bims-metlip 2020-02-23 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2020‒02‒23
38 papers selected by
Sofia Costa
Cold Spring Harbor Laboratory

Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography-mass spectrometry and hydrophilic interaction liquid chromatography-mass spectrometry.
A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay to profile 20 plasma steroids in endocrine disorders.
Addressing Glutathione Redox Status in Clinical Samples by Two-Step Alkylation with N-ethylmaleimide Isotopologues.
Monitoring of the influence of long-term oxidative stress and ischemia on the condition of kidney using solid phase microextraction chemical biopsy coupled with liquid chromatography high resolution mass spectrometry.
Data on the optimisation of a solid phase extraction method for fractionating estrogen metabolites from small urine volumes.
COLMAR Lipids Web Server and Ultrahigh-Resolution Methods for 2D NMR- and MS-Based Lipidomics.
Recent progresses of derivatization approaches in the targeted lipidomics analysis by mass spectrometry.
Validation of an LC-MS/MS-based dilute-and-shoot approach for the quantification of > 500 mycotoxins and other secondary metabolites in food crops: challenges and solutions.
Localization of metabolites of human kidney tissue with infrared laser-based selected reaction monitoring mass spectrometry imaging and silver-109 nanoparticle-based surface assisted laser desorption/ionization mass spectrometry imaging.
Miniaturized liquid chromatography focusing on analytical columns and mass spectrometry: A review.
Quantitative amino acid analysis by liquid chromatography-tandem mass spectrometry using low cost derivatization and an automated liquid handler.
Development and validation of fast UHPLC-MS/MS screening method for 87 NPS and 32 other drugs of abuse in hair and nails: application to real cases.
Triple quadrupole-mass spectrometry protocols for the analysis of NBOMes and NBOHs in blotter papers.
Profiling analysis of phospholipid fatty acids in serum as a complement to the comprehensive fatty acids method.
Measuring Sulforaphane and Its Metabolites in Human Plasma: A High Throughput Method.
A sensitive LC-MS assay using derivatization with boron trifluoride to quantify curcuminoids in biological samples.
Multiplexed therapeutic drug monitoring of antipsychotics in dried plasma spots by LC-MS/MS.
Extraction and Determination of Vitamin K1 in Foods by Ultrasound-Assisted Extraction, SPE, and LC-MS/MS.
Development and validation of a UPLC-MS/MS method for simultaneous determination of LBPT and its metabolites in human plasma.
A sensitive method for the quantification of short-chain fatty acids by benzyl chloroformate derivatization combined with GC-MS.
Enzymatic studies with 3-oxa n-3 DPA.
LC-MS/MS method for the determination of hormones: Validation, application and health risk assessment in various bovine matrices.
Analysis of Phospholipids, Lysophospholipids, and Their Linked Fatty Acyl Chains in Yellow Lupin Seeds (Lupinus luteus L.) by Liquid Chromatography and Tandem Mass Spectrometry.
A Sensitive HPTLC Method for the Estimation of Glibenclamide, Rosiglitazone Maleate and Metformin Hydrochloride from a Multicomponent Dosage Form.
Simultaneous detection of 24 oral antidiabetic drugs and their metabolites in urine by liquid chromatography-tandem mass spectrometry.
Reversed phase UPLC/APCI-MS determination of Vitamin K1 and menaquinone-4 in human plasma: Application to a clinical study.
Simultaneous quantification of free fatty acids and acylcarnitines in plasma samples using dansylhydrazine labeling and liquid chromatography-triple quadrupole mass spectrometry.
LC-MS/MS METHOD FOR THE DETERMINATION OF ANTIDEPRESSANTS AND BENZODIAZEPINES IN MECONIUM.
Untargeted HILIC-MS-Based Metabolomics Approach to Evaluate Coffee Roasting Process: Contributing to an Integrated Metabolomics Multiplatform.
Paper spray mass spectrometry for the analysis of picoliter droplets.
Rapid determination of isoflavones and other bioactive compounds in soybean using SWATH-MS.
The pathway through LC-MS method development: in-house or ready-to-use kit-based methods?
Protein precipitation coupled to paper spray with a tube for one-step analysis of blood.
Non-targeted discovery-based analysis for gas chromatography with mass spectrometry: A comparison of peak table, tile, and pixel-based Fisher ratio analysis.
A computational platform for high-throughput analysis of RNA sequences and modifications by mass spectrometry.
CalibraCurve: A Tool for Calibration of Targeted MS-based Measurements.
Multi-residue Ultra Performance Liquid Chromatography-High resolution mass spectrometric method for the analysis of 21 cyanotoxins in surface water for human consumption.
Quantification of Furanyl Fentanyl and its Metabolites in Human and Rat Plasma using LC-MS/MS.

Anal Bioanal Chem. 2020 Feb 20.

Validation of lipidomic analysis of human plasma and serum by supercritical fluid chromatography-mass spectrometry and hydrophilic interaction liquid chromatography-mass spectrometry.

Denise Wolrab, Michaela Chocholoušková, Robert Jirásko, Ondřej Peterka, Michal Holčapek.

  Ultrahigh-performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) has a great potential for the high-throughput lipidomic quantitation of biological samples; therefore, the full optimization and method validation of UHPSFC/MS is compared here with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) in hydrophilic interaction liquid chromatography (HILIC) mode as the second powerful technique for the lipid class separation. First, the performance of six common extraction protocols is investigated, where the Folch procedure yields the best results with regard to recovery rate, matrix effect, and precision. Then, the full optimization and analytical validation for eight lipid classes using UHPSFC/MS and HILIC-UHPLC/MS methods are performed for the same sample set and applied for the lipidomic characterization of pooled samples of human plasma, human serum, and NIST SRM 1950 human plasma. The choice of appropriate internal standards (IS) for individual lipid classes has a key importance for reliable quantitative workflows illustrated by the selectivity while validation and the calculation of the quantitation error using multiple internal standards per lipid class. Validation results confirm the applicability of both methods, but UHPSFC/MS provides some distinct advantages, such as the successful separation of both non-polar and polar lipid classes unlike to HILIC-UHPLC/MS, shorter total run times (8 vs. 10.5 min), and slightly higher robustness. Various types of correlations between methods (UHPSFC/MS and HILIC-UHPLC/MS), biological material (plasma and serum), IS (laboratory and commercially mixtures), and literature data on the standard reference material show the intra- and inter-laboratory comparison in the quantitation of lipid species from eight lipid classes, the concentration differences in serum and plasma as well as the applicability of non-commercially available internal standard mixtures for lipid quantitation.

Keywords:  Hydrophilic interaction liquid chromatography; Lipidomics; Mass spectrometry; Matrix effect; Plasma; Quantitation; Serum; Supercritical fluid chromatography; Validation

DOI:  https://doi.org/10.1007/s00216-020-02473-3
Clin Chem Lab Med. 2020 Feb 21. pii: /j/cclm.ahead-of-print/cclm-2019-0869/cclm-2019-0869.xml. [Epub ahead of print]

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay to profile 20 plasma steroids in endocrine disorders.

Zhenxin Wang, Hao Wang, Yingfei Peng, Fangjun Chen, Lin Zhao, Xiaomu Li, Jiaqian Qin, Qianqian Li, Beili Wang, Baishen Pan, Wei Guo.

  Background Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assays are employed in more and more clinical laboratories to quantify steroids. The steroid quantification by LC-MS/MS shows great value in screening or diagnosing endocrine disorders; however, the number of functional steroids included in the LC-MS/MS methods is still limited. Methods Here, we describe the performance and validation of a 20-steroid plasma panel by LC-MS/MS. The panel included progestogens (including mineralocorticoids and glucocorticoids), androgens and estrogens biosynthesized in steroid metabolic pathways. The LC-MS/MS method was validated according to guidance documents, and subsequently employed to profile steroid changes in endocrine disorders. Results Using LC-MS/MS, 20 steroids were separated and quantified in 8 min. Coefficients of variation (CVs) of the 20 analytes at the lower limit of quantification (LLoQ) were all less than 15% (ranging from 1.84% to 14.96%). The linearity of the assay was demonstrated by all the R2 values greater than 0.995. Individual plasma steroids changed significantly in patients with subclinical Cushing's syndrome (SCS) and polycystic ovary syndrome (PCOS) - 17-hydroxypregnenolone (17-OH-PR), testosterone (T) and dihydrotestosterone (DHT) were significantly decreased in SCS patients, while in PCOS patients, pregnenolone, corticosterone (CORT), androstenedione (A4) and T were significantly increased and DHT was decreased. Conclusions The LC-MS/MS method we developed for the quantification of 20 plasma steroids is clinical practicable. The steroid profiling data using this assay indicate its screening value for endocrine disorders. To further explore the value of the assay, more investigations are however needed.

Keywords:  endocrine disorders; liquid chromatography-tandem mass spectrometry (LC-MS/MS); panel; pathway; steroids

DOI:  https://doi.org/10.1515/cclm-2019-0869
Metabolites. 2020 Feb 16. pii: E71. [Epub ahead of print]10(2):

Addressing Glutathione Redox Status in Clinical Samples by Two-Step Alkylation with N-ethylmaleimide Isotopologues.

Tamara Tomin, Matthias Schittmayer, Ruth Birner-Gruenberger.

  Determination of the ratio of reduced to oxidized glutathione is of profound clinical interest in assessing the oxidative status of tissues and body fluids. However, this ratio is not yet a routine clinical parameter due to the analytically challenging interconversion of reduced (free) glutathione to oxidized (bound) glutathione. We aimed to facilitate this ratio determination in order to aid its incorporation as a routine clinical parameter. To this end, we developed a simple derivatization route that yields different isotopologues of N-ethylmaleimide alkylated glutathione from reduced and oxidized glutathione (after its chemical reduction) for mass spectrometric analysis. A third isotopologue can be used as isotopic standard for simultaneous absolute quantification. As all isotopologues have similar chromatographic properties, matrix effects arising from different sample origins can only impact method sensitivity but not quantification accuracy. Robustness, simplified data analysis, cost effectiveness by one common standard, and highly improved mass spectrometric sensitivity by conversion of oxidized glutathione to an alkylated glutathione isotopologue are the main advantages of our approach. We present a method fully optimized for blood, plasma, serum, cell, and tissue samples. In addition, we propose production of N-ethylmaleimide customized blood collection tubes to even further facilitate the analysis in a clinical setting.

Keywords:  GSH; GSSG; NEM; d5-NEM; oxidative stress

DOI:  https://doi.org/10.3390/metabo10020071
J Sep Sci. 2020 Feb 18.

Monitoring of the influence of long-term oxidative stress and ischemia on the condition of kidney using solid phase microextraction chemical biopsy coupled with liquid chromatography high resolution mass spectrometry.

Iga Stryjak, Natalia Warmuzińska, Joanna Bogusiewicz, Kamil Łuczykowski, Barbara Bojko.

  The limiting factor in conventional quality assessments of transplanted organs, namely the invasiveness of tissue sample collection, has prompted much research on the field of transplantology to focus on the development of alternative evaluation methods of organ quality. In the present project, we undertake the challenge to address the need for a new analytical solution for graft quality assessments by using a novel metabolomic diagnostic protocol based on low-invasive solid-phase microextraction. Solid-phase microextraction probes of ca. 0.2 mm coated with 4 mm long mixed-mode extraction phase were inserted into rabbit kidneys immediately following euthanasia and after 2, 4, 6, and 21 h of preservation. LC-MS analysis of the extracts was performed with the use of a reversed phase column and a Q-Exactive Focus mass spectrometer operated in positive ionization mode. Statistical analysis of significantly changing compounds revealed metabolic profile changes in kidneys induced by ischemia and oxidative stress as a function of the duration of cold storage. The most pronounced alterations were reflected in levels of essential amino acids and purine nucleosides. Our findings demonstrate that the proposed approach may be successfully used to monitor changes in the metabolic profile of organs over time of preservation. This article is protected by copyright. All rights reserved.

Keywords:  kidney preservation; liquid chromatography; mass spectrometry; metabolomics; solid-phase microextraction

DOI:  https://doi.org/10.1002/jssc.202000032
Data Brief. 2020 Apr;29 105222

Data on the optimisation of a solid phase extraction method for fractionating estrogen metabolites from small urine volumes.

Carien L van der Berg, Gerda Venter, Francois H van der Westhuizen, Elardus Erasmus.

  Certain estrogen metabolites have been implicated in the pathophysiology of breast cancer. Moreover, the estrogen metabolite profiles of healthy women and those with (a high risk of) breast cancer differ significantly. The development of an analytical method to determine the relative levels of all the estrogen biotransformation products has been described in van der Berg et al. [1]. An improvement on previously developed methods was the ability to also detect molecules such as sulphate and glucuronide conjugates as well as progesterone, estradiol precursors, and metabolites from the 16-hydroxylation metabolic pathway of estrogens simultaneously with all other estrogen metabolites. The data presented here describe the optimisation of a solid phase extraction method with different fractionation steps for LC-MS/MS analysis of 27 estrogen-related metabolites from small urine volumes. Conditions that were optimised include the elution and washing solvent concentration, the urine, loading, washing, and elution volumes, as well as pH. All raw data used to construct the bar graphs presented in this article are included in the supplementary data file. The data indicated that fractionation was necessary in order to elute estrogen metabolites with different chemical properties at different eluate compositions. Only one of the fractions (containing the less water-soluble metabolites) underwent derivatisation before LC-MS/MS analysis.

Keywords:  E1, Estrone; E2, Estradiol; E3, Estriol; ESI, Electrospray ionisation; Estrogen metabolism; G, Glucuronide conjugation; LC, Liquid chromatography; LC-MS/MS; M, Methyl conjugation; MS, Mass spectrometry; MS/MS, Tandem mass spectrometry; MeOH, Methanol; Method optimisation; OH, Hydroxy; S, Sulphate conjugation; SPE, Solid phase extraction; Solid phase extraction; v/v, Volume/volume ratio

DOI:  https://doi.org/10.1016/j.dib.2020.105222
J Proteome Res. 2020 Feb 19.

COLMAR Lipids Web Server and Ultrahigh-Resolution Methods for 2D NMR- and MS-Based Lipidomics.

Cheng Wang, Istvan Timari, Bo Zhang, Da-Wei Li, Abigail Leggett, Amal O Amer, Lei Bruschweiler-Li, Rachel E Kopec, Rafael Bruschweiler.

Accurate identification of lipids in biological samples is a key step in lipidomics studies. Multidimensional NMR spectroscopy is a powerful analytical tool for this purpose as it provides comprehensive structural information on lipid composition at atomic resolution. However, the interpretation of NMR spectra of complex lipids mixtures is currently hampered by limited spectral resolution and the absence of a customized lipids NMR databases along with user-friendly spectral analysis tools. We introduce a new 2D HSQC metabolite database "COLMAR Lipids" that was specifically curated for hydrophobic metabolites presently containing 501 compounds with accurate experimental 2D 13C-1H HSQC chemical shift data measured in CDCl3. A new module in the public COLMAR suite of NMR web servers was developed for the semi-automated analysis of complex lipidomics mixtures (http://spin.ccic.osu.edu/index.php/colmarm/index2). To obtain 2D HSQC spectra with the necessary high spectral resolution along both 13C and 1H dimensions, non-uniform sampling (NUS) in combination with pure shift spectroscopy was applied allowing the extraction of an abundance of unique cross-peaks belonging to hydrophobic compounds in complex lipidomics mixtures. As shown here, this information is critical for the unambiguous identification of underlying lipid molecules by means of the new COLMAR Lipids web server as is demonstrated for Caco-2 cell and lung tissue cell extracts.

DOI: https://doi.org/10.1021/acs.jproteome.9b00845
J Sep Sci. 2020 Feb 18.

Recent progresses of derivatization approaches in the targeted lipidomics analysis by mass spectrometry.

Xian-En Zhao, Shuyun Zhu, Huwei Liu.

  Lipidomics plays an essential role in the development of an improved understanding of lipids metabolism and the identification of new biomarkers or therapeutic targets of related diseases. The strong analytical power of mass spectrometry and its rapid developments in the respect of instruments and techniques have significantly accelerated the emerging lipidomics and related application fields in biology, medicine and pharmacy. The strategy of chemical derivatization can remarkably improve the shortcomings of mass spectrometric analytical technologies of shotgun lipidomics and liquid chromatography mass spectrometry, and in the past decade many related studies have been reported for fatty acids, glycerophospholipids, sphingomyelins, monoglycerides, diacylglycerols, long-chain bases, steroids and so on. Therefore, this review will focus on new chemical derivatization approaches about the research progresses of shotgun- and liquid chromatography mass spectrometry-based targeted lipidomics (from 2005 to July 2019, most of reports emerged in the past five years), and put forward the problems and prospects in this field. It is expected to be helpful for the design and synthesis of new derivatization reagents especially the outstanding stable isotope labeling derivatization reagents, and the development and application of new chemical derivatization strategies and matched mass spectrometric analysis methods. This article is protected by copyright. All rights reserved.

Keywords:  chemical derivatization; lipidomics; liquid chromatography mass spectrometry; shotgun lipidomics; stable isotope labeling

DOI:  https://doi.org/10.1002/jssc.201901346
Anal Bioanal Chem. 2020 Feb 20.

Validation of an LC-MS/MS-based dilute-and-shoot approach for the quantification of > 500 mycotoxins and other secondary metabolites in food crops: challenges and solutions.

Michael Sulyok, David Stadler, David Steiner, Rudolf Krska.

  This paper describes the validation of an LC-MS/MS-based method for the quantification of > 500 secondary microbial metabolites. Analytical performance parameters have been determined for seven food matrices using seven individual samples per matrix for spiking. Apparent recoveries ranged from 70 to 120% for 53-83% of all investigated analytes (depending on the matrix). This number increased to 84-94% if the recovery of extraction was considered. The comparison of the fraction of analytes for which the precision criterion of RSD ≤ 20% under repeatability conditions (for 7 replicates derived from different individual samples) and intermediate precision conditions (for 7 technical replicates from one sample), respectively, was met (85-97% vs. 93-94%) highlights the contribution of relative matrix effects to the method uncertainty. Statistical testing of apparent recoveries between pairs of matrices exhibited a significant difference for more than half of the analytes, while recoveries of the extraction showed a much better agreement. Apparent recoveries and matrix effects were found to be constant over 2-3 orders of magnitude of analyte concentrations in figs and maize, whereas the LOQs differed less than by a factor of 2 for 90% of the investigated compounds. Based on these findings, this paper discusses the applicability and practicability of current guidelines for multi-analyte method validation. Investigation of (apparent) recoveries near the LOQ seems to be insufficiently relevant to justify the enormous time-effort for manual inspection of the peaks of hundreds of analytes. Instead, more emphasis should be put on the investigation of relative matrix effects in the validation procedure. Graphical abstract.

Keywords:  LC-MS/MS; Matrix effects; Multi-analyte methods; Mycotoxins; Recovery; Validation

DOI:  https://doi.org/10.1007/s00216-020-02489-9
Anal Chem. 2020 Feb 21.

Localization of metabolites of human kidney tissue with infrared laser-based selected reaction monitoring mass spectrometry imaging and silver-109 nanoparticle-based surface assisted laser desorption/ionization mass spectrometry imaging.

Joanna Niziol, Jan Sunner, Iwona B Beech, Krzysztof Ossoliński, Anna Ossolińska, Tadeusz Ossoliński, Aneta Płaza, Tomasz Ruman.

Infrared (IR) Laser Ablation-remote-Electrospray Ionization (LARESI) platform coupled to a tandem mass spectrometer (MS/MS) operated in selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) modes was developed and employed for targeted imaging of selected metabolites in human kidney cancer tissue. The use of SRM or MRM essentially avoids artifacts in single-compound images that are caused by the inability to filter out chemical noise in full scan MS mode. For comparison purposes, images of the same metabolites were obtained with ultraviolet (UV) desorption/ionization mass spectrometry imaging (UV-LDI-MSI) using monoisotopic silver-109 nanoparticle-enhanced target (109AgNPET) in full-scan MS mode. Tissue samples from patients with renal cell carcinoma (RCC) containing both cancerous and non-cancerous regions were imaged. Sixteen endogenous metabolites that were reported in the literature as varying in abundance between cancerous and non-cancerous areas in various tissues were selected for analysis. They included ten amino acids, four nucleosides and nucleobases, as well as lactate and vitamin E. The obtained MS images vividly illustrate differences in abundances of selected metabolites between cancerous and non-cancerous regions of the kidney tissue. Importantly, the two imaging methods offered similar results. This study demonstrates the applicability of the novel ambient LARESI SRM/MRM MSI method to tissue analyses and its potential for investigating and discovering cancer biomarkers.

DOI: https://doi.org/10.1021/acs.analchem.9b04580
Anal Chim Acta. 2020 Mar 22. pii: S0003-2670(19)31534-X. [Epub ahead of print]1103 11-31

Miniaturized liquid chromatography focusing on analytical columns and mass spectrometry: A review.

Edvaldo Vasconcelos Soares Maciel, Ana Lúcia de Toffoli, Eduardo Sobieski, Carlos Eduardo Domingues Nazário, Fernando Mauro Lanças.

  The technological advances achieved over the last decades boosted the development of suitable benchtop platforms to work at miniaturized liquid chromatography scale (capillary and nano-LC). Under the right conditions, miniaturized LC can offer higher analysis efficiency resulting in superior chromatographic resolution and overall sensitivity than conventional LC. Among the main advantages are the reduced reagents and sample requirement, the decreasing on analytical column dimensions, and consequently flow rates and the easer coupling to mass spectrometry. This review describes fundamental aspects and advances over miniaturized LC technology with a focus on the last decade. Therefore, relevant characteristics of the most common analytical column, covering both filled (packed and monolithic) and open tubular (PLOT and WCOT) columns, are herein discussed. Alternatively, other modern approaches based on microchip separations or 2D configurations aiming for the sample preparation on the first dimension, are also introduced. Likewise, some positive and negative aspects of these systems over HPLC are underscored. Besides, considering the necessity to developed components to work at capillary or nanoscale, without significant dead-volumes, the most critical features of specially designed instrumentation for benchtop instruments are briefly discussed highlighting connectors, pumping, injections, oven and detection systems. Also, a more detailed section is presented focused on mass spectrometry efforts towards its miniaturization and how this trend can be useful working together with miniaturized LC. Finally, applications of capillary and nano-LC involving bioanalytical, environmental, and food methods are discussed to support the miniaturized LC as a powerful and emergent separation technique for the years ahead.

Keywords:  Capillary liquid chromatography; Instrumentation; Mass spectrometry; Miniaturized; Nano liquid chromatography; Open tubular liquid chromatography

DOI:  https://doi.org/10.1016/j.aca.2019.12.064
JIMD Rep. 2020 Jan;51(1): 62-69

Quantitative amino acid analysis by liquid chromatography-tandem mass spectrometry using low cost derivatization and an automated liquid handler.

William S Phipps, Eric Crossley, Richard Boriack, Patricia M Jones, Khushbu Patel.

  Amino acid analysis is central to newborn screening and the investigation of inborn errors of metabolism. Ion-exchange chromatography with ninhydrin derivatization remains the reference method for quantitative amino acid analysis but offers slow chromatography and is susceptible to interference from other co-eluting compounds. Liquid-chromatography tandem mass spectrometry (LC-MS/MS) provides a rapid and highly specific alternative, but sample preparation is frequently laborious and sometimes cost prohibitive. To address these limitations, we validated an LC-MS/MS method using the aTRAQ Reagents Application Kit with a modified protocol consuming only half reagents. Adequate performance for clinical specimen measurement of 26 amino acids with high clinical relevance was achieved. An automated liquid handler and modified calibration and normalization approaches were used to ensure reproducible assay performance. Linear measurement between 5 and 2000 μM was achieved for most analytes despite use of a small, 10 μl sample size. Overall the method achieved near substantially improved throughput and enabled use of smaller samples volumes for batched analyses of clinical samples.

Keywords:  amino acid; inborn errors of metabolism; isotope‐coded derivatization; liquid chromatography; mass spectrometry; triple quadrupole

DOI:  https://doi.org/10.1002/jmd2.12080
Anal Bioanal Chem. 2020 Feb 15.

Development and validation of fast UHPLC-MS/MS screening method for 87 NPS and 32 other drugs of abuse in hair and nails: application to real cases.

Giulio Mannocchi, Annagiulia Di Trana, Anastasio Tini, Simona Zaami, Massimo Gottardi, Simona Pichini, Francesco Paolo Busardò.

  Interest on keratinized matrix analysis for clinical and forensic purposes has been recently grown due to the wide temporary detection window for psychotropic and toxic substances entrapped after repeated consumption. The aim of this study was the development and full validation of an UHPLC-MS/MS screening method to quantify 119 molecules among most abused classic drugs and new psychoactive substances in hair and in nails, to assess the polyconsumption. Twenty-five milligrams of hair or nail samples, added with the internal standard mixture, were cut and incubated with 500 μL M3® buffer reagent at controlled temperature. After cooling, 1 μL supernatant was injected in the chromatographic system equipped with an Oasis HLB column. After the 10 min chromatographic separation through a gradient mobile phase (aqueous ammonium formate, phase A; acetonitrile, phase B), the target compounds were detected in multiple reaction monitoring mode. The method was linear (r2 always better than 0.99) in a calibration range of LOQ 20000 pg compound for milligram hair and of LOQ 1000 pg compound per milligram nail. Process efficiency of analytes under investigation was always better than 65% and no significant ion suppression due to matrix effect was observed. Intra-assay and inter-assay precision and accuracy were always better than 15%. The applicability and trueness of the method were examined by analysing real samples of hair and nail from users of psychoactive drugs in recreational contexts. Both classic drugs and new psychoactive substances could be determined as result of single or repeated use and accumulation in keratin matrices. Graphical abstract.

Keywords:  Alternative matrices; Hair; Nails; New psychoactive substances; UHPLC-MS/MS

DOI:  https://doi.org/10.1007/s00216-020-02462-6
Forensic Sci Int. 2020 Feb 06. pii: S0379-0738(20)30046-3. [Epub ahead of print]309 110184

Triple quadrupole-mass spectrometry protocols for the analysis of NBOMes and NBOHs in blotter papers.

Damila Rodrigues de Morais, Kelly Francisco da Cunha, Taís Betoni Rodrigues, Rafael Lanaro, Lilian de Melo Barbosa, Jorge Jardim Zacca, Marcos Nogueira Eberlin, Jose Luiz Costa.

  NBOMe and NBOH are new psychoactive substances with potent activity on serotonin 5-HT2a receptors causing serious toxic effects, including serotonin toxidrome and death. The aim of this work was to develop a comprehensive MS/MS protocol, using triple quadrupole mass spectrometers coupled to LC and GC, for rapid screening and quantitation of NBOMes and NBOHs in seized blotter papers. Different scan methods (neutral loss, precursor ion or multiple reaction monitoring) were used to obtain structural information of phenylethylamine class. The developed protocol was validated for qualitative and quantitative analysis, showing a satisfactory limit of detection (1 ng/mL), with excellent selectivity, imprecision (intra and interday imprecision lower than 1.2 % RSD) and accuracy (between -7.1 and +5.6 %, n = 15), as well as bias values. The analysis of real samples shown that NBOH compounds were the most frequently detected, with concentrations ranging from 0.1 to 1,929 μg per blotter sample. Triple quadrupole mass spectrometers can be a useful tool for identification of new psychoactive substances. A comprehensive protocol using both LC-MS/MS and GC-MS/MS, with different scanning modes, have been developed and showed to be useful to screening NBOMe and NBOH in blotter papers.

Keywords:  GC–MS/MS; LC–MS/MS; NBOH; NBOMe; New psychoactive substances; Phenethylamine derivatives

DOI:  https://doi.org/10.1016/j.forsciint.2020.110184
J Chromatogr A. 2020 Feb 11. pii: S0021-9673(20)30165-5. [Epub ahead of print] 460965

Profiling analysis of phospholipid fatty acids in serum as a complement to the comprehensive fatty acids method.

Inmaculada Criado-Navarro, Antonio Mena-Bravo, Mónica Calderón-Santiago, Feliciano Priego-Capote.

  Fatty acids (FAs) are mostly found in blood as triglycerides, phospholipids (PLs) and cholesteryl esters. Determination of FAs is typically carried out in serum or plasma by a comprehensive method (known as the classical FAMEs method since FAs are determined as Fatty Acids Methyl Esters), which is based on liquid-liquid extraction, derivatization by transesterification, and determination by gas chromatography (GC) coupled to a suited detection technique. However, this method does not favor the determination of FAs that are chemically conjugated in PLs due to kinetics impediment. For this reason, we have developed a selective method to determine the FAs profile of PLs in serum based on solid-phase extraction (SPE) for isolation of PLs and determination of the FAME derivatives by GC-mass spectrometry (GC-MS). The method was applied to serum samples collected from twenty-five individuals to compare the FAs profile versus that provided by the non-selective protocol based on liquid-liquid extraction of lipid families. Statistical analysis revealed compositional changes in the FAs profile with special emphasis on the content of saturated (SFAs) and monounsaturated FAs (MUFAs). Thus, SFAs passed from 34.0% with the classical method to 49.3% in PLs while MUFAs went from 24.4% to 11.4%. This study proves that the proposed method provides complementary results to the comprehensive method and, therefore, both methods can be combined to evaluate the effect of intervention diets and their connection to metabolic diseases.

Keywords:  FAMEs, solid-phase extraction; Fatty acids; Gas chromatography; Mass spectrometry; Phospholipids

DOI:  https://doi.org/10.1016/j.chroma.2020.460965
Molecules. 2020 Feb 13. pii: E829. [Epub ahead of print]25(4):

Measuring Sulforaphane and Its Metabolites in Human Plasma: A High Throughput Method.

Annie Langston-Cox, Dovile Anderson, Darren J Creek, Kirsten Palmer, Euan M Wallace, Sarah A Marshall.

  (1) Background: There is increasing understanding of the potential health benefits of cruciferous vegetables. In particular sulforaphane (SFN), found in broccoli, and its metabolites sulforaphane-glutathione (SFN-GSH), sulforaphane-cysteine (SFN-Cys), sulforaphane cysteine-glycine (SFN-CG) and sulforaphane-N-acetyl-cysteine (SFN-NAC) have potent antioxidant effects that may offer therapeutic value. Clinical investigation of sulforaphane as a therapeutic antioxidant requires a sensitive and high throughput process for quantification of sulforaphane and metabolites; (2) Methods: We collected plasma samples from healthy human volunteers before and for eight hours after consumption of a commercial broccoli extract supplement rich in sulforaphane. A rapid and sensitive method for quantification of sulforaphane and its metabolites in human plasma using Liquid Chromatography-Mass Spectrometry (LC-MS) has been developed; (3) Results: The LC-MS analytical method was validated at concentrations ranging between 3.9 nM and 1000 nM for SFN-GSH, SFN-CG, SFN-Cys and SFN-NAC and between 7.8 nM and 1000 nM in human plasma for SFN. The method displayed good accuracy (1.85%-14.8% bias) and reproducibility (below 9.53 %RSD) including low concentrations 3.9 nM and 7.8 nM. Four SFN metabolites quantitation was achieved using external standard calibration and in SFN quantitation, SFN-d8 internal standardization was used. The reported method can accurately quantify sulforaphane and its metabolites at low concentrations in plasma; (4) Conclusions: We have established a time- and cost-efficient method of measuring sulforaphane and its metabolites in human plasma suitable for high throughput application to clinical trials.

Keywords:  Liquid Chromatography–Mass Spectrometry; pharmacokinetic; sulforaphane

DOI:  https://doi.org/10.3390/molecules25040829
Anal Biochem. 2020 Feb 17. pii: S0003-2697(19)31256-4. [Epub ahead of print] 113636

A sensitive LC-MS assay using derivatization with boron trifluoride to quantify curcuminoids in biological samples.

Alexander J Yoon, Haiqiang Wu, Roy D Pan, Bruce Teter, Jack Cipolla, Edwin Chang, Luis Z Avila, Saroj K Basak, Eri Srivatsan, Marilene B Wang, Greg M Cole, Sally A Frautschy, Phillip D Hampton, Kym F Faull.

  A procedure is described to measure curcumin (C), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), tetrahydrocurcumim (TC) and their glucuronidated metabolites (CG, DMCG, and BDMCG) in plasma, brain, liver and tumor samples. The procedure involves converting the analytes to their boron difluoride derivatives and analyzing them by combined liquid chromatography coupled to an ion trap mass spectrometer operating in the negative ion MSn scan mode. The method has superb limits of detection of 0.01 nM for all curcuminoids and 0.5 nM for TC and the glucuroniated metabolites, and several representative chromatograms of biological samples containing these analytes are provided. In addition, the pharmacokinetic profile of these compounds in one human who daily consumed an over-the-counter curcuminoid product shows the peak and changes in circulating concentrations achieved by this mode of administration.

Keywords:  Boron trifluoride (BF(3)); Curcumin; Curcuminoids; Liquid chromatography coupled mass spectrometry (LCMS); Pharmacokinetics

DOI:  https://doi.org/10.1016/j.ab.2020.113636
J Sep Sci. 2020 Feb 20.

Multiplexed therapeutic drug monitoring of antipsychotics in dried plasma spots by LC-MS/MS.

C Ruggiero, S Ramirez, E Ramazzotti, R Mancini, R Muratori, M A Raggi, M Conti.

  In this work, a convenient method for the therapeutic monitoring of seven common antipsychotic drugs in "dried plasma spot" samples has been developed. It is based on the LC-MS/MS technique, operating in multiple reaction monitoring mode, and a straightforward procedure for the simultaneous extraction of all antipsychotics in a single step, with high extraction yield. The method was fully validated with proper accuracy, precision, selectivity and sensitivity, for all the drugs. Limits of quantification were 0.12, 1.09, 1.46, 1.47, 5.70, 1.32, 1.33 ug/L for haloperidol, aripiprazole, olanzapine, quetiapine, clozapine, risperidone and paliperidone, respectively. Accuracy, intra-day and inter-day precision values were <10% for all drugs at all concentration levels examined. The method was tested in the analysis of 30 plasma samples from real patients for each drug. The proposed analytical approach, by combining practical and logistical advantages of micro-sampling with LC-MS/MS analytical performance, could offer an ideal strategy for accurate and timely therapeutic drug monitoring of antipsychotic drugs in most clinical settings, even in remote centers and/or in out-patient settings, bringing so many potential improvements in psychiatric patient care. This article is protected by copyright. All rights reserved.

Keywords:  antipsychotic drugs; dried plasma spots; liquid chromatography-mass spectrometry; therapeutic drug monitoring

DOI:  https://doi.org/10.1002/jssc.201901200
Molecules. 2020 Feb 14. pii: E839. [Epub ahead of print]25(4):

Extraction and Determination of Vitamin K1 in Foods by Ultrasound-Assisted Extraction, SPE, and LC-MS/MS.

Yueqing Xu, Liangxiao Zhang, Ruinan Yang, Xu Yu, Li Yu, Fei Ma, Hui Li, Xiupin Wang, Peiwu Li.

  Vitamin K1 is one of the important hydrophobic vitamins in fat-containing foods. Traditionally, lipase is employed in the determination of vitamin K1 to remove the lipids, which makes the detection complex, time-consuming, and insensitive. In this study, the determination of vitamin K1 in fat-containing foods was developed based on ultrasound-assisted extraction (UAE), solid-phase extraction (SPE) combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The optimal conditions for extraction of vitamin K1 were material-liquid ratio of 1:70 (g/mL), extraction temperature of 50 °C, extraction power of 700 W, extraction time of 50 min, material-wash fluid ratio of 1:60 (g/mL), and 8 mL of hexane/anhydrous ether (97:3, v/v) as the elution solvent. Then, vitamin K1 was analyzed on a ZORBAX SB-C18 column (50 mm × 2.1 mm, 1.8 μm) by gradient elution with water (0.01% formic acid) and methanol (0.01 formic acid + 2.5 mmol/L ammonium formate) as the mobile phase. The limit of detection (LOD) and limit of quantification (LOQ) were 0.05 and 0.16 μg/kg, respectively. Calibration curve was linear over the range of 10-500 ng/mL (R2 > 0.9988). The recoveries at three spiked levels were between 80.9% and 119.1%. The validation and application indicated that the proposed method was simple and sensitive in determination of vitamin K1 in fat-containing foods.

Keywords:  LC-MS/MS; foods; solid-phase extraction; ultrasound-assisted extraction; vitamin K1

DOI:  https://doi.org/10.3390/molecules25040839
Bioanalysis. 2020 Feb 21.

Development and validation of a UPLC-MS/MS method for simultaneous determination of LBPT and its metabolites in human plasma.

Mengyi Wu, Aijing Liu, Quankun Zhuang, Ranran Jia, Yinping Zhou, Yali Liu, Hongyun Wang.

  Aim: A Ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method was developed to detfermine LBPT as well as its four metabolites in human plasma to support the clinical study aiming to evaluate the efficacy of LBPT tablet in patients undergoing hip/knee replacement. Methodology: Plasma samples were prepared by protein precipitation and then separated on a C18 analytical column using (A) acetonitrile (B) 0.1% formic acid and 10 mM ammonium formate in water. The detection was performed on a triple quadrupole tandem mass spectrometer in positive electrospray ionization using multiple reactions monitoring mode. Results & conclusion: The method has been validated in accordance with the US FDA guidelines and was applied to the measurement of five analytes in human plasma samples from a Phase II clinical trial.

Keywords:  LBPT; UPLC–MS/MS; pharmacokinetics; platelet-activating factor

DOI:  https://doi.org/10.4155/bio-2019-0289
Analyst. 2020 Feb 19.

A sensitive method for the quantification of short-chain fatty acids by benzyl chloroformate derivatization combined with GC-MS.

Menghan Li, Rongrong Zhu, Xiaoxia Song, Zhijun Wang, Hongbo Weng, Jianying Liang.

Short-chain fatty acids (SCFAs) were identified as critical markers in the diagnosis of chronic and metabolic diseases, but a sensitive and stable method to determine SCFAs in feces is a challenge for analysts due to the high volatility. Herein, a sensitive and accurate method to determine SCFAs adopting precolumn derivatization coupled with gas chromatography-mass spectrometry (GC-MS) has been developed. Benzyl chloroformate (BCF) was chosen as the reaction reagent and emulsified derivatization was applied to homogenize the reaction system. Higher sensitivity, wider application and satisfactory derivatization efficiency were obtained using the developed method. An excellent method validation showed a good linearity ranging from 0.9947 to 0.9998. At the same time, the intra-day and inter-day precision were achieved in the range of 0.56% to 13.07%. The lower limits of detection of all target analytes varied from 0.1 to 5 pg. The recovery ranged from 80.87% to 119.03%, and storage stability under three different conditions was also determined. This method was also successfully applied to the analysis of SCFAs in mice fecal samples to illustrate the significant differences between normal and type 2 diabetes mellitus mice.

DOI: https://doi.org/10.1039/d0an00005a
Bioorg Chem. 2020 Feb 08. pii: S0045-2068(19)31632-3. [Epub ahead of print]96 103653

Enzymatic studies with 3-oxa n-3 DPA.

Maria K Pangopoulos, Jens M N Nolsøe, Simen G Antonsen, Romain A Colas, Jesmond Dalli, Marius Aursnes, Yngve Stenstrøm, Trond Vidar Hansen.

  Cyclooxygenase-2 and several lipoxygenases convert polyunsaturated fatty acids into a large variety of products. During inflammatory processes, these enzymes form several distinct families of specialized pro-resolving lipid mediators possessing potent anti-inflammatory and pro-resolving effects. These mediators have attracted a great interest as leads in drug discovery and have recently been the subject of biosynthetic pathway studies using docosahexaenoic and n-3 docosapentaenoic acid as substrates. Herein we present enzymatic studies with cyclooxygenase-2 and 5-, 12- and 15-lipoxygenase enzymes using 3-oxa n-3 DPA as a synthetic mimic of n-3 docosapentaenoic acid. Structural elucidation based on data from RP-HPLC UV and LC/MS-MS experiments enabled the identification of novel enzymatically formed products. These findings constitute the basis for further biosynthetic studies towards understanding the mechanisms regulating substrate utilization in the biosynthesis of specialized pro-resolving lipid mediators.

Keywords:  3-oxa n-3 DPA; Biosynthesis; Cyclooxygenase-2; Lipids; Lipoxygenases; Oxygenated products; Polyunsaturated fatty acids; Specialized pro-resolving lipid mediators

DOI:  https://doi.org/10.1016/j.bioorg.2020.103653
Food Chem Toxicol. 2020 Feb 17. pii: S0278-6915(20)30092-2. [Epub ahead of print] 111204

LC-MS/MS method for the determination of hormones: Validation, application and health risk assessment in various bovine matrices.

Fadl Moussa, Samia Mokh, Samah Doumiati, Barbara Barboni, Nicola Bernabò, Mohamad Al Iskandarani.

  A new method, using liquid chromatography coupled to mass spectrometry (LC-MS/MS) for the detection of fourteen natural and synthetic hormones in muscles, was validated in other bovine matrices (liver, kidney, bile and hair) according to the Decision Commission 2002/657/EC. As result, this method demonstrates good linearity (R2 > 0.99) as well as accuracy with coefficients of variation for repeatability and reproducibility lower than 23%. Moreover, the values of decision limit (CCα) and detection capability (CCβ) were determined indicating values ranging from 0.13 to 0.86 μg/kg and 0.25-1.72 μg/k for the majority of analytes. Recovery rate in the different matrices varied from 51.5 to 107%. Indeed, this method has been successfully applied to detect anabolic hormones in eighty-eight samples (muscle, liver, kidney, and bile) collected from different local slaughterhouses. Results showed that progesterone was found in 30 samples at concentrations ranging from 0.11 to 11.7 μg/kg, while testosterone was detected in 34 samples at concentrations ranging from 0.5 to 9.52 μg/kg. All bile samples contain epi-testosterone at concentration ranging from 0.89 to 280 μg/kg. These obtained data were used to calculate the estimated daily intake, hazard quotient and hazard index as exposure assessment.

Keywords:  Animal matrices; Hormones; Liquid chromatography tandem mass spectrometry; Matrix effect; QusEChERS; Validation

DOI:  https://doi.org/10.1016/j.fct.2020.111204
Molecules. 2020 Feb 13. pii: E805. [Epub ahead of print]25(4):

Analysis of Phospholipids, Lysophospholipids, and Their Linked Fatty Acyl Chains in Yellow Lupin Seeds (Lupinus luteus L.) by Liquid Chromatography and Tandem Mass Spectrometry.

Cosima Damiana Calvano, Mariachiara Bianco, Giovanni Ventura, Ilario Losito, Francesco Palmisano, Tommaso R I Cataldi.

  Hydrophilic interaction liquid chromatography (HILIC) and electrospray ionization (ESI) coupled to either Fourier-transform (FT) orbital-trap or linear ion-trap tandem mass spectrometry (LIT-MS/MS) was used to characterize the phospholipidome of yellow lupin (Lupinus luteus) seeds. Phosphatidylcholines (PC) were the most abundant species (41 ± 6%), which were followed by lyso-forms LPC (30 ± 11%), phosphatidylethanolamines (PE, 13 ± 4%), phosphatidylglycerols (PG, 5.1 ± 1.7%), phosphatidic acids (PA, 4.9 ± 1.8%), phosphatidylinositols (PI, 4.7 ± 1.1%), and LPE (1.2 ± 0.5%). The occurrence of both isomeric forms of several LPC and LPE was inferred by a well-defined fragmentation pattern observed in negative ion mode. An unprecedented characterization of more than 200 polar lipids including 52 PC, 42 PE, 42 PA, 35 PG, 16 LPC, 13 LPE, and 10 PI, is reported. The most abundant fatty acids (FA) as esterified acyl chains in PL were 18:1 (oleic), 18:2 (linoleic), 16:0 (palmitic), and 18:3 (linolenic) with relatively high contents of long fatty acyl chains such as 22:0 (behenic), 24:0 (lignoceric), 20:1 (gondoic), and 22:1 (erucic). Their occurrence was confirmed by reversed-phase (RP) LC-ESI-FTMS analysis of a chemically hydrolyzed sample extract in acid conditions at 100 °C for 45 min.

Keywords:  LC-ESI-tandem MS; Lupinus luteus; fatty acids; food; phospholipids

DOI:  https://doi.org/10.3390/molecules25040805
J Chromatogr Sci. 2020 Feb 22. pii: bmz124. [Epub ahead of print]

A Sensitive HPTLC Method for the Estimation of Glibenclamide, Rosiglitazone Maleate and Metformin Hydrochloride from a Multicomponent Dosage Form.

Swati D Bhende, Murali Balaram Varanasi, Konde Abbulu.

  A sensitive, rapid and cost-effective method based on HPTLC with UV detection was developed for the quantitation of Glibenclamide (GLIBEN), Rosiglitazone maleate (ROSI) and Metformin hydrochloride (MET) from a combined dosage form. Pre-coated RP-18 F254s aluminum sheets were used as the stationary phase. Methanol-tetrahydrofuran-water-glacial acetic acid (16: 3.6: 4: 0.4, v/v) used as the mobile phase, along with chamber saturation of 10 min offered an optimum migration (Rf = 0.54, 0.62 and 0.80 for GLIBEN, ROSI and MET, respectively). TLC Scanner 3 was used for densitometric evaluation of the chromatograms. DigiStore 2 Documentation System with winCATS software version 1.4.10 was used for the quantitation and photodocumentation. The LOD for GLIBEN, ROSI and MET was found to be 80 ng, 80 ng and 48 ng, respectively. Moreover, the LOQ was 200 ng, 200 ng and 120 ng for GLIBEN, ROSI and MET, respectively. The method was linear for GLIBEN (r = 0.9991), ROSI (r = 0.9993) and MET (r = 0.9988) within the tested range (200-1000, 200-1000 and 120-600 ng/band, respectively). The method was found to be precise and accurate for all the three drugs. The method was applied for the analysis of Triglucored tablets, and it proved to be a reliable quality control tool for the routine analysis of GLIBEN, ROSI and MET in a combined dosage form.

Keywords:  Glibenclamide; HPTLC; Metformin hydrochloride; Method validation; Rosiglitazone maleate

DOI:  https://doi.org/10.1093/chromsci/bmz124
J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Feb 03. pii: S1570-0232(19)30894-3. [Epub ahead of print]1141 122020

Simultaneous detection of 24 oral antidiabetic drugs and their metabolites in urine by liquid chromatography-tandem mass spectrometry.

Ying Hoo Lam, Mei Tik Leung, Chor Kwan Ching, Tony Wing Lai Mak.

  Drugs are the most frequent cause of hypoglycemia. Though the drug history is usually obvious in diabetic patients, the diagnosis could be a challenge in patients without a history of such exposure. Screening for oral antidiabetic drugs has been recommended as part of the hypoglycemia workup in patients without diabetes. Many published analytical methods of oral antidiabetic agents were usually of limited coverage and restricted to parent drugs only. In the current study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical system for the simultaneous detection of 24 oral antidiabetic drugs and their metabolites in urine was established and validated. The method covered both conventional as well as the newer antidiabetic drugs such as dipeptidyl peptidase-4 inhibitors and sodium-glucose cotransporter-2 inhibitors. Following sample preparation by solid phase extraction, analytes were detected by LC-MS/MS with multiple reaction monitoring triggered enhanced product ion scan. The method was successfully applied to 233 cases of unexplained hypoglycemia, with 83 oral antidiabetic drugs detected in 51 of the urine samples.

Keywords:  Drug screening; Drug-induced hypoglycemia; LC-MS/MS; Oral antidiabetic drug; Urine analysis

DOI:  https://doi.org/10.1016/j.jchromb.2020.122020
J Pharm Biomed Anal. 2020 Feb 04. pii: S0731-7085(19)31135-5. [Epub ahead of print]183 113147

Reversed phase UPLC/APCI-MS determination of Vitamin K1 and menaquinone-4 in human plasma: Application to a clinical study.

Yuning Zhang, Yashpal S Chhonker, Veenu Bala, Alex Hagg, Linda G Snetselaar, Terry L Wahls, Daryl J Murry.

  A rapid and sensitive liquid chromatographic-tandem mass spectrometric method was developed and validated to simultaneously quantitate vitamin K1 (PK) and vitamin K2 (MK-4) concentrations in human plasma to evaluate nutritional interventions. Charcoal-stripped human plasma was used for standard and calibration preparation with vitamin E-d6 as the internal standard. Patient plasma samples were extracted using n-hexane and analytes separated using an ACE -PFP C18 column (50 × 2.1 mm, 3 μ) equipped with standard C18 guard. The mobile phase consisted of 0.1 % formic acid in water and 0.1 % formic acid in methanol at a flow rate of 0.5 mL/min. Analyte quantification was achieved by using MS/MS with a positive atmospheric pressure chemical ionization in multiple reaction monitoring (MRM) mode. The lower limit of quantification was 0.01 ng/mL for both PK and MK-4. The assay was linear over the concentration range from 0.01-50 ng/mL for all analytes with a determination coefficient (r2) of 0.998 or better. The intra-and inter-day accuracy and precision were within the acceptable limits per FDA guidance. The validated method was successfully applied to a clinical study for quantification of PK and MK-4 in human plasma to assess nutritional status and dietary interventions in patients with neurodegenerative diseases. Baseline plasma concentrations of PK and MK-4 ranged from 0.23 to 1.81 ng/mL and 0.33-0.57 ng/mL, respectively.

Keywords:  Charcoal-stripped human plasma; Liquid chromatography-tandem mass spectrometry; Liquid-liquid extraction; Menaquinones; Phylloquinone; Vitamin K

DOI:  https://doi.org/10.1016/j.jpba.2020.113147
Anal Bioanal Chem. 2020 Feb 20.

Simultaneous quantification of free fatty acids and acylcarnitines in plasma samples using dansylhydrazine labeling and liquid chromatography-triple quadrupole mass spectrometry.

Guan-Yuan Chen, Qibin Zhang.

  Free fatty acid (FFA) and acylcarnitine (AcCar) are key elements of energy metabolism. Dysregulated levels of FFA and AcCar are associated with genetic defects and other metabolic disorders. Due to differences in the physicochemical properties of these two classes of compounds, it is challenging to quantify FFA and AcCar in human plasma using a single method. In this work, we developed a chemical isotope labeling (CIL)-based liquid chromatography-multiple reaction monitoring (LC-MRM) method to simultaneously quantify FFA and AcCar. Dansylhydrazine (DnsHz) was used to label the carboxylic acid moiety on FFA and AcCar. This resulted in the formation of a permanently charged ammonium ion for facile ionization in positive ionization mode and higher hydrophobicity for enhanced retention of short-chain analogs on reversed-phase LC columns and enabled absolute quantification by using heavy labeled DnsHz analogs as internal standards. Labeling conditions including the concentration and freshness of cross-linker, reaction time, and temperature were optimized. This method can successfully quantify all short-, medium- and long-chain FFAs and AcCars with greatly enhanced sensitivity. Using this method, 25 FFAs and 13 AcCars can be absolutely quantified and validated in human plasma samples within 12 min. Simultaneous quantification of FFA and AcCar enabled by this CIL-based LC-MRM method facilitates the investigation of fatty acid metabolism and has potential in clinical applications.

Keywords:  Acylcarnitine; Chemical isotope labeling; Free fatty acid; Human plasma; LC-MRM-MS; Targeted metabolomics

DOI:  https://doi.org/10.1007/s00216-020-02514-x
J Anal Toxicol. 2020 Feb 12. pii: bkaa012. [Epub ahead of print]

LC-MS/MS METHOD FOR THE DETERMINATION OF ANTIDEPRESSANTS AND BENZODIAZEPINES IN MECONIUM.

A López-Rabuñal, E Lendoiro, M Concheiro, M López-Rivadulla, A Cruz, A de-Castro-Ríos.

  An LC-MS/MS method for the determination of 14 benzodiazepines (alprazolam, α-hydroxyalprazolam, clonazepam, bromazepam, diazepam, nordiazepam, lorazepam, lormetazepam, oxazepam, flunitrazepam, 7-aminoflunitrazepam, triazolam, midazolam and zolpidem) and 15 antidepressants (amitriptyline, nortriptyline, imipramine, desipramine, clomipramine, norclomipramine, fluoxetine, norfluoxetine, sertraline, norsertraline, paroxetine, venlafaxine, desmethylvenlafaxine, citalopram and desmethylcitalopram) in meconium was developed and validated. Meconium samples (0.25 ± 0.02 g) were homogenized in methanol and subjected to mixed-mode cation exchange solid-phase extraction. Chromatographic separation was performed in reversed-phase, with a gradient of 0.1% formic acid in 2 mM ammonium formate and acetonitrile. Two different chromatographic gradient methods were employed, one for the separation of antidepressants and another for benzodiazepines. Analytes were monitored by tandem mass spectrometry employing ESI+ in MRM mode (2 transitions per compound). Method validation included: linearity (n = 5, LOQ to 400 ng/g), limits of detection (n = 6, 1-20 ng/g), limits of quantification (n = 9, 5-20 ng/g), selectivity (no endogenous or exogenous interferences), accuracy (n = 15, 90.6-111.5%), imprecision (n = 15, 0-14.6%), matrix effect (n = 10, -73% to 194.9%), extraction efficiency (n = 6, 35.9-91.2%), process efficiency (n = 6, 20.1-188.2%), stability 72 h in the autosampler (n = 3, -8.5% to 9%) and freeze/thaw stability (n = 3, -1.2 to -47%). The method was applied to 4 meconium specimens, which were analysed with and without hydrolysis (enzymatic and alkaline). The authentic meconium samples tested positive for alprazolam, α-hydroxyalprazolam, clonazepam, diazepam, nordiazepam, fluoxetine, norfluoxetine, clomipramine and norclomipramine. Therefore, the present LC-MS/MS method allows a high throughput determination of the most common benzodiazepines and antidepressants in meconium, which could be useful in clinical and forensic settings.

Keywords:  Antidepressant; LC-MS/MS; benzodiazepine; in utero exposure; meconium; pregnancy

DOI:  https://doi.org/10.1093/jat/bkaa012
Molecules. 2020 Feb 17. pii: E887. [Epub ahead of print]25(4):

Untargeted HILIC-MS-Based Metabolomics Approach to Evaluate Coffee Roasting Process: Contributing to an Integrated Metabolomics Multiplatform.

Raquel Pérez-Míguez, María Castro-Puyana, Elena Sánchez-López, Merichel Plaza, María Luisa Marina.

  An untargeted metabolomics strategy using hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) was developed in this work enabling the study of the coffee roasting process. Green coffee beans and coffee beans submitted to three different roasting degrees (light, medium, and strong) were analyzed. Chromatographic separation was carried out using water containing 10 mM ammonium formate with 0.2 % formic acid (mobile phase A) and acetonitrile containing 10 mM ammonium formate with 0.2 % formic acid (mobile phase B). A total of 93 molecular features were considered from which 31 were chosen as the most statistically significant using variable in the projection values. 13 metabolites were tentatively identified as potential biomarkers of the coffee roasting process using this metabolomic platform. Results obtained in this work were complementary to those achieved using orthogonal techniques such as reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) and capillary electrophoresis-mass spectrometry (CE-MS) since only one metabolite was found to be common between HILIC-MS and RPLC-MS platforms (caffeoylshikimic acid isomer) and other between HILIC-MS and CE-MS platforms (choline). On the basis of these results, an untargeted metabolomics multiplatform is proposed in this work based on the integration of the three orthogonal techniques as a powerful tool to expand the coverage of the roasted coffee metabolome.

Keywords:  HILIC; coffee roasting process; mass spectrometry; multiplatform; untargeted metabolomics

DOI:  https://doi.org/10.3390/molecules25040887
Analyst. 2020 Feb 17.

Paper spray mass spectrometry for the analysis of picoliter droplets.

Ravleen Kaur Kohli, James F Davies.

Recent experimental efforts have shown that single particle levitation methods may be effectively coupled with mass spectrometry (MS) using paper spray (PS) ionization for compositional analysis of picoliter droplets. In this work, we characterize the response of PS-MS to analytes delivered in the form of picoliter droplets and explore its potential for identification and quantification of these samples. Using a microdroplet dispenser to generate droplets, we demonstrate sensitivity to a range of oxygenated organic molecules typical of compounds found in atmospheric secondary organic aerosol. We assess experimental factors that influence the reproducibility and sensitivity of the method and explore the linearity of the system response to increasing analyte mass in droplets containing single or multicomponent analytes. We show that the ratio of analyte signal from multicomponent samples may be used to characterize the relative composition of the system. These measurements demonstrate that the droplet PS-MS method is an effective tool for qualitative and quantitative analysis of single picoliter droplets containing picogram levels of analyte. The potential applications of this technique for characterizing the composition of levitated particles will be discussed.

DOI: https://doi.org/10.1039/c9an02534k
Anal Chim Acta. 2020 Mar 22. pii: S0003-2670(19)31524-7. [Epub ahead of print]1103 122-133

Rapid determination of isoflavones and other bioactive compounds in soybean using SWATH-MS.

Han-Ju Chien, Chang-Sheng Wang, Yu-Hsun Chen, Jie-Teng Toh, Yi-Feng Zheng, Xiang-Gui Hong, Hung-Yu Lin, Chien-Chen Lai.

  Isoflavones are the major bioactive components in soybeans. Sequential window acquisition of all theoretical fragment ions (SWATH) is a kind of data-independent acquisition (DIA), such that all fragments of each precursor will be preserved in a SWATH-Mass Spectrometry (SWATH-MS) run. In this study, a high-throughput SWATH-MS method for the determination of 12 isoflavones in soybeans was established. Furthermore, amino acids, saponins can be semi-quantitated from the same SWATH-MS data. Combination of targeted quantification and untargeted profiling with SWATH, all bioactive compounds were analyzed within 5 min in 10 min run time, and the method had good linear regression with r2 > 0.99. The precisions (RSD %) of the intra-day and inter-day analyses ranged from 2.11% to 18.7%, and the accuracies (RE%) ranged from -14.39% to 17.48%. The matrix effect ranged from 88.66% to 114.82%. Moreover, 7 varieties of soybeans were analyzed and compared with this robust screening method.

Keywords:  Amino acid; High-throughput; Isoflavone; SWATH-MS; Saponin; Soybean

DOI:  https://doi.org/10.1016/j.aca.2019.12.054
Clin Chem Lab Med. 2020 Feb 18. pii: /j/cclm.ahead-of-print/cclm-2019-0916/cclm-2019-0916.xml. [Epub ahead of print]

The pathway through LC-MS method development: in-house or ready-to-use kit-based methods?

Caroline Le Goff, Jordi Farre-Segura, Violeta Stojkovic, Patrice Dufour, Stéphanie Peeters, Justine Courtois, Adrien Nizet, Nathalie De Vos, Etienne Cavalier.

  Historically, the determination of low concentration analytes was initially made possible by the development of rapid and easy-to-perform immunoassays (IAs). Unfortunately, typical problems inherent to IA technologies rapidly appeared (e.g. elevated cost, cross-reactivity, lot-to-lot variability, etc.). In turn, liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are sensitive and specific enough for such analyses. Therefore, they would seem to be the most promising candidates to replace IAs. There are two main choices when implementing a new LC-MS/MS method in a clinical laboratory: (1) Developing an in-house method or (2) purchasing ready-to-use kits. In this paper, we discuss some of the respective advantages, disadvantages and mandatory requirements of each choice. Additionally, we also share our experiences when developing an in-house method for cortisol determination and the implementation of an "ready-to-use" (RTU) kit for steroids analysis.

Keywords:  in-house method; kit ready to use; mass spectrometry

DOI:  https://doi.org/10.1515/cclm-2019-0916
Rapid Commun Mass Spectrom. 2020 Feb 17. e8759

Protein precipitation coupled to paper spray with a tube for one-step analysis of blood.

Mengjie Dai, Guangming Huang.

RATIONALE: Accurate measurement of trace compounds in blood sample is of great importance in clinical diagnosis and life science. Ambient ionization mass spectrometry, however, suffers from a matrix effect when dealing with complex samples such as blood. Hence, it is of great importance to reduce the matrix effects in blood sample, as is described in this study.METHODS: A low-cost and disposable Teflon tube was used as a platform to precipitate the protein in blood. The analytes were extracted into organic solvent and the precipitated protein can be adsorbed by the chromatography paper inserted. Hence, the Teflon tube after precipitating can be directly subjected to paper spray ionization mass spectrometry, achieving one-step analysis of blood.
RESULTS: High sensitivity and satisfactory stability were achieved when analyzing pharmaceuticals, acids and endogenic metabolites in blood. The absolute signal intensities of characteristic product ions of the tested analytes were 8-20 times higher with a tube than those obtained from paper spray. Detection limits and quantitative performance were evaluated for three drugs: carbamazepine, metformin and tioconazole. In addition, the limits of detection and quantitation were improved 9-14 and 8-12 fold, respectively.
CONCLUSIONS: Protein precipitation coupled to paper spray with a tube and then to mass spectrometry has been successfully achieved and applied in the one-step analysis of trace compounds in blood sample. The experimental results showed that this method was sensitive, stable, convenient and economic for the direct analysis of blood.

DOI: https://doi.org/10.1002/rcm.8759
Talanta. 2020 May 01. pii: S0039-9140(19)31301-3. [Epub ahead of print]211 120668

Non-targeted discovery-based analysis for gas chromatography with mass spectrometry: A comparison of peak table, tile, and pixel-based Fisher ratio analysis.

Chris E Freye, Patrick R Bowden, Margo T Greenfield, Bryce C Tappan.

  The ability to discover minute differences between samples or sample classes for gas chromatography coupled to mass spectrometry (GC-MS) can be a challenging endeavor, especially when those differences are not a priori. Fisher ratio (F-ratio) analysis is an apt technique to probe the differences between GC-MS chromatograms. F-ratio analysis is a supervised, non-targeted, discovery-based method that compares two different samples (or sample classes) to reduce the GC-MS dataset into a hit list composed of class distinguishing compounds. Three different F-ratio techniques, peak table, tile, and pixel-based were used to "discover" nine non-native analytes that were spiked into gasoline at four different nominal concentrations of 250, 85, 25, 5 parts-per-million (ppm). For the tile and pixel-based F-ratio calculations, a novel methodology is introduced to improve the sensitivity of the F-ratio calculations while reducing false positives. Furthermore, we use a combinatorial technique using null class comparisons, termed null distribution analysis, to determine a statistical F-ratio cutoff for analysis of the hit lists. The pixel-based algorithm was the most sensitive method and was able to "discover" all nine spiked analytes at a nominal concentration of 250 ppm albeit with one false positive interspersed towards the bottom of the hit list. The pixel-based software was also able to "discover" more of the spiked analytes at the lower concentrations with seven of the spiked analytes "discovered" at 85 ppm, four of the spiked analytes "discovered" at 25 ppm, and one analyte "discovered" at 5 ppm.

Keywords:  ANOVA; Fisher ratio; GC-MS; Gas chromatography; Mass spectrometry

DOI:  https://doi.org/10.1016/j.talanta.2019.120668
Nat Commun. 2020 Feb 17. 11(1): 926

A computational platform for high-throughput analysis of RNA sequences and modifications by mass spectrometry.

Samuel Wein, Byron Andrews, Timo Sachsenberg, Helena Santos-Rosa, Oliver Kohlbacher, Tony Kouzarides, Benjamin A Garcia, Hendrik Weisser.

The field of epitranscriptomics continues to reveal how post-transcriptional modification of RNA affects a wide variety of biological phenomena. A pivotal challenge in this area is the identification of modified RNA residues within their sequence contexts. Mass spectrometry (MS) offers a comprehensive solution by using analogous approaches to shotgun proteomics. However, software support for the analysis of RNA MS data is inadequate at present and does not allow high-throughput processing. Existing software solutions lack the raw performance and statistical grounding to efficiently handle the numerous modifications found on RNA. We present a free and open-source database search engine for RNA MS data, called NucleicAcidSearchEngine (NASE), that addresses these shortcomings. We demonstrate the capability of NASE to reliably identify a wide range of modified RNA sequences in four original datasets of varying complexity. In human tRNA, we characterize over 20 different modification types simultaneously and find many cases of incomplete modification.

DOI: https://doi.org/10.1038/s41467-020-14665-7
Proteomics. 2020 Feb 22. e1900143

CalibraCurve: A Tool for Calibration of Targeted MS-based Measurements.

Michael Kohl, Markus Stepath, Thilo Bracht, Dominik A Megger, Barbara Sitek, Katrin Marcus, Martin Eisenacher.

  Targeted proteomics techniques allow accurate quantitative measurements of analytes in complex matrices with dynamic linear ranges that span up to 4-5 orders of magnitude. Hence, targeted methods are promising for the development of robust protein assays in several sensitive areas, e.g. in health care. However, exploiting the full method potential requires reliable determination of the dynamic range along with related quantification limits for each analyte. Here, we present a software named CalibraCurve that enables an automated batch-mode determination of dynamic linear ranges and quantification limits for both targeted proteomics and similar assays. The software uses a variety of measures to assess the accuracy of the calibration, namely precision and trueness. Two different kinds of customizable graphs are created (calibration curves and response factor plots). The accuracy measures and the graphs offer an intuitive, detailed and reliable opportunity to assess the quality of the model fit. Thus, we deem CalibraCurve a highly useful and flexible tool to facilitate the development and control of reliable SRM/MRM-MS-based proteomics assays. This article is protected by copyright. All rights reserved.

Keywords:  MRM; calibration; limit of quantification; linear range; targeted proteomics

DOI:  https://doi.org/10.1002/pmic.201900143
Talanta. 2020 May 01. pii: S0039-9140(20)30029-1. [Epub ahead of print]211 120738

Multi-residue Ultra Performance Liquid Chromatography-High resolution mass spectrometric method for the analysis of 21 cyanotoxins in surface water for human consumption.

Giorgia Di Pofi, Gabriele Favero, Federica Nigro Di Gregorio, Emanuele Ferretti, Emanuela Viaggiu, Luca Lucentini.

  The presence of cyanobacteria and their toxins in water used as drinking water or for recreational purposes may represent a risk for human health. This work describes the development of an advanced analytical method for simultaneous determination of 21 cyanotoxins (including Microcystins, Cyanopeptolins, Anabaenopeptins and Microginins) in drinking water based on Ultra Performance Liquid Chromatography coupled with a Q-TOF mass spectrometer. Water samples, spiked with Nodularin as internal standard at 1 μg/L, were extracted using Carbograph 4 SPE cartridge and 10 μL of the extracted sample were injected into the UPLC-HRMS/MS system. Analytes separation was obtained using a UPLC C18 column, acetonitrile and water as mobile phases, both containing 10 mM formic acid, and operating in positive ionization mode and sensitivity mode. The method has been proven to be robust, precise and accurate with recovery percentages above 85% and with relative standard deviations ≤16% and LODs between 0.002 and 0.047 μg/L, fitting for the intended purposes at the concentrations of interest. This method was applied during a monitoring activity in an Italian volcanic lake in Viterbo (Lazio Region, Italy), due to a severe algal proliferation in January 2018-March 2019 period and for the assessment of cyanobacteria proliferation risk and of cyanotoxin production in drinking water chain.

Keywords:  Cyanotoxins; Drinking water; High resolution mass spectrometry; Microcystins; Water sample

DOI:  https://doi.org/10.1016/j.talanta.2020.120738
J Anal Toxicol. 2020 Feb 12. pii: bkaa013. [Epub ahead of print]

Quantification of Furanyl Fentanyl and its Metabolites in Human and Rat Plasma using LC-MS/MS.

Kaitlyn B Palmquist, Madeleine Swortwood.

  Fentanyl analogs (novel and traditional) continue to impact the ever-growing opioid epidemic. Furanyl fentanyl is one analog equipotent to fentanyl that has documented involvement in thousands of intoxication and fatality cases around the world. Due to its prevalence, toxicologists need to improve detection and understanding of this analog. A method for the quantification of furanyl fentanyl (FuF) and its metabolites (4-ANPP, furanyl norfentanyl (FuNorF)) in a small volume (100 μL) of human plasma by LC-MS/MS was developed and validated according to ANSI/ASB Standard. The method was cross-validated in rat plasma for a future pharmacokinetic/pharmacodynamic study. In human plasma, calibration ranges were 0.025-25 ng/mL (FuF and 4-ANPP) and 0.5-25 ng/mL (FuNorF). LOD were 0.0125 ng/mL (FuF and 4-ANPP) and 0.25 ng/mL (FuNorF). LLOQ coincided with lowest calibrator concentrations of 0.025 ng/mL (FuF and 4-ANPP) and 0.5 ng/mL (FuNorF). Precision and bias values were determined to be acceptable for all analytes. Matrix effects were acceptable for all analytes (-8.6-25.0%), except FuNorF with suppression > 25%. Extraction recoveries ranged from 84.5-98.1%. No carryover or endogenous interferences were observed. Qualitative interferences with 4-ANPP were observed from some n-acyl substituted fentanyl analogs predicted to be low concentration standard impurities. Analytes were stable under all conditions and dilution integrity was sustained. The method was successfully cross-validated in rat plasma with acceptable bias (-7.4-4.4%), precision (within-run < 19% CV and between-run < 12.6% CV), matrix effects (-9.3-17.2%, except FuNorF with > 25% suppression), recoveries (79.2-94.5%), and dilution integrity (1/2 and 1/10).

Keywords:  Fentanyl analogs; Furanyl fentanyl; LC-MS/MS; Novel Synthetic Opioids

DOI:  https://doi.org/10.1093/jat/bkaa013