bims-metlip 2021-11-07 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–11–07
sixteen papers selected by
Sofia Costa, Icahn School of Medicine at Mount Sinai

Simultaneous Analysis of the Metabolome and Lipidome Using Polarity Partition Two-Dimensional Liquid Chromatography-Mass Spectrometry.
HERMES: a molecular-formula-oriented method to target the metabolome.
SLAW: A Scalable and Self-Optimizing Processing Workflow for Untargeted LC-MS.
Fully Automatized Detection of Phosphocholine-Containing Lipids through an Isotopically Labeled Buffer Modification Workflow.
Comparison of two LC-MS/MS methods for the quantification of 24,25-dihydroxyvitamin D3 in patients and external quality assurance samples.
Fast Sampling of the Cellular Metabolome.
Rapid quantitative analysis of hormones in serum by multilayer paper spray MS: Free MS from HPLC.
Validation of a rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of vistusertib.
Metabolite Profiling of Meridianin C In Vivo of Rat by UHPLC/Q-TOF MS.
Estimation of ZYKR1 in human urine and plasma utilizing LC-MS/MS positive electrospray ionization; a kappa opioid receptor (KOR) agonist.
Parallel Accelerator and Molecular Mass Spectrometry Measurement of Carbon-14-Labeled Analytes.
Metabolomics workflow as a driven tool for rapid detection of metabolites in doping analysis. Development and validation.
Quantitative measurement of pioglitazone in neoplastic and normal tissues by AP-MALDI mass spectrometry imaging.
Enantioselective analyses of chloroquine and hydroxychloroquine in rat liver microsomes through chiral liquid chromatography-tandem mass spectrometry.
Performance of ion pairing chromatography and hydrophilic interaction liquid chromatography coupled to charged aerosol detection for the analysis of underivatized amino acids.
Rapid analysis of quinones in complex matrices by derivatization-based wooden-tip electrospray ionization mass spectrometry.

Anal Chem. 2021 Nov 05.

Simultaneous Analysis of the Metabolome and Lipidome Using Polarity Partition Two-Dimensional Liquid Chromatography-Mass Spectrometry.

Jieqing Feng, Qisheng Zhong, Jiangmeng Kuang, Jiaqi Liu, Taohong Huang, Ting Zhou.

Comprehensive metabolic profiling is a considerable challenge for systems biology since the metabolites in biological samples have significant polarity differences. A heart-cutting two-dimensional liquid chromatography-mass spectrometry (2D-LC-MS) method-based polarity partition was established to analyze both the metabolome and lipidome in a single run. Based on the polarity partition strategy, metabolites with high polarity were retained and separated by one-dimensional hydrophilic chromatography, while low- and medium-polarity lipids were collected into a sample loop and injected into two-dimensional reversed-phase chromatography for separation. A simple online dilution strategy realized the online coupling of the 2D-LC-MS, which effectively solved band broadening and peak distortion caused by solvent incompatibility. Moreover, a dual gradient elution procedure was introduced to further broaden the coverage of low-polarity lipids. The metabolites' log P values, which this 2D-LC-MS method could analyze, ranged from -8.79 to 26.86. The feasibility of the 2D-LC-MS system was demonstrated by simultaneous analysis of the metabolome and lipidome in rat plasma related to depression. A total of 319 metabolites were determined within 40 min, including organic acids, nucleosides, carbohydrate derivatives, amino acids, lipids, and other organic compounds. Finally, 44 depression-related differential metabolites were screened. Compared with conventional LC-MS-based methods, the 2D-LC method covered over 99% of features obtained by two conventional methods. In addition, the selectivity and resolution of the hydrophilic metabolites were improved, and the matrix effects of the hydrophobic metabolites were reduced in the developed method. The results indicated that the established 2D-LC system is a powerful tool for comprehensive metabolomics studies.

DOI: https://doi.org/10.1021/acs.analchem.1c03905
Nat Methods. 2021 Nov;18(11): 1370-1376

HERMES: a molecular-formula-oriented method to target the metabolome.

Roger Giné, Jordi Capellades, Josep M Badia, Dennis Vughs, Michaela Schwaiger-Haber, Theodore Alexandrov, Maria Vinaixa, Andrea M Brunner, Gary J Patti, Oscar Yanes.

Comprehensive metabolome analyses are essential for biomedical, environmental, and biotechnological research. However, current MS1- and MS2-based acquisition and data analysis strategies in untargeted metabolomics result in low identification rates of metabolites. Here we present HERMES, a molecular-formula-oriented and peak-detection-free method that uses raw LC/MS1 information to optimize MS2 acquisition. Investigating environmental water, Escherichia coli, and human plasma extracts with HERMES, we achieved an increased biological specificity of MS2 scans, leading to improved mass spectral similarity scoring and identification rates when compared with a state-of-the-art data-dependent acquisition (DDA) approach. Thus, HERMES improves sensitivity, selectivity, and annotation of metabolites. HERMES is available as an R package with a user-friendly graphical interface for data analysis and visualization.

DOI: https://doi.org/10.1038/s41592-021-01307-z
Anal Chem. 2021 Nov 04.

SLAW: A Scalable and Self-Optimizing Processing Workflow for Untargeted LC-MS.

Alexis Delabriere, Philipp Warmer, Vincenth Brennsteiner, Nicola Zamboni.

Metabolomics has been shown to be promising for diverse applications in basic, applied, and clinical research. These applications often require large-scale data, and while the technology to perform such experiments exists, downstream analysis remains challenging. Different tools exist in a variety of ecosystems, but they often do not scale to large data and are not integrated into a single coherent workflow. Moreover, the outcome of processing is very sensitive to a multitude of algorithmic parameters. Hence, parameter optimization is not only critical but also challenging. We present SLAW, a scalable and yet easy-to-use workflow for processing untargeted LC-MS data in metabolomics and lipidomics. The capabilities of SLAW include (1) state-of-the-art peak-picking algorithms, (2) a new automated parameter optimization routine, (3) an efficient sample alignment procedure, (4) gap filling by data recursion, and (5) the extraction of consolidated MS2 and an isotopic pattern across all samples. Importantly, both the workflow and the parameter optimization were designed for robust analysis of untargeted studies with thousands of individual LC-MSn runs. We compared SLAW to two state-of-the-art workflows based on openMS and XCMS. SLAW was able to detect and align more reproducible features in all data sets considered. SLAW scaled well, and its analysis of a data set with 2500 LC-MS files consumed 40% less memory and was 6 times faster than that using the XCMS-based workflow. SLAW also extracted 2-fold more isotopic patterns and MS2 spectra, which in 60% of the cases led to positive matches against a spectral library.

DOI: https://doi.org/10.1021/acs.analchem.1c02687
Anal Chem. 2021 Nov 02.

Fully Automatized Detection of Phosphocholine-Containing Lipids through an Isotopically Labeled Buffer Modification Workflow.

Andrea Cerrato, Sara Elsa Aita, Anna Laura Capriotti, Chiara Cavaliere, Carmela Maria Montone, Susy Piovesana, Aldo Laganà.

High-resolution mass spectrometry is the foremost technique for qualitative and quantitative lipidomics analyses. Glycerophospholipids and sphingolipids, collectively termed polar lipids, are commonly investigated by hyphenated liquid chromatography-mass spectrometry (LC-MS) techniques that reduce aggregation effects and provide a greater dynamic range of detection sensitivity compared to shotgun lipidomics. However, automatic polar lipid identification is hindered by several isobaric and isomer mass overlaps, which cause software programs to often fail to correctly annotate the lipid species. In the present paper, a buffer modification workflow based on the use of labeled and unlabeled acetate ions in the chromatographic buffers was optimized by Box-Behnken design of the experiments and applied to the characterization of phosphocholine-containing lipids in human plasma samples. The contemporary generation of [M + CH3COO]-, [M + CD3COO]-, and [M - CH3]- coupled with a dedicated data processing workflow, which was specifically set up on Compound Discoverer software, allowed us to correctly determine adduct composition, molecular formulas, and grouping, as well as granting a lower false-positive rate and streamlining the manual validation step compared to commonly employed lipidomics platforms. The proposed workflow represents a robust yet easier alternative to the existing approaches for improving lipid annotation, as it does not require extensive sample pretreatment or prior isotopic enrichment or derivatization.

DOI: https://doi.org/10.1021/acs.analchem.1c02944
Clin Chem Lab Med. 2021 Nov 02.

Comparison of two LC-MS/MS methods for the quantification of 24,25-dihydroxyvitamin D3 in patients and external quality assurance samples.

Sieglinde Zelzer, Caroline Le Goff, Stéphanie Peeters, Chiara Calaprice, Andreas Meinitzer, Dietmar Enko, Walter Goessler, Markus Herrmann, Etienne Cavalier.

   OBJECTIVES: In-house developed liquid-chromatography mass spectrometry (LC-MS/MS) methods are used more and more frequently for the simultaneous quantification of vitamin D metabolites. Among these, 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) is of clinical interest. This study assessed the agreement of this metabolite in two validated in-house LC-MS/MS methods.
METHODS: 24,25(OH)2D3 was measured in 20 samples from the vitamin D external quality assurance (DEQAS) program and in a mixed cohort of hospital patients samples (n=195) with the LC-MS/MS method at the Medical University of Graz (LC-MS/MS 1) and at the University of Liège (LC-MS/MS 2).
RESULTS: In DEQAS samples, 24,25(OH)2D3 results with LC-MS/MS 1 had a proportional bias of 1.0% and a negative systemic difference of -0.05%. LC-MS/MS 2 also showed a proportional bias of 1.0% and the negative systemic bias was -0.22%. Comparing the EQA samples with both methods, no systemic bias was found (0.0%) and the slope was 1%. The mean difference of 195 serum sample measurements between the two LC-MS/MS methods was minimal (-0.2%). Both LC-MS/MS methods showed a constant bias of 0.31 nmol/L and a positive proportional bias of 0.90%, respectively.
CONCLUSIONS: This study is the first to assess the comparability of 24,25(OH)2D3 concentrations in a mixed cohort of hospitalized patients with two fully validated in-house LC-MS/MS methods. Despite different sample preparation, chromatographic separation and ionization, both methods showed high precision measurements of 24,25(OH)2D3. Furthermore, we demonstrate the improvement of accuracy and precision measurements of 24,25(OH)2D3 in serum samples and in the DEQAS program.

Keywords:  24,25-dihydroxyvitamin D (24,25(OH)2D); liquid-chromatography mass spectrometry (LC-MS/MS); method comparison; patient samples; vitamin D external quality assurance scheme (DEQAS)

DOI:  https://doi.org/10.1515/cclm-2021-0792
Methods Mol Biol. 2022 ;2349 11-39

Fast Sampling of the Cellular Metabolome.

Walter M van Gulik, Andre B Canelas, Hilal Taymaz-Nikerel, Rutger D Douma, Lodewijk P de Jonge, Joseph J Heijnen.

  Obtaining meaningful snapshots of the metabolome of microorganisms requires rapid sampling and immediate quenching of all metabolic activity, to prevent any changes in metabolite levels after sampling. Furthermore, a suitable extraction method is required ensuring complete extraction of metabolites from the cells and inactivation of enzymatic activity, with minimal degradation of labile compounds. Finally, a sensitive, high-throughput analysis platform is needed to quantify a large number of metabolites in a small amount of sample. An issue which has often been overlooked in microbial metabolomics is the fact that many intracellular metabolites are also present in significant amounts outside the cells and may interfere with the quantification of the endo metabolome. Attempts to remove the extracellular metabolites with dedicated quenching methods often induce release of intracellular metabolites into the quenching solution. For eukaryotic microorganisms, this release can be minimized by adaptation of the quenching method. For prokaryotic cells, this has not yet been accomplished, so the application of a differential method whereby metabolites are measured in the culture supernatant as well as in total broth samples, to calculate the intracellular levels by subtraction, seems to be the most suitable approach. Here we present an overview of different sampling, quenching, and extraction methods developed for microbial metabolomics, described in the literature. Detailed protocols are provided for rapid sampling, quenching, and extraction, for measurement of metabolites in total broth samples, washed cell samples, and supernatant, to be applied for quantitative metabolomics of both eukaryotic and prokaryotic microorganisms.

Keywords:  Endometabolome; Exometabolome; Fast sampling; Isotope dilution mass spectrometry; Microbial metabolomics; Quenching

DOI:  https://doi.org/10.1007/978-1-0716-1585-0_2
Talanta. 2022 Jan 15. pii: S0039-9140(21)00822-5. [Epub ahead of print]237 122900

Rapid quantitative analysis of hormones in serum by multilayer paper spray MS: Free MS from HPLC.

Tianhao Wu, Gongwei Sun, Mingying Ma, Xingyu Pan, Sichun Zhang, Xinrong Zhang.

  Developing rapid and reliable method for simultaneous hormones quantitation is of great significant because of important roles of hormones in metabolism. However, current methods are faced with problems of low throughput or complicated operation procedure to remove matrices from serum samples in routine clinical diagnosis. In the present work, a multilayer PS-MS method was developed for rapid and simple detection of hormones. In the strategy, multilayer filter paper acted as the Liquid Chromatography in LC-MS/MS for separation of hormones and biological matrices. Qualitative and quantitative analysis of three hormones, testosterone (T), androsterone (ADT) and androstenedione (4-AD) were realized through MS/MS spectra. The method exhibited linearity in the range of 0.02-2 μg/L and the results of recovery and repeatability were satisfactory for standard samples and spiked serum. The time-cost of a whole detection process was less than 3 min. The established multilayer PS-MS realized rapid, simple and reliable quantitative analysis of various hormones and provided broad prospect for clinical analysis of small molecules in different biological samples. Moreover, it provides a novel MS approach with high through-put and free HPLC, meeting the requirements of point-of-care testing (POCT).

Keywords:  Hormones; Multilayer; PS-MS; Quantitative analysis; Rapid; Serum

DOI:  https://doi.org/10.1016/j.talanta.2021.122900
J Pharm Biomed Anal. 2021 Oct 22. pii: S0731-7085(21)00547-1. [Epub ahead of print]208 114436

Validation of a rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of vistusertib.

Susan C Scott, Nicole M Anders, Morgan Scardina, Christine L Hann, Michelle A Rudek.

  Vistusertib is an orally bioavailable mTOR inhibitor that is being studied in clinical trials. A novel reliable method was developed to quantitate vistusertib using LC-MS/MS to explore drug exposure-response relationships. Sample preparation involved protein precipitation using acetonitrile. Separation of vistusertib and the internal standard, AZD8055, was achieved with a Waters Acquity UPLC BEH C18 column utilizing isocratic elution over a 3 min total analytical run time. A SCIEX 4500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of vistusertib. The assay range was 5-5000 ng/mL and proved to be accurate (98.7-105.7%) and precise (CV ≤ 10.5%). A 40,000 ng/mL sample that was diluted 1:10 (v/v) with plasma was accurately quantitated. Long-term frozen plasma stability for vistusertib at -70 °C has been determined for at least 29 months. The method was applied for the measurement of plasma concentrations of vistusertib in a patient a solid tumor receiving 35 mg twice daily dose orally.

Keywords:  Assay; Tandem mass spectrometry; Validation; Vistusertib

DOI:  https://doi.org/10.1016/j.jpba.2021.114436
J Anal Methods Chem. 2021 ;2021 1382421

Metabolite Profiling of Meridianin C In Vivo of Rat by UHPLC/Q-TOF MS.

Guozhe Zhang, Linxia Xiao, Liang Qi.

Meridianin C (MC), as a marine alkaloid, is a potent protein kinase inhibitor which exhibits good anticancer activity. However, the in vivo metabolism of MC has not been described to date. In this study, an ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF MS) method is employed to investigate the in vivo metabolites of MC in rats. Plasma, bile, urine, and feces are collected after a single oral dose of MC. Protein precipitation, solid phase extraction (SPE), and ultrasonic extraction methods are used to prepare samples. Based on the mass spectral fragmentation patterns, elution order, and retrieving literatures, a total of 13 metabolites of MC were detected and tentatively identified, utilizing MetaboLynx software. The metabolic pathways of MC in rats include N- or O-glucuronidation, O-sulfation, N-hydroxylation, dihydroxylation, and trihydroxylation. The relative content of the metabolites in each kinds of biological samples is also evaluated. This study will help to understand the in vivo properties of MC for the future usage.

DOI: https://doi.org/10.1155/2021/1382421
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Oct 24. pii: S1570-0232(21)00463-3. [Epub ahead of print]1185 122982

Estimation of ZYKR1 in human urine and plasma utilizing LC-MS/MS positive electrospray ionization; a kappa opioid receptor (KOR) agonist.

Ashok Ghoghari, Chandrakant Bhatt, Kuldip Patel, Anil Jha, Harilal Patel, Mukul Jain, Taufik Momin, Deven Parmar, Pankaj Patel.

  ZYKR1, a short chain novel peptide with selective kappa opioid receptor agonist activity used as analgesics for the treatment of pain management. A sensitive and selective LC-MS/MS assay was developed and validated for estimation of ZYKR1 in human urine and plasma. ZY17258, an analogue compound was used as an internal standard. ZYKR1 was quantified using a selective reaction monitoring in electrospray ionization positive mode. The chromatographic separation was performed using mobile phase consisted of 0.05% v/v formic acid in water and methanol in gradient elution by analytical column Kinetex C8, 100 A°, 5 µm, 100 × 4.6 mm with 8.0 min analytical run time. Solid Phase extraction technique was used for purification of ZYKR1 and IS from human urine and plasma. The calibration curves were linear over range of 0.300 ng/mL to 300 ng/mL and 0.500 ng/mL to 500 ng/mL for human urine and plasma, respectively. No matrix effect and no significant carryover were observed. The extraction recovery was consistent and ranged from about 85% to 93% in human urine and in plasma respectively. Inter-day and intra-day accuracy (bias, %) and precision (CV, %) was -11.11 to 5.91 % and -2.25 to 6.65 % in human urine and -2.74 to 7.17 % and 2.24 to 15.18 % in plasma respectively were well within the acceptance criteria. Both the assays were devoid of endogenous matrix interference and commonly used concomitant drug interference. The validated assays were used for estimation of ZYKR1 from clinical pharmacokinetic study sample bioanalysis in healthy human subjects.

Keywords:  LC-MS/MS; Validation; ZY17258; ZYKR1; kappa opioid receptor (KOR)

DOI:  https://doi.org/10.1016/j.jchromb.2021.122982
Methods Mol Biol. 2022 ;2349 1-10

Parallel Accelerator and Molecular Mass Spectrometry Measurement of Carbon-14-Labeled Analytes.

Benjamin J Stewart, Ted J Ognibene.

  Parallel accelerator and molecular mass spectrometry (PAMMS) is a powerful analytical technique capable of simultaneous quantitation of carbon-14 tracer and structural characterization of 14C-labeled biomolecules. Here we describe the use of PAMMS for the analysis of biological molecules separated by high-performance liquid chromatography. This protocol is intended to serve as a guide for researchers who need to perform PAMMS experiments using instrumentation available at resource centers such as the National User Resource for Biological Accelerator Mass Spectrometry at Lawrence Livermore National Laboratory.

Keywords:  Accelerator mass spectrometry; Isotope ratio mass spectrometry; Liquid sample interface; Radiocarbon

DOI:  https://doi.org/10.1007/978-1-0716-1585-0_1
Rapid Commun Mass Spectrom. 2021 Nov 04. e9217

Metabolomics workflow as a driven tool for rapid detection of metabolites in doping analysis. Development and validation.

P Leogrande, D Jardines, D Martinez-Brito, E Domenici, X de la Torre, M K Parr, F Botrè.

RATIONALE: This work demonstrated the high potential of combining high-resolution mass spectrometry with chemometric tools, using metabolomics as a guided tool for anti-doping analysis. The administration of 7-keto-DHEA was studied as a proof-of-concept of the effectiveness of the combination of knowledge-based and machine-learning approaches to differentiate the changes due to the athletic activities from those due to the recourse to doping substances and methods.
METHODS: Urine samples were collected from 5 healthy volunteers before and after an oral administration by identifying three-time intervals. Raw data were acquired by injecting less than one microliter of derivatized samples into an Agilent Technologies 8890 Gas Chromatograph coupled to an Agilent Technologies 7250 Accurate-Mass Quadrupole Time-of-Flight, by using a low energy electron ionization source, and then they were preprocessed to align peak retention times with the same accurate mass. The resulting data table was subjected to multivariate analysis.
RESULTS: Multivariate analysis showed a high similarity between the samples belonging to the same collection interval and a clear separation between the different excretion intervals. The discrimination between blank and long excretion groups may suggest the presence of long excretion markers, which are particularly significant in anti-doping analysis. Furthermore, matching the most significant features with some of the metabolites reported in the literature data demonstrated the rationality of the proposed metabolomics-based approach.
CONCLUSIONS: The application of metabolomics tools as an investigation strategy could reduce the time and resources required to identify and characterize intake markers maximizing the information that can be extracted from the data and extending the research field by avoiding a priori bias. Therefore, metabolic fingerprinting of prohibited substance intakes could be an appropriate analytical approach to reduce the risk of false-positive/negative results, aiding in the interpretation of "abnormal" profiles and discrimination of pseudo-endogenous steroid intake in the anti-doping field.

DOI: https://doi.org/10.1002/rcm.9217
Talanta. 2022 Jan 15. pii: S0039-9140(21)00840-7. [Epub ahead of print]237 122918

Quantitative measurement of pioglitazone in neoplastic and normal tissues by AP-MALDI mass spectrometry imaging.

Lavinia Morosi, Cristina Matteo, Marina Meroni, Tommaso Ceruti, Ilaria Fuso Nerini, Ezia Bello, Roberta Frapolli, Maurizio D'Incalci, Massimo Zucchetti, Enrico Davoli.

  Pioglitazone is a Peroxisome Proliferator-Activated Receptor (PPAR) agonist of the thiazolidinedione class of compounds with promising anticancer activity. An innovative quantitative mass spectrometry imaging (MSI) method and a HPLC-UV method were developed and validated to investigate its distribution in tumor and liver tissues. The MSI method is based on stable isotope normalization and resulted highly specific and sensitive (0.2 pmol/spot). The correct identification of the drug ion signal is confirmed by MS/MS analysis on tissue. The method shows an optimal lateral resolution (25 μm) relying on the ionization efficiency and fine laser diameter of the atmospheric pressure MALDI source. The HPLC-UV method is simple and straightforward involving quick protein precipitation and shows good sensitivity (50ng/sample) using a small starting volume of biological sample. Thus, it is applicable to samples obtained from both preclinical models and clinical surgical procedures. MSI and HPLC-UV assays were validated assessing linearity, intra- and inter-day precision and accuracy, limit of quantification, selectivity and recovery. These are the first methods developed and validated for the analysis of pioglitazone in tissues, and they were applied successfully to myxoid liposarcoma xenograft-bearing mice, which received clinically relevant drug doses. Pioglitazone was measured by either method in sections of tumor and liver 2, 6 and 24 h post-treatment. Drug distribution was relatively homogeneous.

Keywords:  Drug distribution; Mass spectrometry imaging; Pioglitazone; Quantitative molecular imaging; Solid tumors

DOI:  https://doi.org/10.1016/j.talanta.2021.122918
Chirality. 2021 Nov 04.

Enantioselective analyses of chloroquine and hydroxychloroquine in rat liver microsomes through chiral liquid chromatography-tandem mass spectrometry.

Dong Guo, Rujian He, Wenxia Su, Ziqing Liang, Weiguang Zhang, Jun Fan.

  An efficient, sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) chiral analysis method was established for determination of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes. Effects of polysaccharide chiral stationary phases and basic additives on chiral separations of two analytes were discussed in detail. Amylose tris(3, 5-dimethylphenylcarbamate)-coated chiral stationary phase showed the best separation performance for them with acetonitrile-diethylamine-ethanol-diethylamine mixture (90:0.1:10:0.1, v/v/v/v) among four chiral stationary phases. Then, multiple reaction monitoring mode was selected as the data acquisition for determination of two pairs of enantiomers. The proposed LC-MS/MS chiral analysis method was validated in terms of linearity, accuracy, precision, and specificity. Good linearity with correlation coefficient over 0.998 was obtained in the concentration range of 0.05-5 μM. Limits of quantification for chloroquine and hydroxychloroquine enantiomers were 5.0 and 1.0 nM, respectively. The recoveries ranged from 81.14% to 111.09%. The intra-day and inter-day relative standard deviation were less than 6.5%. Moreover, concentrations of chloroquine and hydroxychloroquine enantiomers in rat liver microsomes were determined through the proposed LC-MS/MS analysis method. After incubated with rat liver microsomes for 10 min, the enantiomeric factor of hydroxychloroquine decreased from 0.50 to 0.45 (p < 0.001). In brief, our developed determination method for chloroquine and hydroxychloroquine enantiomers through LC-MS/MS spectrometry showed the characteristics of high-efficiency, fast speed, and very low detection limit, and would be greatly beneficial for screening and quantitation of them in biological matrices.

Keywords:  LC-MS/MS chiral analysis; chloroquine; hydroxychloroquine; metabolism; rat liver microsomes

DOI:  https://doi.org/10.1002/chir.23384
J Chromatogr A. 2021 Oct 13. pii: S0021-9673(21)00737-8. [Epub ahead of print]1659 462613

Performance of ion pairing chromatography and hydrophilic interaction liquid chromatography coupled to charged aerosol detection for the analysis of underivatized amino acids.

Ruben Pawellek, Ulrike Holzgrabe.

  The charged aerosol detector (CAD) is frequently employed in liquid chromatography for the analysis of small polar and ionizable compounds such as amino acids and amino sugars, which provide a weak chromophore only. Separation of these compounds is achieved by means of ion pair chromatography (IPC), and, more recently, hydrophilic interaction chromatography (HILIC) techniques. However, as the CAD's response is highly dependent on the mobile phase composition, the substantial differences in the mobile phase composition of IPC and HILIC have a distinct impact on the detector's performance. This study was aimed at systematically comparing the performance of IPC and HILIC when coupled to the CAD. Therefore, the separation techniques characterized by their specific mobile phase compositions were evaluated for their influence on the CAD response and the signal-to-noise ratio (S/N) of the amino acids L-alanine, L-leucine, and L-phenylalanine applying the response surface methodology (RSM). The RSM results derived from flow injection analysis (FIA) indicated that the CAD response and thus the obtainable S/N are significantly higher in HILIC compared to IPC where the S/N decreased with the chain length of the applied ion-pairing reagent. In addition, an IPC and a HILIC method, respectively, were developed for the impurity profiling of the branched-chain amino acids (BCAAs) L-leucine, L-isoleucine, and L-valine. The beneficial effects of the HILIC conditions on the S/N observed under FIA conditions were partly offset by moderate column bleed effects when using an amide functionalized column, which facilitates the separation in the HILIC method. Satisfactory LOQs (3-10 ng on column) were obtained with both methods; however, the HILIC method was found to be slightly superior in terms of sensitivity and separation efficiency.

Keywords:  Branched-chain amino acid (BCAA); Charged aerosol detector (CAD); High-performance liquid chromatography (HPLC); Hydrophilic interaction chromatography (HILIC); Ion pair chromatography (IPC); Response surface methodology (RSM)

DOI:  https://doi.org/10.1016/j.chroma.2021.462613
Talanta. 2022 Jan 15. pii: S0039-9140(21)00834-1. [Epub ahead of print]237 122912

Rapid analysis of quinones in complex matrices by derivatization-based wooden-tip electrospray ionization mass spectrometry.

Chen Ling, Qiaofang Shi, Zhanpeng Wei, Jingjing Zhang, Junjie Hu, Jiying Pei.

  Quinones are important components participating in various biological processes as well as hazardous substances to human health. Rapid determination of quinones in environmental samples and biofluids is the basis for assessing their health effect. Here, we presented a rapid, straightforward, highly sensitive and environmental-friendly wooden-tip electrospray ionization mass spectrometry (ESI-MS) method for the determination of quinones in PM2.5, urine and serum. An amine group "tag" was introduced to the quinone structure through in situ derivatization with cysteamine to improve ionization efficiency of quinones in wooden-tip ESI-MS. The toothpicks were treated by sharpening and acidification with HNO3. Experimental parameters, including sample volume, spray voltage, and spray solvent composition were optimized to be 1 μL, 3.5 kV, and ACN/CH3COOC2H5 (v/v, 9:1), respectively. The limits of detection for the determination of 1,4-benzoquinone, methyl-p-benzoquinone, 1,4-naphthoquinone and 1,4-anthraquinone in ACN under the optimal conditions were 1.00, 0.96, 0.13, 0.16 ng (1.00, 0.96, 0.13, 0.16 μg/mL, sample volume, 1 μL), respectively. This approach was successfully applied to the determination of 1,4-naphthoquinone and 1,4-anthraquinone in complex matrices, including PM2.5, urine and serum without or with minimal sample preparation (LOD range: 0.22-1.48 ng).

Keywords:  Complex matrices; Derivatization; Mass spectrometry; Quinones; Wooden-tip electrospray ionization

DOI:  https://doi.org/10.1016/j.talanta.2021.122912