Principal component analysis (PCA) showed that the samples were separated based on their type (legume and tempe) along PC1 with a 36.3% variance, in which sugars and amino acids significantly contributed to the separation. In addition, samples were separated along PC2 with a 17.2% variance based on the different production places. The combination of 75% soybean and 25% red kidney bean (75S:25R) could increase the concentrations of amino acids that were initially low in soybean tempe. The 75S:25R tempe showed no significant differences in taste and texture preference compared to the 100% soybean tempe, based on sensory result. These findings provide evidence that metabolomics could be used to identify the differences between tempes from different legumes.

Keywords:  Fermented legumes-based; Gas chromatography-mass spectrometry; Metabolomics; Sensory evaluation; Tempe

DOI:  https://doi.org/10.1016/j.jbiosc.2021.07.001
Anal Bioanal Chem. 2021 Sep 02.

Investigation of stationary phases performance for eicosanoids profiling in RP-HPLC.

Kodjo Nouwade, Sana Tfaili, Pierre Chaminade.

  Eicosanoids - oxidative derivatives from arachidonic acid - represent biologically active lipid mediators in inflammatory processes. Different analytical methods treat eicosanoid analysis. Among which, reverse phase liquid chromatography figures as the appropriate method for eicosanoid profiling. RP-HPLC for eicosanoid analysis is often conducted on C18 columns. Some studies focused on profiling one family of eicosanoids; others considered all eicosanoid families. In both cases, co-elution remained a major issue and detection in mass spectrometry partially resolves this problem. In fact, the mass transitions used to monitor eicosanoid species are not specific enough and many isobars can be listed. For this, optimizing the RP-HPLC separation remains important. Based on the parameter Fs - deriving from the hydrophobic-subtraction model - and radar plots, we chose columns with different selectivities. The hydrophobic-subtraction model guided our interpretation of molecular interactions between eicosanoids and stationary phases. We founded our approach for selectivity optimization on peak capacity per minute and time needed values. Herein, we screened seven stationary phases and evaluated their chromatographic performances in RP-HPLC. Stationary phases presented different chemistry, type of silica, length, and particle size. Superficially porous particle columns registered better chromatographic profiles than classical stationary phases; and columns with embedded polar group did not serve our purpose. The stationary phase Accucore C30 - even being the least retentive - revealed the best selectivity and efficiency, and recorded the shorter duration for eicosanoid analysis.

Keywords:  Accucore C30; Eicosanoids; Hydrophobic-subtraction model; Peak capacity per minute; Selectivity; Time needed

DOI:  https://doi.org/10.1007/s00216-021-03618-8
Food Chem. 2021 Aug 18. pii: S0308-8146(21)01892-6. [Epub ahead of print]369 130886

Surface molecularly imprinted solid-phase extraction for the determination of vancomycin and norvancomycin in milk by liquid chromatography coupled to tandem mass spectrometry.

Hao Zhou, Rong Liu, Qianqian Chen, Xiantong Zheng, Jingli Qiu, Tongyan Ding, Limin He.

  A simple and sensitive method based on surface molecularly imprinted solid-phase extraction (SMISPE) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed to determine the residues of vancomycin (VCM) and norvancomycin (NVCM) in milk samples. The imprinted polymer prepared with teicoplanin as a virtual template can specifically recognize VCM and NVCM. The samples were purified with SMISPE and analyzed by LC-MS/MS in positive ionization mode. The results showed that the VCM and NVCM had a good linear correlation in the range of 0.5 μg/kg to 50 μg/kg. The recoveries of target analytes were from 83.3% to 92.1%, and the limits of quantification were both 1.0 μg/kg. The matrix effects of VCM and NVCM were -11.0% and -3.43%, respectively. The proposed method can efficiently eliminate the interference from matrix compounds and reduce baseline noise, which is useful for the monitoring of the residues of VCM and NVCM in milk samples.

Keywords:  LC–MS/MS; Milk; Molecular imprinting solid-phase extraction; Norvancomycin; Vancomycin

DOI:  https://doi.org/10.1016/j.foodchem.2021.130886
Bioinformatics. 2021 Sep 03. pii: btab636. [Epub ahead of print]

mspack: efficient lossless and lossy mass spectrometry data compression.

Felix Hanau, Hannes Röst, Idoia Ochoa.

MOTIVATION: Mass spectrometry data, used for proteomics and metabolomics analyses, have seen considerable growth in the last years. Aiming at reducing the associated storage costs, dedicated compression algorithms for Mass Spectrometry (MS) data have been proposed, such as MassComp and MSNumpress. However, these algorithms focus on either lossless or lossy compression, respectively, and do not exploit the additional redundancy existing across scans contained in a single file. We introduce mspack, a compression algorithm for MS data that exploits this additional redundancy and that supports both lossless and lossy compression, as well as the mzML and the legacy mzXML formats. mspack applies several preprocessing lossless transforms and optional lossy transforms with a configurable error, followed by the general purpose compressors gzip or bsc to achieve a higher compression ratio.
RESULTS: We tested mspack on several datasets generated by commonly used mass spectrometry instruments. When used with the bsc compression backend, mspack achieves on average 76% smaller file sizes for lossless compression and 94% smaller file sizes for lossy compression, as compared to the original files. Lossless mspack achieves 10 - 60% lower file sizes than MassComp, and lossy mspack compresses 36 - 60% better than the lossy MSNumpress, for the same error, while exhibiting comparable accuracy and running time.
AVAILABILITY: mspack is implemented in C ++ and freely available at https://github.com/fhanau/mspack under the Apache license.
SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI: https://doi.org/10.1093/bioinformatics/btab636
Nat Protoc. 2021 Sep 03.

An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in E. coli.

Emily J Geddes, Zhong Li, Paul J Hergenrother.

Novel classes of broad-spectrum antibiotics have been extremely difficult to discover, largely due to the impermeability of the Gram-negative membranes coupled with a poor understanding of the physicochemical properties a compound should possess to promote its accumulation inside the cell. To address this challenge, numerous methodologies for assessing intracellular compound accumulation in Gram-negative bacteria have been established, including classic radiometric and fluorescence-based methods. The recent development of accumulation assays that utilize liquid chromatography-tandem mass spectrometry (LC-MS/MS) have circumvented the requirement for labeled compounds, enabling assessment of a substantially broader range of small molecules. Our unbiased study of accumulation trends in Escherichia coli using an LC-MS/MS-based assay led to the development of the eNTRy rules, which stipulate that a compound is most likely to accumulate in E. coli if it has an ionizable Nitrogen, has low Three-dimensionality and is relatively Rigid. To aid in the implementation of the eNTRy rules, we developed a complementary web tool, eNTRyway, which calculates relevant properties and predicts compound accumulation. Here we provide a comprehensive protocol for analysis and prediction of intracellular accumulation of small molecules in E. coli using an LC-MS/MS-based assay (which takes ~2 d) and eNTRyway, a workflow that is readily adoptable by any microbiology, biochemistry or chemical biology laboratory.

DOI: https://doi.org/10.1038/s41596-021-00598-y
J Vis Exp. 2021 Aug 13.

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry.

Danye Qiu, Verena B Eisenbeis, Adolfo Saiardi, Henning J Jessen.

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented.

DOI: https://doi.org/10.3791/62847