bims-metlip 2020-04-12 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2020‒04‒12
forty papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

Metabolomics Benefits from Orbitrap GC-MS-Comparison of Low- and High-Resolution GC-MS.
Chemical Discovery in the Era of Metabolomics.
Pre-column derivatization method for determining phenylephrine in human plasma and its application in a pharmacokinetic study.
Quantitative Imprint Mass Spectrometry Imaging of Endogenous Ceramides in Rat Brain Tissue with Kinetic Calibration.
Development of a high-performance liquid chromatography-tandem mass spectrometric method for the determination of Methimazole in human blood matrices.
Temperature-programmed capillary-HPLC/atmospheric pressure chemical ionization-MS for analysis of fatty acid methyl esters.
Development and validation of a liquid chromatography-tandem mass spectrometry method to quantify metabolites of phthalates, including di-2-ethylhexyl terephthalate (DEHTP) and bisphenol A in human urine.
Rapid Determination of 13 Sedative Substances and Their Metabolites in Blood by LC-MS/MS.
Development and validation of a fast gas chromatography combustion isotope ratio mass spectrometry method for the detection of epiandrosterone sulfate in urine.
Lipid profile of fish species by liquid chromatography coupled to mass spectrometry and a novel linear retention index database.
Development of a high-coverage metabolome relative quantitative method for large-scale sample analysis.
Development of a simple and rapid LC-MS/MS method for the simultaneous quantification of five Alternaria mycotoxins in human urine.
Comparison of dimethylated and methylchlorinated amylose stationary phases, coated and covalently immobilized on silica, for the separation of some chiral compounds in supercritical fluid chromatography.
Comparative Routine Therapeutic Drug Monitoring of Mycophenolic Acid in human Plasma with HPLC-UV and Isotope Dilution LC-MS/MS.
Dual laser and desorption electrospray ionization mass spectrometry imaging using the same interface.
Characterization of wines with liquid chromatography electrospray ionization mass spectrometry: Quantification of amino acids via ionization efficiency values.
Differentiation of soluble aqueous humor metabolites in primary open angle glaucoma and controls.
Enhanced phospholipid isomer analysis by online photochemical derivatization and RPLC-MS.
Mouse single oocyte imaging by MALDI-TOF MS for lipidomics.
A Mass Spectral Library of Acylcarnitines Derived from Human Urine.
A background subtraction approach for determination of endogenous cortisol and 6β-hydroxycortisol in urine by UPLC-MS/MS with application in a within-day variability study in HIV-infected pregnant women.
Data normalization strategies in metabolomics: Current challenges, approaches, and tools.
MOTA: Network-Based Multi-Omic Data Integration for Biomarker Discovery.
Expanding the applicability of magnetic ionic liquids for multiclass determination in biological matrices based on dispersive liquid-liquid microextraction and HPLC with diode array detector analysis.
High-Performance Liquid Chromatography and Liquid Chromatography/Mass Spectrometry Studies on Stress Degradation Behavior of Sulfapyridine and Development of a Validated, Specific, Stability-Indicating HPLC Assay Method.
Rapid Determination of Ammonia in Biological Samples by GC-MS Derivatization Method.
Schirmer Paper Noninvasive Microsampling for Direct Mass Spectrometry Analysis of Human Tears.
ANNALS EXPRESS: A method for measuring glycerol-blanked triglyceride concentrations by using gas chromatography-isotope dilution mass spectrometry.
Development an UPLC-ESI-Orbitrap MS method for determination of xanthopurpurin in rat plasma and its application to pharmacokinetic study.
Chemical compositional analysis of soil fulvic acids using Fourier transform ion cyclotron resonance mass spectrometry.
Impurities identification and quantification for arginine vasopressin by liquid chromatography-high resolution mass spectrometry.
Micellar high performance liquid chromatographic method for separation and validation of two anti-hepatitis C- virus drugs in pure form, human plasma and human urine.
Enhancing the selectivity of liquid chromatography-mass spectrometry by using trilinear decomposition on LC-MS data: an application to three-way calibration of coeluting analytes in human plasma.
A Multivariate Metabolomics Method for Estimating Platelet Mitochondrial Oxygen Consumption Rates in Patients with Sepsis.
In vitro evaluation of reactive nature of E- and Z-guggulsterones and their metabolites in human liver microsomes using UHPLC-Orbitrap mass spectrometer.
Linear calibrations in chromatography: the incorrect use of ordinary least squares for determinations at low levels, and the need to redefine the limit of quantification with this regression model.
Investigation of inter- and intra-tumoral heterogeneity of glioblastoma using TOF-SIMS.
Detection system of the intracellular nitric oxide in yeast by HPLC with a fluorescence detector.
Breast cancer diagnosis by analysis of serum N-glycans using MALDI-TOF mass spectroscopy.
Steroid hormone analysis of adolescents and young women with polycystic ovarian syndrome and adrenocortical dysfunction using UPC2-MS/MS.

Metabolites. 2020 Apr 04. pii: E143. [Epub ahead of print]10(4):

Metabolomics Benefits from Orbitrap GC-MS-Comparison of Low- and High-Resolution GC-MS.

Daniel Stettin, Remington X Poulin, Georg Pohnert.

  The development of improved mass spectrometers and supporting computational tools is expected to enable the rapid annotation of whole metabolomes. Essential for the progress is the identification of strengths and weaknesses of novel instrumentation in direct comparison to previous instruments. Orbitrap liquid chromatography (LC)-mass spectrometry (MS) technology is now widely in use, while Orbitrap gas chromatography (GC)-MS introduced in 2015 has remained fairly unexplored in its potential for metabolomics research. This study aims to evaluate the additional knowledge gained in a metabolomics experiment when using the high-resolution Orbitrap GC-MS in comparison to a commonly used unit-mass resolution single-quadrupole GC-MS. Samples from an osmotic stress treatment of a non-model organism, the microalga Skeletonema costatum, were investigated using comparative metabolomics with low- and high-resolution methods. Resulting datasets were compared on a statistical level and on the level of individual compound annotation. Both MS approaches resulted in successful classification of stressed vs. non-stressed microalgae but did so using different sets of significantly dysregulated metabolites. High-resolution data only slightly improved conventional library matching but enabled the correct annotation of an unknown. While computational support that utilizes high-resolution GC-MS data is still underdeveloped, clear benefits in terms of sensitivity, metabolic coverage, and support in structure elucidation of the Orbitrap GC-MS technology for metabolomics studies are shown here.

Keywords:  Orbitrap Gas Chromatography–Mass Spectrometry (Orbitrap GC–MS), high-resolution mass spectrometry (HRMS); comparative metabolomics; diatom; instrument comparison; metabolite identification; osmotic stress

DOI:  https://doi.org/10.3390/metabo10040143
J Am Chem Soc. 2020 Apr 10.

Chemical Discovery in the Era of Metabolomics.

Miriam Sindelar, Gary J Patti.

Untargeted metabolomics aims to quantify the complete set of metabolites within a biological system, most commonly by liquid chromatography/mass spectrometry (LC/MS). Since nearly the inception of the field, compound identification has been widely recognized as the rate-limiting step of the experimental workflow. In spite of exponential increases in the size of metabolomic databases, which now contain experimental MS/MS spectra for over a half million reference compounds, chemical structures still cannot be confidently assigned to many signals in a typical LC/MS dataset. The purpose of this Perspective is to consider why identification rates continue to be low in untargeted metabolomics. One rationalization is that many naturally occurring metabolites detected by LC/MS are true "novel" compounds that have yet to be incorporated into metabolomic databases. An alternative possibility, however, is that research data do not provide database matches because of informatic artifacts, chemical contaminants, and signal redundancies. Increasing evidence suggests that, for at least some sample types, many unidentifiable signals in untargeted metabolomics result from the latter rather than new compounds originating from the specimen being measured. The implications of these observations on chemical discovery in untargeted metabolomics is discussed.

DOI: https://doi.org/10.1021/jacs.9b13198
Biomed Chromatogr. 2020 Apr 08. e4843

Pre-column derivatization method for determining phenylephrine in human plasma and its application in a pharmacokinetic study.

Li Li, Yong Wang, Guanglei Chen, Kelly Dong, Haifeng Song.

  In the present study, a rapid derivatization liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated to evaluate phenylephrine in human plasma. The plasma samples were processed to precipitate the proteins, followed by derivatization of the phenylephrine in the plasma with dansyl-chloride solution and extraction with methyl tert-butyl ether:n-hexane (2:1; (v/v)). The treated samples were analyzed on a Gemini C18 column with 3-min gradient elution, and sensitive detection was achieved with a Waters TQ-s. The method gave linear results over a concentration range from 0.020 to 10.0 ng/mL. The lower limit of quantification was 0.020 ng/mL. Intra- and inter-day precision was <15%, and accuracy was 95.0-105.3%. The validated LC-MS/MS method was successfully applied in the pharmacokinetic analysis of phenylephrine in Chinese subjects with common cold after a single-dose administration of 5, 10, or 20 mg phenylephrine. This pre-column derivatization method may also be applied in the analysis of endogenous hormones such as norepinephrine and adrenaline in a biological matrix.

Keywords:  derivatization; liquid chromatography-tandem mass spectrometry; pharmacokinetics; phenylephrine

DOI:  https://doi.org/10.1002/bmc.4843
Anal Chem. 2020 Apr 07.

Quantitative Imprint Mass Spectrometry Imaging of Endogenous Ceramides in Rat Brain Tissue with Kinetic Calibration.

Qian Wu, Stanislav S Rubakhin, Jonathan V Sweedler.

Quantitative mass spectrometry imaging (MSI) is an effective technique for determining the spatial distribution of molecules in a variety of sample types; however, the quality of the ion signals is related to the chemical and morphological properties of both the same matrix and targeted analyte(s). Issues may arise with the incorporation of standards into the sample at repeatable, well-defined concentrations, as well as with the extraction and incorporation of endogenous analytes versus standards from tissue into the matrix. To address these concerns, we combine imprint MSI (iMSI) with kinetic calibration, and use it to quantify lipids in rat brain tissue samples. Briefly, tissues were imprinted on slides coated with a dopamine-modified TiO2 monolith pretreated with analyte standards, resulting in the adsorption of endogenous analytes onto the coating and desorption of standards into the tissue. The incorporation of standards into the tissue enabled quantification of the measured analytes using kinetic calibration. Moreover, matrix effects were reduced, and the intensities of analyte standard signals became more uniform. The symmetry of the adsorption of endogenous ceramides and the desorption of ceramide standards suggests that the content of adsorbed endogenous ceramide can be determined by measuring the content of desorbed ceramide standard. Using kinetic calibration, endogenous ceramide concentrations were calculated for a range of dry and wet tissue imprinting conditions and compared with quantitative MSI using a standard spiking approach. We validated quantitative iMSI using liquid chromatography tandem mass spectrometry (LC-MS/MS) and found that the concentrations determined using iMSI compared with LC-MS/MS were in the range of 70 to 200% over the concentration range of endogenous ceramides; the correlation coefficient between iMSI and LC-MS/MS was over 0.9 (Pearson's r), while the recoveries via traditional standard spiking were between 200% and 5000% depending on the brain region and sample preparation conditions. .

DOI: https://doi.org/10.1021/acs.analchem.0c00392
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Mar 27. pii: S1570-0232(19)31356-X. [Epub ahead of print]1144 122083

Development of a high-performance liquid chromatography-tandem mass spectrometric method for the determination of Methimazole in human blood matrices.

Yusuke Ito, Hiroyuki Sakai, Hidenori Nagao, Ryota Suzuki, Masato Odawara, Kouichi Minato.

  Methimazole (MMI, 1-methyl-2-mercaptoimidazole) is widely used for the treatment of hyperthyroidisms. There are methods available for the measurement of MMI concentration in human serum or plasma from the past, but none meet the current regulatory standards for bioanalytical method validations. In this paper, we developed and validated a total MMI measurement method using liquid chromatography-tandem mass spectrometry (LC-MS/MS), a technique that conforms to current bioanalytical method validation. To form a free sulfhydryl group on MMI, sodium bisulfite was added to 50 µl of plasma or serum samples containing MMI before the derivatization step. The internal standard (MMI-D3) was spiked into samples, then these samples were derivatized with 4-chloro-7-nitro-2,1,3-benzoxadiazole. After derivatization, these samples were extracted by supported liquid extraction. Then, the organic solvent was evaporated and the residue was dissolved in 50% methanol and injected into the LC-MS/MS system. A calibration curve was plotted over the concentration range 1-1000 ng/mL (r2 = 0.999). The intra-day and inter-day precisions were less than 10.2% and 9.8%, respectively. The intra-day and inter-day accuracies were between 89.5% and 101.1%, and 96.0% and 99.7%, respectively. The long-term stability of samples showed good precision and accuracy. The validated method was successfully applied to determine serum total MMI concentration in Graves' disease patients after oral administration of 5, 10 or 15 mg MMI. The range of circulating total MMI concentrations was found to be between 2.69 and 304.27 ng/mL in this study. It was shown that the measured serum total MMI concentrations changed in a dose-dependent manner.

Keywords:  Derivatization; LC-MS/MS; Methimazole; Sample stability; Supported liquid extraction

DOI:  https://doi.org/10.1016/j.jchromb.2020.122083
J Sep Sci. 2020 Apr 11.

Temperature-programmed capillary-HPLC/atmospheric pressure chemical ionization-MS for analysis of fatty acid methyl esters.

Vladimír Vrkoslav, Barbora Rumlová, Timotej Strmeň, Josef Cvačka.

  A new capillary HPLC method with the atmospheric pressure chemical ionization-MS was developed for the analysis of fatty acid methyl esters and long-chain alcohols. The chromatographic separation was achieved using a Zorbax SB-C18 HPLC column (0.3 × 150 mm, 3.5 μm) with a mobile phase composed of acetonitrile and formic acid and delivered isocratically at a flow rate of 10 μL/min. The column temperature was programmed simply, using a common column oven. Good reproducibility of the temperature profile and retention times were achieved. The temperature programming during the isocratic HPLC run had a similar effect as a solvent gradient; it reduced retention times of later eluting analytes and improved their detection limits. Two atmospheric pressure chemical ionization sources of the MS detector were compared: an enclosed conventional ion source and an in-house made ion source with a glass microchip nebulizer. The enclosed source provided better detectability of saturated fatty acid methyl esters and made it possible to determine the double bond positions using acetonitrile-related adducts, while the open chip-based source provided better analytical figures of merit for unsaturated fatty acid methyl esters. Temperature-programmed capillary HPLC is a promising method for analyzing neutral lipids in lipidomics and other applications. This article is protected by copyright. All rights reserved.

Keywords:  double bond; lipidomics; lipids; temperature programming

DOI:  https://doi.org/10.1002/jssc.201901235
Rapid Commun Mass Spectrom. 2020 Apr 04.

Development and validation of a liquid chromatography-tandem mass spectrometry method to quantify metabolites of phthalates, including di-2-ethylhexyl terephthalate (DEHTP) and bisphenol A in human urine.

Gianfranco Frigerio, Laura Campo, Rosa Mercadante, Patricia Martín Santos, Pasquale Missineo, Elisa Polledri, Silvia Fustinoni.

RATIONALE: Several phthalates and bisphenol A are endocrine-disrupting chemicals (EDCs). Recently, their use has been partially restricted, and less toxic compounds, such as di-2-ethylhexyl terephthalate (DEHTP), have been placed on the market. The aim of this work was to develop and validate a method for the simultaneous quantitation of bisphenol A and urinary metabolites of phthalates, including DEHTP.METHODS: An isotopic dilution HPLC/ESI-MS/MS method for the determination of bisphenol A (BPA), monobenzyl phthalate (MBzP), mono-2-ethyl-5-carboxypentyl phthalate (MECPP), mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP), mono-2-ethyl-5-hydroxyhexyl terephthalate (MEHHTP), monoethyl phthalate (MEP), and mono-n/i-butyl phthalates (MnBP/MiBP) in human urine was developed. A complete validation was carried out and the method was applied to 36 non-occupationally exposed adults.
RESULTS: Limits of quantitation ranged from 0.02 (MECPP) to 1 μg/L (MnBP and MiBP). Relative standard deviations below 10% indicated a suitable precision; accuracy, evaluated using a standard reference material, ranged from 74.3% to 117.5%; isotopically labelled internal standards were suitable for correcting the matrix effect. The accuracy was confirmed by the successful participation in an external verification exercise. However, for terephthalates, the validation was incomplete due to the lack of reference material and external validation. Levels of the investigated chemicals in subjects were in line with those previously reported.
CONCLUSIONS: An LC/MS/MS assay for the simultaneous measurement of BPA and phthalate metabolites in human urine was developed and validated; it is useful to investigate exposure in epidemiological studies involving the general population.

DOI: https://doi.org/10.1002/rcm.8796
Fa Yi Xue Za Zhi. 2020 Feb;36(1): 45-51

Rapid Determination of 13 Sedative Substances and Their Metabolites in Blood by LC-MS/MS.

Y T Shi, H J Wang, J W Wang, J Zheng, Y H Wu.

  Abstract: Objective To establish an analysis method for simultaneous determination of 13 sedative substances and their metabolites in blood by liquid-liquid extraction and liquid chromatography-tandem mass spectrometry （LC-MS/MS） technology and to apply the method to actual cases. Methods The samples were extracted with ethyl acetate after an internal standard was added. The extract was condensed until it was nearly dry and then its residues were dissolved with methanol, filtered through 0.22 μm filter and finally determined. The 13 sedative substances and their metabolites were separated through the C18 chromatographic column, then gradient elution was performed on them with methanol and 20 mmol/L ammonium formate （containing 0.1% formic acid） solution. After that, they were determined in the electrospray positive ion mode and quantified by internal standard method. Results The 13 sedative substances and their metabolites in blood showed good linearity in the range of 5-200 μg/L with correlation coefficients ranging from 0.990 3 to 0.999 8. The detection limits were 0.1-1.0 μg/L. Recovery rates of sedative substances were in the range of 71.2%-93.4% when solutions with concentrations of 10, 50 and 200 μg/L were added. The deviations of intra-day and inter-day relative standard deviations （RSD） were not more than 8.6%. Accuracies （bias） were within ±9.8%. Conclusion This method is rapid, simple, effective and sensitive, and can be applied to analysis of 13 sedative substances and their metabolites in blood in forensic toxicology.

Keywords:  forensic toxicology; hypnotics and sedative; chromatography, liquid; mass spectrometry; liquid-liquid extraction; blood

DOI:  https://doi.org/10.12116/j.issn.1004-5619.2020.01.010
Drug Test Anal. 2020 Apr 08.

Development and validation of a fast gas chromatography combustion isotope ratio mass spectrometry method for the detection of epiandrosterone sulfate in urine.

Laurie De Wilde, Pieter Van Renterghem, Peter Van Eenoo, Michaël Polet.

  In doping control, to confirm the exogenous origin of exogenously administered anabolic androgenic steroids (AAS), a gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) analysis is performed. Recently published work suggests epiandrosterone sulfate (EpiAS) as a promising IRMS target compound for the detection of AAS, capable of prolonging the detection window. However, EpiAS is only excreted in urine in its sulfoconjugated form while all other IRMS target compounds are excreted glucuronidated, meaning that EpiAS cannot be incorporated in the existing IRMS methods. A separate extensive sample preparation needs to be performed on this compound with a different hydrolysis and extraction procedure and a different liquid chromatography (LC) clean-up. The current work presents a new, fast and easy to implement EpiAS IRMS method. The approach was based on the direct GC analysis of non-hydrolysed EpiAS, making the solid phase extraction, hydrolysis and acetylation step redundant. Sample preparation consisted of a simple liquid liquid extraction, followed by LC fraction collection. A population study was performed to check the compliance with the criteria drafted by the World Anti-Doping Agency (WADA). To verify the applicability of the developed approach, the method was applied to the samples of four administration studies (i.e., dehydroepiandrosterone (DHEA), testosterone gel (T gel), androstenedione (ADION) and intramuscular testosterone undecanoate. In contrast to previously published data, the strength of EpiAS as target compound and the prolongation of the detection window in comparison with the conventional IRMS target compounds was less pronounced.

Keywords:  Doping; Epiandrosterone sulfate; Isotope ratio mass spectrometry; Steroids; Urine

DOI:  https://doi.org/10.1002/dta.2801
J Sep Sci. 2020 Apr 07.

Lipid profile of fish species by liquid chromatography coupled to mass spectrometry and a novel linear retention index database.

Francesca Rigano, Marianna Oteri, Giuseppe Micalizzi, Domenica Mangraviti, Paola Dugo, Luigi Mondello.

  In a previous paper, Rigano et al. established a new linear retention index system for the identification of triacylglycerols by LC methods only on the basis of the retention behavior and independently from many experimental parameters. In that work, a database of 209 compounds was built, but only 54 of them, typical of vegetable oils, were confirmed by MS. The aim of the present research is to extend the applicability of the novel approach to more complex samples, such as fish lipid extracts, and assess the complementarity between MS and retention information to achieve univocal identification. To this purpose, a new software was implemented to make the identification process easy and automatic as in GC-MS where the retention index filter is added in the spectral search to discriminate between compounds with similar MS spectra. A total of 69 species were identified and, thanks to their baseline separation obtained by an ultra HPLC method, a semi-quantification was also performed. The species under investigation were Dicentrarchus labrax, coming from aquaculture and the wild. Some differences in their native lipid composition were observed, probably related to a different diet. A major number of samples would be necessary to confirm such a preliminary finding. This article is protected by copyright. All rights reserved.

Keywords:  linear retention Index; lipids; sea bass; spectral library; triacylglycerols

DOI:  https://doi.org/10.1002/jssc.202000171
Anal Chim Acta. 2020 May 01. pii: S0003-2670(20)30256-7. [Epub ahead of print]1109 44-52

Development of a high-coverage metabolome relative quantitative method for large-scale sample analysis.

Zhi Zhou, Yanhua Chen, Yang Gao, Nan Bi, Xiaofei Yue, Jiuming He, Ruiping Zhang, Luhua Wang, Zeper Abliz.

  The development of quantitative metabolomics approaches for future standardized and translational applications has become increasingly important. Data-independent targeted quantitative metabolomics (DITQM) is a newly proposed method providing ion pair information on 1324 metabolites. However, the quantification of more than 1000 metabolites in large sample sizes has still not been implemented. In this study, on the basis of the DITQM concept, scheduled multiple reaction monitoring (MRM) methods for both high-abundant and low-abundant metabolites were established to broaden the quantification coverage, and an open-source program "Quanter_1.0" was coded to facilitate efficient data handling. Our results demonstrated that 1015 metabolites in human plasma met the quantitative requirements and could be relatively determined in an effective manner. The method was then applied to a large-scale sample study of lung cancer consisting of three distinct analytical batches. It was obvious that data quality that originated from quantitative metabolomics was improved, with substantially lower intra- and inter-batch data variation, resulting in a more effective multivariate statistical model. Finally, 26 potential biomarkers of lung cancer were discovered. Collectively, our approach provides a promising tool for quantitative metabolomics research involving large-scale sample sizes and clinical application.

Keywords:  High-coverage analysis; Large-scale sample size; Liquid chromatography-tandem mass spectrometry; Relative quantitative metabolomics

DOI:  https://doi.org/10.1016/j.aca.2020.02.049
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Mar 30. pii: S1570-0232(20)30018-0. [Epub ahead of print]1144 122096

Development of a simple and rapid LC-MS/MS method for the simultaneous quantification of five Alternaria mycotoxins in human urine.

Xiaoting Qiao, Jing Zhang, Yunjia Yang, Jie Yin, Hong Li, Yang Xing, Bing Shao.

  Alternaria mycotoxins, such as tenuazonic acid (TeA), altenuene (ALT), alternariol (AOH), tentoxin (TEN) and alternariol monomethyl ether (AME) are frequently found in foods and may pose a potential risk to human health. Human biomonitoring can help measure our exposure to these mycotoxins, and help us determine if the exposure is changing over time. In this study, a simple liquid-liquid extraction sample preparation procedure followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous analysis of five Alternaria mycotoxins in human urine. High recoveries (92.7-103.2%) were obtained for all the tested mycotoxins with relative standard deviations (RSDs, %) of less than 6.4%. The limits of quantification (LOQs) for the analytes in urine ranged from 0.001 to 0.05 ng/mL. The method was successfully applied to investigate the levels of five Alternaria mycotoxins from 135 volunteers. In all of the samples, at least one Alternaria mycotoxin was detected. TeA, AME and AOH were the predominant Alternaria mycotoxins, and the detection rates were 85.9%, 96.3% and 51.9%, respectively.

Keywords:  Alternaria mycotoxin; Human urine; Liquid-liquid extraction; UPLC-MS/MS; β-glucuronidase/arylsulfatase

DOI:  https://doi.org/10.1016/j.jchromb.2020.122096
J Chromatogr A. 2020 Mar 18. pii: S0021-9673(20)30265-X. [Epub ahead of print] 461053

Comparison of dimethylated and methylchlorinated amylose stationary phases, coated and covalently immobilized on silica, for the separation of some chiral compounds in supercritical fluid chromatography.

Anca-Elena Dascalu, Alina Ghinet, Bezhan Chankvetadze, Emmanuelle Lipka.

  The chromatographic properties of a new coated amylose tris(3-chloro-5-methylphenylcarbamate) were evaluated in supercritical fluid chromatography for the separation of enantiomers of chiral 1-aryl-5-aryl-pyrrolidin-2-one derivatives, potential anticancer agents, and some commercial drugs. The mobile phase consisted of CO2-modifier mixtures with 30% of either methanol or ethanol, the flow rate was 3 mL/min. The column oven temperature was 40 °C and the outlet pressure was 15 MPa, in order to limit the compressibility of the CO2, thus limiting density variation along the column. The obtained results were then compared to those observed toward 3 other stationary phases: the coated amylose tris(3,5-dimethylphenylcarbamate), the immobilized amylose tris(3,5-dimethylphenylcarbamate) and the coated amylose tris(5-chloro-2-methylphenylcarbamate). It was shown that the new coated amylose tris(3-chloro-5-methylphenylcarbamate) was the most retentive column whatever the studied compounds, particularly for thalidomide and omeprazole with retention factors up to 73.3 and 29.5for the second enantiomer, respectively. Concerning the enantioselectivity, even most of the compounds are separated on all the four columns, the coated amylose tris(3-chloro-5-methylphenylcarbamate) allows the best resolution for most of the ten studied analytes (except omeprazole for which the resolution values are equal to 7.8 and 9.7 on the coated amylose tris(3-chloro-5-methylphenylcarbamate) and amylose tris(3,5-dimethylphenylcarbamate), respectively). Acting in complementary ways, the two chlorinated stationary phases permitted the complete separation of enantiomers of nine compounds out of the ten.

Keywords:  1-aryl-5-aryl-pyrrolidin-2-ones; Amylose tris(3,5-dimethylphenylcarbamate); Amylose tris(3-chloro-5-methylphenylcarbamate); Amylose tris(5-chloro-2-methylphenylcarbamate); Chirality

DOI:  https://doi.org/10.1016/j.chroma.2020.461053
Clin Lab. 2020 Apr 01. 66(4):

Comparative Routine Therapeutic Drug Monitoring of Mycophenolic Acid in human Plasma with HPLC-UV and Isotope Dilution LC-MS/MS.

Michael Paal, Katharina Habler, Bernd Northoff, Michael Vogeser.

BACKGROUND: Therapeutic drug monitoring (TDM) of the immunosuppressant mycophenolic acid (MPA) is especially recommended for the control of personalized immunosuppressive therapy. Various test systems are available for MPA monitoring, including high performance liquid chromatography combined with UV detection (HPLC-UV) and isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS).METHODS: In the present work, commercially available kits for MPA monitoring with HPLC-UV and ID-LC-MS/ MS were subjected to routine use TDM. Following method verification according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, 105 native sample duplicates from patients under therapy with mycophenolate mofetil were assayed with both procedures for comparative testing.
RESULTS: Using bi-level quality controls, the estimate of repeatability, within-laboratory imprecision and inaccuracy were ≤ 5.18%, ≤ 5.95% and ≤ 3.86% for all MPA measurements. Weighted Deming regression analysis yielded a slope of 0.93, an intercept of 0.04, and Pearson's correlation coefficient (r) of 0.99, while Bland-Altman analysis showed a combined relative bias of 4.93% (± 1.96 SD: -16.68 - 26.54%). Plasma samples taken from a patient re-peatedly showed the presence of an interferent only in HPLC-UV analysis.
CONCLUSIONS: Based on these results, HPLC-UV testing can be considered suitable for routine TDM of MPA in the clinical setting with high precision. Due to the risk of unforeseen analytical interference in ever-increasing multimorbidity and polypharmacy, highly selective ID-LC-MS/MS methodology should be given preference over HPLC-UV analysis whenever feasible.

DOI: https://doi.org/10.7754/Clin.Lab.2019.190820
Anal Chem. 2020 Apr 10.

Dual laser and desorption electrospray ionization mass spectrometry imaging using the same interface.

Lauren Katz, Michael Woolman, Francis Talbot, Siham Amara-Belgadi, Megan Wu, Sara Tortorella, Sunit Das, Howard J Ginsberg, Arash Zarrine-Afsar.

For a more comprehensive characterization of molecular heterogeneities of matter, multi-modal mass spectrometry imaging must be developed to take advantage of the complementarity of information available through different ionization mechanisms. We report the design, implementation, and performance validation of a laser desorption imaging interface comprised of add-on components that adapt a commercial Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging interface for dual imaging of Picosecond Infrared Laser Mass Spectrometry (PIRL-MS) with DESI-MS. The interface utilizes hardware elements and data analysis pipelines already established for DESI-MS imaging, and was further validated in cancer margin assessments using human medulloblastoma cancers. The PIRL-MS images were robust and reproducible across multiple experimental runs on independently prepared xenograft tumours, and could be segmented into cancer and healthy regions in concordance with pathology using a variety of supervised and unsupervised clustering methods. The spectral quality and complexity obtained with this interface were examined with infiltrating and non-infiltrating tumours, and were comparable to other mass spectrometry analysis interfaces. The average PIRL-MS spectra from spatially resolved images could be used for robust cancer m/z model building to classify medulloblastoma cancer from healthy tissue without any misclassifications, an observation that held true over close to 70 sampling data points. While the unsupervised spectral analysis methods suggested a slight suppression of signal in the phospholipid range compared to the hand-held configuration, these changes were insufficient to hamper utility in cancer margin assessment with spatially resolved data obtained with our interface. Dual PIRL-MS and DESI-MS imaging of consecutive sections, as suggested by multivariate loading plots, revealed highly complementary molecular information with m/z values identifiable with one desorption method sufficient to reveal cancer regions being absent in another, further emphasising the need for effective hardware and software interfaces for dual mass spectrometry imaging.

DOI: https://doi.org/10.1021/acs.analchem.9b05340
J Chromatogr A. 2020 Mar 06. pii: S0021-9673(20)30218-1. [Epub ahead of print] 461012

Characterization of wines with liquid chromatography electrospray ionization mass spectrometry: Quantification of amino acids via ionization efficiency values.

Artur Gornischeff, Anneli Kruve, Riin Rebane.

  Quantification of analysis results for the suspect and non-targeted screening is essential for obtaining meaningful insight from the measurements. Ionization efficiency predictions is a possible approach to enable quantitation without standard substances. This is, however, especially challenging for the analysis carried out by combining the full scan mode either with fragmentation experiments in data-dependent or data-independent acquisition mode. Here we investigate the correlation of ionization efficiency values measured in full scan mode with the response factors measured in multiple reaction monitoring (MRM) mode for derivatized amino acids. We observe good correlation (R2 of 0.80) for 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids. This encourages the use of the measured ionization efficiency values to estimate amino acid concentrations in different beverages. We apply the measured ionization efficiency values for estimating the concentration of amino acids for measurements done both in full scan as well as in MRM mode in wines and beers. We show that the calculated concentrations are in very good correlation with measured values (R2 of 0.71 to 1.00). The method possesses average trueness of 70.5% and shows an insignificant matrix effect.

Keywords:  Amino acids; Derivatization; Ionization efficiency; Liquid chromatography; Mass spectrometry; Standard-free quantification

DOI:  https://doi.org/10.1016/j.chroma.2020.461012
Exp Eye Res. 2020 Apr 01. pii: S0014-4835(19)30870-X. [Epub ahead of print]194 108024

Differentiation of soluble aqueous humor metabolites in primary open angle glaucoma and controls.

Ciara Myer, Jordan Perez, Leila Abdelrahman, Roberto Mendez, Ram B Khattri, Anna K Junk, Sanjoy K Bhattacharya.

  We report an analysis of the aqueous humor (AH) metabolome of primary open angle glaucoma (POAG) in comparison to normal controls. The AH samples were obtained from human donors [control (n = 35), POAG (n = 23)]. The AH samples were subjected to one-dimensional 1H nuclear magnetic resonance (NMR) analyses on a Bruker Avance 600 MHz instrument with a 1.7 mM NMR probe. The same samples were then subjected to isotopic ratio outlier analysis (IROA) using a Q Exactive orbitrap mass spectrometer after chromatography on an Accela 600 HPLC. Clusterfinder Build 3.1.10 was used for identification and quantification based on long-term metabolite matrix standards. In total, 278 metabolites were identified in control samples and 273 in POAG AH. The metabolites identified were fed into previously reported proteome and genome information and the OmicsNet interaction network generator to construct a protein-metabolite interactions network with an embedded protein-protein network. Significant differences in metabolite composition in POAG compared to controls were identified indicating potential protein/gene pathways associated with these metabolites. These results will expand our previous understanding of the impeded AH metabolite composition, provide new insight into the regulation of AH outflow, and likely aid in future AH and trabecular meshwork multi-omics network analyses.

Keywords:  Aqueous humor; Glaucoma; IROA; Metabolomics; NMR; POAG

DOI:  https://doi.org/10.1016/j.exer.2020.108024
Anal Chem. 2020 Apr 09.

Enhanced phospholipid isomer analysis by online photochemical derivatization and RPLC-MS.

Wenpeng Zhang, Bing Shang, Zheng Ouyang, Yu Xia.

Mapping the complete molecular composition of a lipidome is considered as an important goal of lipidomics for unraveling pathways and mechanisms behind lipid homeostasis. Conventional dissociation methods of mass spectrometry (MS) usually cannot give detailed structural information of lipids such as locations of carbon-carbon double bond (C=C) in acyl chains. Double bond derivatization via the Paternò-Büchi (PB) reaction has been demonstrated as a simple and highly efficient method for identification of C=C locations of different classes of lipids when paired with tandem mass spectrometry (MS/MS). In this work, reverse-phase lipid chromatography (RPLC)-MS was coupled with online PB reaction to achieve enhanced analysis of isomers and isobars of phospholipids. A new acetone-containing mobile phase was developed, which showed good elution performance for the separation of phospholipids by C18 columns. An improved flow microreactor was developed, enabling online derivatization of phospholipid C=C in 20 s. The workflow of RPLC-PB-MS/MS was developed and optimized for identification of C=C locations in isobaric ether-linked and diacyl phospholipids, 13C isobars, and acyl chain isomers in biological lipid extracts. Separation and identification of C=C locations of cis/trans phospholipid isomers were achieved for lipid standards. The incorporation of the PB reaction into RPLC-MS workflow enabled analysis of phospholipid isomers and isobars with high confidence, demonstrating its potential for high-throughput phospholipid identification from complex mixtures.

DOI: https://doi.org/10.1021/acs.analchem.0c00690
Cytotechnology. 2020 Apr 09.

Mouse single oocyte imaging by MALDI-TOF MS for lipidomics.

Anna Bodzon-Kulakowska, Roberta Arena, Przemyslaw Mielczarek, Kinga Hartman, Paulina Kozoł, Ewa Gibuła-Tarlowska, Tomasz P Wrobel, Łukasz Gąsior, Zbigniew Polański, Grazyna E Ptak, Piotr Suder.

  Reproductive cells are a very special kind of material for the analysis. Depending on the species, their dimensions allow for the application of mass spectrometry imaging-based techniques to receive a reasonable data for interpretation of their condition without any additional sample preparation steps, except for typical sample preparation characteristic for IMS protocols. A comparison between lipid profiles of oocytes could answer the question of the overall quality of the cells in the function of time or conditions of storage. Even tiny differences in the lipid profiles, but still detectable by bioinformatic analysis, could be crucial for the estimation of the conditions of the cells in various stages of development or aging. In our study, MALDI-TOF/TOF MSI was used to analyze and visualize the single oocytes. We deposited the cells on the transparent indium-tin-oxide (ITO) glass and marked their positions, which allowed for the fast localization of the cells and precise laser targeting in the ion source. We also optimized the usage of different MALDI matrices and different approaches. The proposed way of measurement allows analyzing quite a significant quantity of oocytes in a reasonably short time. During the analysis, the lipid composition of the single cell was successfully estimated in a conventional usage of the MALDI ion source, and the localization of lipids was confirmed by imaging mass spectrometry (IMS) analysis. The observed quantity of the lipids allowed for the application of the LIFT™ technique to obtain MS/MS spectra sufficient for lipids' unambiguous identification. We hope that our idea of the oocyte analysis will help to elucidate chemical changes that accompany different processes in which oocytes are involved. There could be such fascinating phenomena as the oocyte maturation, changes in the lipid components during their storage, and much more.

Keywords:  MALDI-imaging; Mink oocyte; Mouse oocyte; Sample preparation methods; Single cell analysis

DOI:  https://doi.org/10.1007/s10616-020-00393-9
Anal Chem. 2020 Apr 09.

A Mass Spectral Library of Acylcarnitines Derived from Human Urine.

Xinjian Yan, Sanford P Markey, Ramesh Marupaka, Qian Dong, Brian T Cooper, Yuri A Mirokhin, William Edward Wallace, Stephen E Stein.

We describe the creation of a mass spectral library of acylcarnitines and conjugated acylcarnitines from the LC-MS/MS analysis of six NIST urine reference materials. To recognize acylcarnitines, we conducted in-depth analyses of fragmentation patterns of acylcarnitines and developed a set of rules, derived from spectra in the NIST17 Tandem MS Library and those identified in urine, using the newly developed hybrid search method. Acylcarnitine tandem spectra were annotated with fragments from carnitine and acyl moieties as well as neutral loss peaks from precursors. Consensus spectra were derived from spectra having similar retention time, fragmentation pattern and the same precursor m/z and collision energy. The library contains 157 different precursor masses, 586 unique acylcarnitines, and 4,332 acylcarnitine consensus spectra. Furthermore, from spectra that partially satisfied the fragmentation rules of acylcarnitines, we identified 125 conjugated acylcarnitines represented by 987 consensus spectra, which appear to originate from Phase II biotransformation reactions. To our knowledge, this is the first report of conjugated acylcarnitines. The mass spectra provided by this work may be useful for clinical screening of acylcarnitines as well as for studying relationships among fragmentation patterns, collision energies, structures, and retention times of acylcarnitines. Further, these methods are extensible to other classes of metabolites.

DOI: https://doi.org/10.1021/acs.analchem.0c00129
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Mar 19. pii: S1570-0232(19)31814-8. [Epub ahead of print]1144 122074

A background subtraction approach for determination of endogenous cortisol and 6β-hydroxycortisol in urine by UPLC-MS/MS with application in a within-day variability study in HIV-infected pregnant women.

Jhohann Richard de Lima Benzi, Fernanda de Lima Moreira, Maria Paula Marques, Geraldo Duarte, Guilherme Suarez-Kurtz, Vera Lucia Lanchote.

  Different methods have been used for CYP3A phenotyping, such as probe drugs or the urinary index 6β-hydroxycortisol/cortisol ratio (6β-OHF:C). This work describes a simple and affordable method for the simultaneous determination of the endogenous compounds cortisol and 6β-hydroxycortisol in urine using a background subtraction approach. The method was applied to investigate the CYP3A activity in HIV-infected pregnant women (n = 9) in the third trimester and postpartum periods. Also, the within-day variability in the 6β-OHF:C index was also evaluated. The sample preparation consists of a pre-cleanup with acetonitrile followed by liquid-liquid extraction with ethyl acetate. The analytes were resolved by employing an Acquity UPLC®BEH C18 column with a mobile phase that consisted of a mixture of acetonitrile containing 0.1% formic acid and 0.1% formic acid in gradient mode. The method presented linearities of 1-1.000 ng/mL and 2-1.000 ng/mL for C and 6β-OHF, respectively, and presented acceptable precision and accuracy. Qualitative and quantitative matrix effects tests were also performed. A high 6β-OHF:C within-day variability was observed in both phases. In the third trimester period, the 6β-OHF:C ranged from 2.57 to 51.69, with a mean ± standard deviation (SD) of 15.12 ± 5.41 (n = 9). Similar values were obtained in the postpartum period, with 6β-OHF:C ranging from 3.48 to 44.54 with a mean ± SD of 14.37 ± 5.73 (n = 7). Even though the 6β-OHF:C is a non-invasive index for CYP3A phenotyping, its use is susceptible to high within-day variability.

Keywords:  6β-hydroxycortisol; Background subtraction; Cortisol; Pregnancy; UPLC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2020.122074
Eur J Mass Spectrom (Chichester). 2020 Apr 10. 1469066720918446

Data normalization strategies in metabolomics: Current challenges, approaches, and tools.

Biswapriya B Misra.



Keywords:  Metabolite; normalization; quantification; sample; software

DOI:  https://doi.org/10.1177/1469066720918446
Metabolites. 2020 Apr 08. pii: E144. [Epub ahead of print]10(4):

MOTA: Network-Based Multi-Omic Data Integration for Biomarker Discovery.

Ziling Fan, Yuan Zhou, Habtom W Ressom.

  The recent advancement of omic technologies provides researchers with the possibility to search for disease-associated biomarkers at the system level. The integrative analysis of data from a large number of molecules involved at various layers of the biological system offers a great opportunity to rank disease biomarker candidates. In this paper, we propose MOTA, a network-based method that uses data acquired at multiple layers to rank candidate disease biomarkers. The networks constructed by MOTA allow users to investigate the biological significance of the top-ranked biomarker candidates. We evaluated the performance of MOTA in ranking disease-associated molecules from three sets of multi-omic data representing three cohorts of hepatocellular carcinoma (HCC) cases and controls with liver cirrhosis. The results demonstrate that MOTA allows the identification of more top-ranked metabolite biomarker candidates that are shared by two different cohorts compared to traditional statistical methods. Moreover, the mRNA candidates top-ranked by MOTA comprise more cancer driver genes compared to those ranked by traditional differential expression methods.

Keywords:  differential network; metabolomics; multi-omic integration; transcriptomics

DOI:  https://doi.org/10.3390/metabo10040144
J Sep Sci. 2020 Apr 10.

Expanding the applicability of magnetic ionic liquids for multiclass determination in biological matrices based on dispersive liquid-liquid microextraction and HPLC with diode array detector analysis.

Camila Will, Eduarda Omena, Gabriela Corazza, Gabrieli Bernardi, Josias Merib, Eduardo Carasek.

  Monitoring biological samples at trace levels of chemicals from anthropogenic actions such as pesticides, pharmaceuticals and hormones has become a very important subject. This work describes a method for the determination of 8 compounds of different chemical classes in human urine samples. Dispersive liquid-liquid microextraction based on magnetic ionic liquids was used as the sample preparation procedure. The main parameters of the method, such as sample dilution, type and volume of disperser solvent, amount of magnetic ionic liquids, extraction time and pH were optimized by univariate and multivariate procedures. Validation was performed using a urine sample of a male volunteer in order to obtain a calibration curve and the main analytical parameters of merit such as limits of detection and quantification. Values varied from 3.0 μg L-1 to 7.5 μg L-1 and from 10 to 25 μg L-1 , respectively. Satisfactory precisions of 21% for intraday (n = 3) and 16% for interday (n = 9) were achieved. Accuracy was evaluated by relative recovery assays using different urine samples and ranged from 75 to 130%. Robustness was assured by the Lenth method. The validated procedure was applied to 5 urine samples from different volunteers and the hormone estrone was found in one sample. This article is protected by copyright. All rights reserved.

Keywords:  hormones; magnetic ionic liquids; multiclass; pesticides; pharmaceuticals; urine

DOI:  https://doi.org/10.1002/jssc.202000143
Assay Drug Dev Technol. 2020 Apr 08.

High-Performance Liquid Chromatography and Liquid Chromatography/Mass Spectrometry Studies on Stress Degradation Behavior of Sulfapyridine and Development of a Validated, Specific, Stability-Indicating HPLC Assay Method.

Bhupinder Kapoor, Reena Gupta, Monica Gulati, Sachin Kumar Singh, Gopal Lal Khatik, Manish Chawla, Krishna Veni Nagappan, Rubiya Khursheed, Rajan Kumar.

  The objective of the current investigation was to develop a simple, rapid, and stability-indicating high-performance liquid chromatography method and to study the degradation behavior of sulfapyridine (SP) under different International Conference on Harmonization (ICH)-recommended conditions. The chromatographic method was developed using C18 (250 × 4.6 mm, 5 μ) column, and mobile phase consisting of acetonitrile-0.1% formic acid (30:70 v/v) at ambient temperature, at a flow rate of 1 mL/min. The elution was monitored at 265 nm using a photodiode array detector. The developed method was subsequently validated as per ICH Q2 (R1) guidelines. The retention time of SP was observed as 4.56 min with the linearity range between 2 to 10 μg/mL. Limit of detection and limit of quantitation for SP were 0.115 and 0.35 μg/mL, respectively. Forced degradation studies were carried out on bulk samples of SP using prescribed acidic, basic, oxidative, thermal, and photolytic conditions. Extent of degradation in 0.1 M hydrochloric acid and under photolytic conditions was found to be 21.56% and 28.57%, respectively. The degradation products formed in stress conditions were identified by liquid chromatography-mass spectrometry (LC-MS). The utility of the method was verified by quantification of SP in different laboratory-made pharmaceutical preparations. The proposed method could be successfully used to quantify SP in different pharmaceutical dosage forms.

Keywords:  HPLC; LCMS; forced degradation study; stability-indicating method; sulfapyridine

DOI:  https://doi.org/10.1089/adt.2019.959
Fa Yi Xue Za Zhi. 2020 Feb;36(1): 41-44

Rapid Determination of Ammonia in Biological Samples by GC-MS Derivatization Method.

H Y Yuan, J T Liu, E Y Xu, L N Gao.

  Abstract: Objective To establish a qualitative and quantitative method to determine ammonia in biological samples by gas chromatography-mass spectrometry （GC-MS）. Methods A heptafluorobutyryl chloride derivatization method was used. GC-MS was used for determination. The effects of different pH conditions, derivatization temperature, time and different extraction solvents on the test results were investigated. The pretreatment conditions were optimized. Results This method could accurately detect the ammonia content in blood, and the limit of detection was determined to be 0.1 μg/mL. The target component showed good linearity in the range of 0.5-200.0 μg/mL （R2=0.987 7）. The relative standard deviation range of intra-day precision was 2.59%-3.88%. The relative standard deviation range of inter-day precision was 3.21%-3.76%. Conclusion The method showed good sensitivity, stability and specificity, therefore can be used for forensic toxicology analysis and clinical biochemical detection.

Keywords:  forensic toxicology; gas chromatography-mass spectrometry; ammonia; plasma; derivatization

DOI:  https://doi.org/10.12116/j.issn.1004-5619.2020.01.009
Anal Chem. 2020 Apr 06.

Schirmer Paper Noninvasive Microsampling for Direct Mass Spectrometry Analysis of Human Tears.

Ya-Nan Yao, Dandan Di, Zi-Cheng Yuan, Lin Wu, Bin Hu.

Rapid and sensitive detection of metabolites and chemical residues in human tears is highly beneficial for understanding eye health. In this study, Schirmer paper was used for noninvasive microsampling of human tears, and was then performed paper spray mass spectrometry (PSMS) for direct analysis of human tears. Schirmer PSMS was successfully used for rapid diagnosis of dry-eye syndrome by detecting the volume and metabolites of human tears. Drugs of abuse, therapeutic drugs, and pharmacodynamics in human tears were also investigated by Schirmer PSMS. Furthermore, specific markers of environmental exposures in the air to human eyes, including volatile organic compounds, aerosol, and smoke, were unambiguously sampled and detected in human tears using Schirmer PSMS. Excellent analytical performances were achieved, including single-use, low-sample consumption (1.0 μL), rapid analysis (the whole analytical procedure completed within 3 min), high sensitivity (absolute limit of detection less than or equal to 0.5 pg, S/N ≥ 3), good reproducibility (relative standard deviation less than 10 %, n = 3), and accurate quantitation (average deviation less than 3 %, n = 3). Overall, our results showed that Schirmer PSMS is a highly effective method for direct tear analysis and is expected to be a convenient tool for human tear analysis in significant clinical applications.

DOI: https://doi.org/10.1021/acs.analchem.9b05078
Ann Clin Biochem. 2020 Apr 07. 4563220921884

ANNALS EXPRESS: A method for measuring glycerol-blanked triglyceride concentrations by using gas chromatography-isotope dilution mass spectrometry.

Isao Koyama, Hironori Imano, Masakazu Nakamura, Akihiko Kitamura, Masahiko Kiyama, Yoshihiro Miyamoto, Hiroyasu Iso.

DOI: https://doi.org/10.1177/0004563220921884
Biomed Chromatogr. 2020 Apr 04. e4838

Development an UPLC-ESI-Orbitrap MS method for determination of xanthopurpurin in rat plasma and its application to pharmacokinetic study.

De-En Han, Yanmei Shi, Ping Tian, Hengchao Wei, Mingsan Miao, Xiu-Min Li.

  A rapid and sensitive method was developed and validated for the quantitative determination of xanthopurpurin (XPP) in rat plasma by UPLC-ESI-Orbitrap mass spectrometry (UPLC-ESI-Orbitrap MS). XPP inhibits IgE production and prevents peanut-induced anaphylaxis. The XPP and emodin (IS) were determined in negative ion mode with m/z 239.0350→211.0400 and 269.0455→241.0507, respectively. The separation process was achieved by ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm, Waters Corp., Milford, MA, USA) with acetonitrile and 0.1% formic acid in water (85:15). The linear range was 0.5 ~ 100 ng/mL, and the correlation coefficient (r2 ) was greater than 0.993. The inter-day and intra-day precision was measured within an acceptable range of 15%. The extraction recovery and matrix effect were 78.9 to 87.2% and 94.3 to 98.5%, respectively. Under different conditions, the XPP was stable in the range of 5.6 ~ 10.6%. This method has been successfully applied to study the pharmacokinetics of XPP with oral dose of 10.0 mg/kg and intravenous dose of 2.0 mg/kg in rats, respectively. The absolute oral bioavailability of XPP was 4.6%.

Keywords:  IgE; UPLC-ESI-Orbitrap MS; anthraquinone; pharmacokinetics; xanthopurpurin

DOI:  https://doi.org/10.1002/bmc.4838
Rapid Commun Mass Spectrom. 2020 Apr 04.

Chemical compositional analysis of soil fulvic acids using Fourier transform ion cyclotron resonance mass spectrometry.

Kosuke Ikeya, Rachel L Sleighter, Patrick G Hatcher, Akira Watanabe.

RATIONALE: Soil fulvic acids (FAs) are considered to be a highly reactive pool of soil organic matter. The functions of FAs are related to thire chemical structures, the details of which are largely unidentified. To better understand them, Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) must be a useful but generally unused tool.METHODS: The structural properties of the components of five FA samples from a variety of soils were determined using FTICR-MS with negative-mode electrospray ionization. The peaks were assigned to molecular formulae, which were categorized into seven compound groups based on the H/C-O/C van Krevelen diagram. Ramp 13 C cross polarization/magic angle spinning nuclear magnetic resonance (NMR) spectra with phase-adjusted spinning side bands were also measured to estimate the C composition.
RESULTS: From FTICR-MS, molecular formulae were assigned to 1746-2605 peaks across the m/z range of 200-700. Those aligned in the lignin-like, tannin-like, and condensed aromatic regions of the van Krevelen diagram accounted for 49-58%, 4-20%, and 18-39% of the total peak magnitude, respectively. The proportion of the summed peak magnitudes that were detected in the lignin-like and condensed aromatic regions correlated positively to the aromatic C% as estimated by 13 C NMR. From Kendrick mass defect analysis using a carboxyl group, 94 molecular formulae were assigned to condensed aromatic acids, of which the maximum ring number was 4-7, as potential structures.
CONCLUSIONS: A high proportion of lignin-like formulas and condensed aromatics, including those probably condensed aromatic acids with small ring numbers, as well as the existence of tannin-like formulae, which were generally lacking in soil humic acids, was suggested as a common feature of soil FAs.

DOI: https://doi.org/10.1002/rcm.8801
Rapid Commun Mass Spectrom. 2020 Apr 04.

Impurities identification and quantification for arginine vasopressin by liquid chromatography-high resolution mass spectrometry.

Peize Wu, Sijian Ye, Ming Li, Hongmei Li, Ying Kan, Zecheng Yang.

RATIONALE: For pharmaceutical quality control, impurities may have unexpected pharmacological or toxicological effects that seriously impact on safety and efficacy. Arginine vasopressin (AVP) is an important cyclic peptide drug, which is mainly used for the treatment of diabetes insipidus and esophageal varices bleeding. With the improvement of analytical techniques, liquid chromatography-high resolution mass spectrometry (LC/hrMS) has become a critical technique for the challenging task of the. identification and quantification of peptide impurities structurally related to AVP.METHODS: An LC/hrMS/MS-based method using a quadruple ion trap-Orbitrap mass spectrometer operated in positive ion ESI mode was developed for the determination and quantification of structurally related peptide impurities in AVP.
RESULTS: Under optimized experimental conditions, 3 deamidation products, ([Glu4 ]AVP, [Asp5 ]AVP and AVP acid), 2 amino acid deletion impurities (des-Pro7 -AVP and des-Gly9 -AVP), 1 amino acid insertion impurity (endo-Gly10a -AVP), 1 end chain reaction product (N-acetyl-AVP) and 1 AVP isomer were detected. Subsequent quantification using an external standard method allowed the total mass fraction of all structurally related peptide impurities in the AVP study material to be estimated as 30.3 mg/g with an expanded uncertainty of 3.0 mg/g (k=2).
CONCLUSION: This work complements the AVP impurity profile and facilitates improved separation and discovery of other potential impurities in vasopressin analogues.

DOI: https://doi.org/10.1002/rcm.8799
Ann Pharm Fr. 2020 Feb 09. pii: S0003-4509(20)30012-2. [Epub ahead of print]

Micellar high performance liquid chromatographic method for separation and validation of two anti-hepatitis C- virus drugs in pure form, human plasma and human urine.

W S Hassan, M S Elmasry, A Shalaby, H M El-Sayed, D W Zidan.

  OBJECTIVES: In the present study, an eco- friendly micellar liquid chromatographic technique was validated for separation and quantification of two drugs; namely ribavirin (RIV), and sofosbuvir (SBV) in pure form, pharmaceuticals containing them, human plasma and human urine. These drugs are administered co-administered for treatment of Hepatitis C virus (HCV) that causes hepatitis C in humans.MATERIAL AND METHODS: These drugs were separated using Nucleosil 100-5 phenyl column. Sodium dodecyl sulphate (SDS) solution (0.05M, pH 7.0) containing triethylamine (0.3%) and n-butanol (10%) was used as a mobile phase with 1.2 mLmin-1 ﬂow rate and 215nm detection wavelength. Nine minutes were required for resolving the two drugs from the matrix.
RESULTS: The method showed good linearity for RIV and SBV with correlation coefficients (r2) more than 0.9996 within the concentration ranges of (20-400) and (40-400) ngmL-1 in pure form, (30-300) and (50-300) ngmL-1 in human plasma and (20-400) and (40-400) ngmL-1 in human urine, respectively.
CONCLUSION: The recommended method was applied for examination of RIV and SBV in pure and pharmaceuticals. The obtained results were statistically matched with reported methods with no significant differences. Also, the recommended method was effectively applied for estimation of both drugs in spiked human urine and plasma without purification or extraction steps and real samples of plasma and urine of humans having therapy of RIV and SBV, as well as, performing tablets dissolution-rate tests with satisfactory results.

Keywords:  Dissolution test; Human plasma; Human urine; La ribavirine; L’urine humaine; Plasma humain; Ribavirin; Sofosbuvir; Test de dissolution

DOI:  https://doi.org/10.1016/j.pharma.2020.01.007
J Sep Sci. 2020 Apr 06.

Enhancing the selectivity of liquid chromatography-mass spectrometry by using trilinear decomposition on LC-MS data: an application to three-way calibration of coeluting analytes in human plasma.

Xiu-Fang Yan, Yan-Mei Liang, Bo Zhou, Jun Bin, Chao Kang.

  The high selectivities of liquid chromatography and mass spectrometry make liquid chromatography - mass spectrometry (LC-MS) become one of the most popular tools for quantitative analysis in complex chemical, biological, and environmental systems. While, the potential mathematical selectivity of LC-MS is rarely explored. This work explored the mathematical selectivity of LC-MS by three-way calibration based on the trilinear model, with an application to quantitative analysis of coeluting aromatic amino acids in human plasma. By the trilinear decomposition of the constructed LC-MS-Sample trilinear model and individual regression of the decomposed relative intensity versus concentration, the proposed three-way calibration method successfully achieved quantitative analysis of coeluting aromatic amino acids in human plasma, even in the presence of uncalibrated interferent(s) and a varying background. This analytical method can ease the requirements for sample preparation and complete chromatographic separation of components, reduce the use of organic solvents, decrease the time of chromatographic separation, and increase the peak capacity of LC-MS. As a "green analytical method", the LC-MS three-way calibration method can provide a promising tool for direct and fast quantitative analysis in complex systems containing uncalibrated spectral interferents, especially for the situation where the coelution problem is hard to overcome. This article is protected by copyright. All rights reserved.

Keywords:  aromatic amino acids; mathematical selectivity; second-order advantage; three-way calibration; trilinear model

DOI:  https://doi.org/10.1002/jssc.202000151
Metabolites. 2020 Apr 02. pii: E139. [Epub ahead of print]10(4):

A Multivariate Metabolomics Method for Estimating Platelet Mitochondrial Oxygen Consumption Rates in Patients with Sepsis.

Marc R McCann, Cora E McHugh, Maggie Kirby, Theodore S Jennaro, Alan E Jones, Kathleen A Stringer, Michael A Puskarich.

  BACKGROUND: Sepsis-induced alterations in mitochondrial function contribute to organ dysfunction and mortality. Measuring mitochondrial function in vital organs is neither feasible nor practical, highlighting the need for non-invasive approaches. Mitochondrial function may be reflected in the concentrations of metabolites found in platelets and whole blood (WB) samples. We proposed to use these as alternates to indirectly estimate platelet mitochondrial oxygen consumption rate (mOCR) in sepsis patients.METHODS: We determined the relationships between platelet mOCR and metabolites in both platelets and WB, as measured by quantitative 1H-NMR metabolomics. The associations were identified by building multiple linear regression models with stepwise forward-backward variable selection. We considered the models to be significant with an ANOVA test (p-value ≤ 0.05) and a positive predicted-R2.
RESULTS: The differences in adjusted-R2 and ANOVA p-values (platelet adj-R2: 0.836 (0.0003), 0.711 (0.0004) vs. WB adj-R2: 0.428 (0.0079)) from the significant models indicate the platelet models were more associated with platelet mOCR.
CONCLUSIONS: Our data suggest there are groups of metabolites in WB (leucine, acetylcarnitine) and platelets (creatine, ADP, glucose, taurine) that are associated with platelet mOCR. Thus, WB and platelet metabolites could be used to estimate platelet mOCR.

Keywords:  acetylcarnitine; bioenergetics; metabolism; mitochondria; mitochondrial function; mitochondrial respiration; nuclear magnetic resonance spectroscopy

DOI:  https://doi.org/10.3390/metabo10040139
J Pharm Biomed Anal. 2020 Mar 23. pii: S0731-7085(19)33119-X. [Epub ahead of print]186 113275

In vitro evaluation of reactive nature of E- and Z-guggulsterones and their metabolites in human liver microsomes using UHPLC-Orbitrap mass spectrometer.

Ankit Balhara, Mayur Ladumor, Dilip Kumar Singh, Pammi Praneetha, Jalvadi Preethi, Sunil Pokharkar, Abhijeet Yashwantrao Deshpande, Sanjeev Giri, Saranjit Singh.

  Guggulipid is known to be useful for hypercholesterolemia, arthritis, acne, and obesity. These activities are attributed to its two principal isomeric active constituents, viz., E- and Z-guggulsterones. There are several side effects reported for guggulipid, which include widespread erythematous papules in a morbilliform pattern and macules localized to the arms; swelling and erythema of the face with burning sensation; pruritis; and bullous lesions on the lower legs with associated headaches, myalgia and itching. We hypothesized that one probable reason for these toxic reactions could be the formation of electrophilic reactive metabolites (RMs) of guggulsterones and their subsequent reaction with cellular proteins. Unfortunately, no report exists in the literature highlighting detection of RMs of guggulsterone isomers. Accordingly, the present study was undertaken to investigate the potential of E- and Z-guggulsterones to form RMs in human liver microsomes (HLM) using glutathione (GSH) and N-acetylcysteine (NAC) as trapping agents. The generated samples were analysed using ultra-high performance liquid chromatography (UHPLC) coupled to an Orbitrap mass spectrometer. The analysis of incubations with trapping agents highlighted that hydroxylated metabolites of guggulsterone isomers showed adduction with GSH and NAC. Even direct adducts of guggulsterone isomers were observed with both the trapping agents. The in silico toxicity potential of E- and Z-guggulsterones and their RMs was predicted using ADMET Predictor™ software and comparison was made against reported toxicities of guggulipid.

Keywords:  E-guggulsterone; Glutathione; Guggulipid; N-acetylcysteine; Reactive metabolite; Z-guggulsterone

DOI:  https://doi.org/10.1016/j.jpba.2020.113275
J Sep Sci. 2020 Apr 06.

Linear calibrations in chromatography: the incorrect use of ordinary least squares for determinations at low levels, and the need to redefine the limit of quantification with this regression model.

Juan M Sanchez.

  Ordinary least squares is widely applied as the standard regression method for analytical calibrations, and it is usually accepted that this regression method can be used for quantification starting at the limit of quantification. However, it requires calibration being homoscedastic and this is not common. Different calibrations have been evaluated to assess whether ordinary least squares is adequate to quantify estimates at low levels. All calibrations evaluated were linear and heteroscedastic. Despite acceptable values for precision at limit of quantification levels were obtained, ordinary least squares fitting resulted in significant and unacceptable bias at low levels. When weighted least squares regression was applied, bias at low levels were solved and accurate estimates were obtained. With heteroscedastic calibrations, limit values determined by conventional methods are only appropriate if weighted least squares is used. A "practical limit of quantification" can be determined with ordinary least squares in heteroscedastic calibrations, which should be fixed at a minimum of 20 times the value calculated with conventional methods. Biases obtained above this "practical limit" were acceptable applying ordinary least squares and no significant differences were obtained between the estimates measured using weighted and ordinary least squares when analyzing real-world samples. This article is protected by copyright. All rights reserved.

Keywords:  accuracy profiles; analytical calibrations; least squares; quantification limit

DOI:  https://doi.org/10.1002/jssc.202000094
Mol Cell Proteomics. 2020 Apr 06. pii: mcp.RA120.001986. [Epub ahead of print]

Investigation of inter- and intra-tumoral heterogeneity of glioblastoma using TOF-SIMS.

Samvel K Gularyan, Alexander A Gulin, Ksenia S Anufrieva, Victoria Shender, Michail I Shakhparonov, Soniya Bastola, Nadezhda V Antipova, Tatiana F Kovalenko, Yury P Rubtsov, Yaroslav A Latyshev, Alexander A Potapov, Marat S Pavlyukov.

  Glioblastoma (GBM) is one of the most aggressive human cancers with a median survival of less than two years. A distinguishing pathological feature of GBM is a high degree of inter- and intratumoral heterogeneity. Intertumoral heterogeneity of GBM has been extensively investigated on genomic, methylomic, transcriptomic, proteomic and metabolomics levels, however only a few studies describe intratumoral heterogeneity due to the lack of methods allowing to analyze GBM samples with high spatial resolution. Here, we applied TOF-SIMS (Time-of-flight secondary ion mass spectrometry) for the analysis of single cells and clinical samples such as paraffin and frozen tumor sections obtained from 57 patients. We developed a technique that allows us to simultaneously detect the distribution of proteins and metabolites in glioma tissue with 800 nm spatial resolution. Our results demonstrate that according to TOF-SIMS data glioma samples can be subdivided into clinically relevant groups and distinguished from the normal brain tissue. In addition, TOF-SIMS was able to elucidate differences between morphologically distinct regions of GBM within the same tumor. By staining GBM sections with gold-conjugated antibodies against Caveolin-1 we could visualize border between zones of necrotic and cellular tumor and subdivide glioma samples into groups characterized by different survival of the patients. Finally, we demonstrated that GBM contains cells that are characterized by high levels of Caveolin-1 protein and cholesterol. This population may partly represent a glioma stem cells. Collectively, our results show that the technique described here allows to analyze glioma tissues with a spatial resolution beyond reach of most of other omics approaches and the obtained data may be used to predict clinical behavior of the tumor.

Keywords:  Cancer biomarker(s); Caveolin-1; Glioblastoma; Imaging Visualization Tools; Mass Spectrometry; Stem cells*; TOF-SIMS; glioma

DOI:  https://doi.org/10.1074/mcp.RA120.001986
Anal Biochem. 2020 Apr 03. pii: S0003-2697(20)30239-6. [Epub ahead of print] 113707

Detection system of the intracellular nitric oxide in yeast by HPLC with a fluorescence detector.

Ryo Nasuno, Seiya Shino, Yuki Yoshikawa, Natsuko Yoshioka, Yuichi Sato, Kohei Kamiya, Hiroshi Takagi.

  Nitric oxide (NO) is an important signaling molecule involved in various biological phenomena in many organisms. The physiological functions and metabolism of NO in yeast, a unicellular microorganism, are still unknown, mainly because it is difficult to analyze the intracellular NO levels accurately. Here, we developed a new method of more accurately measuring NO content in yeast cells with the detection limit of 6 nM, by treating the cells with an NO-specific fluorescence probe followed by high-performance liquid chromatography with fluorescence detection (HPLC/FLD). This approach successfully detected and quantified the NO content inside yeast cells treated with an NO donor. Moreover, the HPLC/FLD analysis indicates that the fluorescence induced under some environmental stress conditions, such as ethanol, vanillin, and heat-shock, was not derived from NO. The HPLC/FLD method developed in this study provides a new strategy for measuring the intracellular NO concentration with higher accuracy.

Keywords:  Environmental stress; Fluorescence probe; Methodology; Nitric oxide; Yeast

DOI:  https://doi.org/10.1016/j.ab.2020.113707
PLoS One. 2020 ;15(4): e0231004

Breast cancer diagnosis by analysis of serum N-glycans using MALDI-TOF mass spectroscopy.

Sae Byul Lee, Shambhunath Bose, Sei Hyun Ahn, Byung Ho Son, Beom Seok Ko, Hee Jeong Kim, Il Yong Chung, Jisun Kim, Woochang Lee, Myung-Su Ko, Kyungsoo Lee, Suhwan Chang, Hyoung Soon Park, Jong Won Lee, Dong-Chan Kim.

Blood and serum N-glycans can be used as markers for cancer diagnosis, as alterations in protein glycosylation are associated with cancer pathogenesis and progression. We aimed to develop a platform for breast cancer (BrC) diagnosis based on serum N-glycan profiles using MALDI-TOF mass spectroscopy. Serum N-glycans from BrC patients and healthy volunteers were evaluated using NosQuest's software "NosIDsys." BrC-associated "NosID" N-glycan biomarkers were selected based on abundance and NosIDsys analysis, and their diagnostic potential was determined using NosIDsys and receiver operating characteristic curves. Results showed an efficient pattern recognition of invasive ductal carcinoma patients, with very high diagnostic performance [area under the curve (AUC): 0.93 and 95% confidence interval (CI): 0.917-0.947]. We achieved effective stage-specific differentiation of BrC patients from healthy controls with 82.3% specificity, 84.1% sensitivity, and 82.8% accuracy for stage 1 BrC and recognized hormone receptor-2 and lymph node invasion subtypes based on N-glycan profiles. Our novel technique supplements conventional diagnostic strategies for BrC detection and can be developed as an independent platform for BrC screening.

DOI: https://doi.org/10.1371/journal.pone.0231004
Pediatr Res. 2020 Apr 04.

Steroid hormone analysis of adolescents and young women with polycystic ovarian syndrome and adrenocortical dysfunction using UPC2-MS/MS.

Amanda C Swart, Therina du Toit, Evgenia Gourgari, Martin Kidd, Meg Keil, Fabio R Faucz, Constantine A Stratakis.

BACKGROUND: We recently identified 35 women with polycystic ovarian syndrome (PCOS) who exhibited features of micronodular adrenocortical hyperplasia. Steroid hormone analysis can be more accurate using state-of-the-art ultra-performance convergence chromatography-tandem mass spectrometry (UPC2-MS/MS). We hypothesized that UPC2-MS/MS may be used to better define hormonally this distinct subgroup of patients with PCOS.METHODS: Plasma from PCOS patients (n = 35) and healthy volunteers (HVs, n = 19) who all received dexamethasone testing was analyzed. Samples were grouped per dexamethasone responses and followed by UPC2-MS/MS analysis. When insufficient, samples were pooled from patients with similar responses to allow quantification over the low end of the assay.
RESULTS: The C11-oxy C19 (11β-hydroxyandrostenedione, 11keto-androstenedione, 11β-hydroxytestosterone, 11keto-testosterone):C19 (androstenedione, testosterone) steroid ratio was decreased by 1.75-fold in PCOS patients compared to HVs. Downstream steroid metabolites 11β-hydroxyandrosterone and 11keto-androsterone were also measurable. The C11-oxy C21 steroids, 11-hydroxyprogesterone and 11keto-dihydroprogesterone levels, were 1.2- and 1.7-fold higher in PCOS patients compared to HVs, respectively.
CONCLUSIONS: We hypothesized that UPC2-MS/MS may accurately quantify steroids, in vivo, and identify novel metabolites in a subgroup of patients with PCOS and adrenal abnormalities. Indeed, it appears that adrenal C11-oxy steroids have the potential of being used diagnostically to identify younger women and adolescents with PCOS who also have some evidence of micronodular adrenocortical hyperplasia.
IMPACT: Adrenal C11-oxy steroids may be clinically important in identifying young patients with PCOS and adrenal abnormalities.The steroids presented in our manuscript have not yet been considered in the clinical setting so far, and we believe that this study could represent a first focused step towards the characterization of a distinct subgroup of women with PCOS who may in fact be treated differently than the average patient with PCOS.This paper can change the understanding of PCOS as one disorder: it is in fact a heterogeneous condition. In addition, for the subgroup of patients with PCOS associated with adrenocortical dysfunction, our paper provides novel hormonal markers that can be used diagnostically. Finally, the paper also adds to the basic pathophysiological understanding of adrenocortical-ovarian interactions in steroidogenesis of young women and adolescent girls with PCOS.

DOI: https://doi.org/10.1038/s41390-020-0870-1