bims-metlip 2023-01-15 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–01–15
33 papers selected by
Sofia Costa, Matterworks

Novel Matrix Normalization Technique for Accurate LC-ESI-MS/MS Detection and Quantification of Drugs and Their Metabolites in Toxicology Research and Practice.
Systematic analysis of in-source modifications of primary metabolites during flow-injection time-of-flight mass spectrometry.
Semi-automated serum steroid profiling with tandem mass spectrometry.
SiMeEx, a simplified method for metabolite extraction of adherent mammalian cells.
Inclusion of 11-Oxygenated Androgens in a Clinical Routine LC-MS/MS Setup for Steroid Hormone Profiling.
Analytical Performance Evaluation for Estradiol using Liquid Chromatography-Tandem Mass Spectrometry.
On-Tissue Chemical Oxidation Followed by Derivatization for Mass Spectrometry Imaging Enables Visualization of Primary and Secondary Hydroxyl-Containing Metabolites in Biological Tissues.
Rapid quantification of seven major neonicotinoids and neonicotinoid-like compounds and their key metabolites in human urine.
Comparison of Workflows for Milk Lipid Analysis: Phospholipids.
Segmented Flow Strategies for Integrating Liquid Chromatography-Mass Spectrometry with Nuclear Magnetic Resonance for Lipidomics.
Development of a matrix-based candidate reference material for human glycated albumin using isotope-dilution liquid chromatography/tandem mass spectrometry.
NMR Metabolomics Methods for Investigating Disease.
Comprehensive Chemical Characterization of Chia (Salvia hispanica L.) Seed Oil with a Focus on Minor Lipid Components.
Establishment of Korean Pediatric Reference Intervals for Estradiol using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.
[A Method for Quantitative Determination of Amino Acids and Organic Acids in Trace Random Urine and Preliminary Application in Research on Vitamin B12 Nutritional Status and Related Metabolism].
[Determination of organophosphate diesters in facility vegetable soils using ultra-high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry].
[Determination of five amanita peptide toxins in poisonous mushrooms by ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometry].
A strategy for screening and identification of new amino acid-conjugated bile acids with high coverage by liquid chromatography-mass spectrometry.
Examination of human osteoarchaeological remains as a feasible source of polar and apolar metabolites to study past conditions.
Simultaneous Measurement of Cortisol, Cortisone, Dexamethasone and Additional Exogenous Corticosteroids by Rapid and Sensitive LC-MS/MS Analysis.
The Development of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Biogenic Amines in Fish Samples.
Tandem Mass Spectrometry in the Analysis of Petroleum-Based Compounds.
Sample preparation and chromatographic methods for the determination of protein-bound uremic retention solutes in human biological samples: An overview.
Application of High-Performance Liquid Chromatography Combined with Fluorescence Detector and Dispersive Liquid-Liquid Microextraction to Quantification of Selected Bisphenols in Human Amniotic Fluid Samples.
Screening method for the simultaneous determination of allantoin and uric acid from dried blood spots.
Technical note: Simultaneous determination of amino thiols in pig tissue by ultra-high performance liquid chromatography with fluorescence detection.
A Comparative Study on the Reduction Modes for Quinone to Determine Ubiquinone by HPLC with Luminol Chemiluminescence Detection Based on the Redox Reaction.
Simultaneous quantification of thalidomide, lenalidomide and pomadomide in plasma by LC-MS/MS.
[Simultaneous determination of 28 fragrance components in cosmetics by gas chromatography-tandem mass spectrometry].
Selective Schiff base formation via gas-phase ion/ion reactions to enable differentiation of isobaric lipids in imaging mass spectrometry.
Online coupling of matrix solid-phase dispersion to direct analysis in real time mass spectrometry for high-throughput analysis of regulated chemicals in consumer products.
Development of a 3D-Printed Ionization Source for Single-Cell Analysis.
Development of the Chromatographic Method for Simultaneous Determination of Azaperone and Azaperol in Animal Kidneys and Livers.

Curr Protoc. 2023 Jan;3(1): e644

Novel Matrix Normalization Technique for Accurate LC-ESI-MS/MS Detection and Quantification of Drugs and Their Metabolites in Toxicology Research and Practice.

Thomas G Rosano, John M Rumberger, Kiley L Scholz, Robert M Konetchy, Michelle Wood.

  Accurate identification and quantification of drugs and their metabolites (analytes) in biological matrices is an analytical foundation of clinical and forensic toxicology. For decades, liquid chromatography interfaced by electrospray ionization with tandem mass spectrometry (LC-ESI-MS/MS) has been a widely used technology for analysis in the field of toxicology, as well as in many other fields of bioscience. It is also known that ion response in LC-ESI-MS/MS analysis is influenced by coeluting biological compounds and that preanalytical sample clean-up is often insufficient in removing these interferences. As a result, a normalization technique is commonly used for assessment and compensation of matrix effects encountered in routine analysis. Internal standardization with a stable isotope analog of the analyte is the predominant normalization technique used in LC-ESI-MS/MS analysis. The technique, however, requires commercial availability or costly custom synthesis of an isotopic analog specific for each analyte. Here we describe an alternative technique for matrix normalization for use in high-volume, multianalyte testing without the need for isotope analogs. The technique involves analysis of the original sample (neat analysis) followed by analysis of a second sample aliquot (spike analysis) that has been fortified with a controlled amount of reference analyte. A calibration procedure similar to internal standardization is employed, using an ion response ratio of neat to fortified analyte. As a demonstration of the technique in multianalyte testing, we provide a detailed protocol for simultaneous detection and quantification of 102 drugs and drug metabolites in human urine. We also provide a support protocol for addition of new analytes to the multianalyte panel, allowing convenient collection of the validation data during routine testing. The matrix normalization technique and testing principles may be applicable to a wide range of analytes and biological matrices, not only those encountered in toxicology but also in other fields of bioscience. © 2023 Wiley Periodicals LLC. Basic Protocol: Detection and quantification of 102 toxicology analytes in urine by LC-ESI-MS/MS analysis using the threshold accurate calibration technique Support Protocol: Method for addition and validation of new analytes to expand the Basic Protocol.

Keywords:  TAC; electrospray ionization; liquid chromatography; matrix effect normalization; tandem mass spectrometry; threshold accurate calibration

DOI:  https://doi.org/10.1002/cpz1.644
Anal Biochem. 2023 Jan 07. pii: S0003-2697(23)00001-5. [Epub ahead of print] 115036

Systematic analysis of in-source modifications of primary metabolites during flow-injection time-of-flight mass spectrometry.

Niklas Farke, Thorben Schramm, Andreas Verhülsdonk, Johanna Rapp, Hannes Link.

  Flow-injection mass spectrometry (FI-MS) enables metabolomics studies with a very high sample-throughput. However, FI-MS is prone to in-source modifications of analytes because samples are directly injected into the electrospray ionization source of a mass spectrometer without prior chromatographic separation. Here, we spiked authentic standards of 160 primary metabolites individually into an Escherichia coli metabolite extract and measured the thus derived 160 spike-in samples by FI-MS. Our results demonstrate that FI-MS can capture a wide range of chemically diverse analytes within 30 seconds measurement time. However, the data also revealed extensive in-source modifications. Across all 160 spike-in samples, we identified significant increases of 11,013 ion peaks in positive and negative mode combined. To explain these unknown m/z features, we connected them to the m/z feature of the (de-)protonated metabolite using information about mass differences and MS2 spectra. This resulted in networks that explained on average 49% of all significant features. The networks showed that a single metabolite undergoes compound specific and often sequential in-source modifications like adductions, chemical reactions, and fragmentations. Our results show that FI-MS generates complex MS1 spectra, which leads to an overestimation of significant features, but neutral losses and MS2 spectra explain many of these features.

Keywords:  Electrospray ionization; Feature network; Flow-injection mass spectrometry; In-source modifications; Metabolomics

DOI:  https://doi.org/10.1016/j.ab.2023.115036
J Mass Spectrom Adv Clin Lab. 2023 Jan;27 40-48

Semi-automated serum steroid profiling with tandem mass spectrometry.

Sophie Rakete, Tom Schubert, Michael Vogeser.

   Objectives: Highly selective and sensitive multi-analyte methods for the analysis of steroids are attractive for the diagnosis of endocrine diseases. Commercially available kits are increasingly used for this purpose. These methods involve laborious solid phase extraction, and the respective panels of target analytes are incomplete. We wanted to investigate whether an improvement of kit solutions is possible by introducing automated on-line solid phase extraction (SPE) and combining originally separate analyte panels.
Methods: Sample preparation was performed using automated on-line SPE on a high-pressure stable extraction column. Chromatographic separation, including isobaric compounds, was achieved using a 0.25 mM ammonium fluoride-methanol gradient on a small particle size biphenyl column. Standard compounds and internal standard mixtures of two panels of a commercially available kit were combined to achieve an optimized and straightforward detection of 15 endogenous steroids. Validation was performed according to the European Medicines Agency (EMA) guidelines with slight modifications.
Results: Validation was successfully performed for all steroids over a clinically relevant calibration range. Deviations of intra- and inter-assay accuracy and precision results passed the criteria and no relevant matrix effects were detected due to highly effective sample preparation. External quality assessment samples showed the applicability as a routine diagnostic method, which was affirmed by the analyses of anonymized clinical samples.
Conclusions: It was found possible to complement a commercially available kit for quantitative serum steroid profiling based on isotope dilution LC-MS/MS by implementing automated on-line SPE, thereby improving the practicality and robustness of the measurement procedure.

Keywords:  A4, Androstendione; ALDO, Aldosterone; APCI, Atmospheric pressure chemical ionization; CAH, Congenital adrenal hyperplasia; CE, Collision energy; CE-IVD, Certified in-vitro-diagnostic device; CV, Coefficient of variation; DHEA, Dehydro-epiandrosterone; DHEA-S, Dehydro-epiandrosterone sulfate; DHT, Dihydrotestosterone; DOC, 11-deoxycorticosterone; E, Cortisone; E2, Estradiol; EMA, European Medicines Agency; EQA, External quality assessment; ESI, Electrospray ionisation; F, Cortisol; IVD, In-vitro-diagnostic; IVDR, EU In vitro Diagnostic Regulation; LC, Liquid chromatography; LC–MS/MS, Liquid chromatography tandem mass spectrometry; LDT, Laboratory developed test; LLOQ, Lower limit of quantification; MRM, Multiple reaction monitoring; On-line solid phase extraction (SPE); P4, Progesterone; QC, Quality control; Robustness; S/N, Signal-to-noise ratio; SID, Stable-isotope dilution; SPE, Solid phase extraction; SST, System suitability test; Serum steroid profiling; Stable-isotope dilution liquid chromatography-tandem mass spectrometry (SID LC-MS/MS); UHPLC, Ultra high performance liquid chromatography; ULOQ, Upper limit of quantification

DOI:  https://doi.org/10.1016/j.jmsacl.2022.12.006
Front Mol Biosci. 2022 ;9 1084060

SiMeEx, a simplified method for metabolite extraction of adherent mammalian cells.

Antonia Henne, Anna Vigh, Andre Märtens, Yannic Nonnenmacher, Melanie Ohm, Shirin Hosseini, Tushar H More, Mario A Lauterbach, Hendrikus Garritsen, Martin Korte, Wei He, Karsten Hiller.

  A reliable method for metabolite extraction is central to mass spectrometry-based metabolomics. However, existing methods are lengthy, mostly due to the step of scraping cells from cell culture vessels, which restricts metabolomics in broader application such as lower cell numbers and high-throughput studies. Here, we present a simplified metabolite extraction (SiMeEx) method, to efficiently and quickly extract metabolites from adherent mammalian cells. Our method excludes the cell scraping step and therefore allows for a more efficient extraction of polar metabolites in less than 30 min per 12-well plate. We demonstrate that SiMeEx achieves the same metabolite recovery as using a standard method containing a scraping step, in various immortalized and primary cells. Omitting cell scraping does not compromise the performance of non-targeted and targeted GC-MS analysis, but enables metabolome analysis of cell culture on smaller well sizes down to 96-well plates. Therefore, SiMeEx demonstrates advantages not only on time and resources, but also on the applicability in high-throughput studies.

Keywords:  GC-MS; mammalian cells; metabolite extraction; metabolomics; stable isotope labeling

DOI:  https://doi.org/10.3389/fmolb.2022.1084060
Int J Mol Sci. 2022 Dec 29. pii: 539. [Epub ahead of print]24(1):

Inclusion of 11-Oxygenated Androgens in a Clinical Routine LC-MS/MS Setup for Steroid Hormone Profiling.

Robert Zeidler, Ronald Biemann, Uta Ceglarek, Jürgen Kratzsch, Berend Isermann, Alexander Gaudl.

  11-Oxygenated androgens (11-OAs) are being discussed as potential biomarkers in diagnosis and therapy control of disorders with androgen excess such as congenital adrenal hyperplasia and polycystic ovary syndrome. However, quantification of 11-OAs by liquid chromatography-tandem mass spectrometry (LC-MS/MS) still relies on extensive sample preparation including liquid-liquid extraction, derivatization and partial long runtimes, which is unsuitable for high-throughput analysis under routine laboratory settings. For the first time, an established online-solid-phase extraction-LC-MS/MS (online-SPE-LC-MS/MS) method for the quantitation of seven serum steroids in daily routine use was extended and validated to include 11-ketoandrostenedione, 11-ketotestosterone, 11β-hydroxyandrostenedione and 11β-hydroxytestosterone. Combining a simple protein precipitation step with fast chromatographic separation and ammonium fluoride-modified ionization resulted in a high-throughput method (6.6 min run time) featuring lower limits of quantification well below endogenous ranges (63-320 pmol/L) with recoveries between 85% and 117% (CVs ≤ 15%). Furthermore, the ability of this method to distinguish between adrenal and gonadal androgens was shown by comparing 11-OAs in patients with hyperandrogenemia to healthy controls. Due to the single shot multiplex design of the method, potential clinically relevant ratios of 11-OAs and corresponding androgens were readily available. The fully validated method covering endogenous concentration levels is ready to investigate the diagnostic values of 11-OAs in prospective studies and clinical applications.

Keywords:  11-oxygenated androgens; LC-MS/MS; androgens; method validation; steroid hormones

DOI:  https://doi.org/10.3390/ijms24010539
Clin Biochem. 2023 Jan 07. pii: S0009-9120(23)00003-6. [Epub ahead of print]113 59-63

Analytical Performance Evaluation for Estradiol using Liquid Chromatography-Tandem Mass Spectrometry.

Eun Jeong Won, Ahram Yi, Young Jin Ko.

   BACKGROUND: There is an increased need for the sensitive and accurate measurement of estradiol levels in patients with estradiol-related endocrine disorders. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a reliable and accurate method for measuring steroid hormone levels. Here, we aimed to establish an LC-MS/MS-based method to quantify estradiol levels without sample derivatization and evaluated its analytical performance.
METHODS: Sciex Triple Quad 6500 + LC-MS/MS was used for estradiol analysis. We evaluated its analytical performance, including linearity, precision, the lower limit of detection and quantification (LoD and LoQ, respectively), accuracy, and carryover. The estradiol results determined by LC-MS/MS were compared with those obtained using a chemiluminescent microparticle immunoassay.
RESULTS: The LC-MS/MS output was linear for serum estradiol concentrations in the range of 0.2-10311.6 pmol/L. The intra-laboratory precision (coefficient of variation) was 3.0-10.1 %. The LoD and LoQ were 2.8 and 7.5 pmol/L, respectively. The overall accuracy was within 15 % of bias, and the carryover was within the acceptable range (<1.0 %). The results of the estradiol analysis determined by LC-MS/MS were comparable to those obtained by the chemiluminescent microparticle immunoassay (r2 = 0.9843), although there was a negative bias of - 17.82 (95 % confidence interval, -27.21 to - 8.44).
CONCLUSIONS: A highly sensitive, derivatization-free LC-MS/MS method was successfully developed in this study. This may be beneficial for estradiol measurements in patients with estradiol-related endocrine disorders.

Keywords:  Estradiol; Tandem mass spectrometry; Ultra-performance liquid chromatography

DOI:  https://doi.org/10.1016/j.clinbiochem.2023.01.003
Anal Chem. 2023 Jan 11.

On-Tissue Chemical Oxidation Followed by Derivatization for Mass Spectrometry Imaging Enables Visualization of Primary and Secondary Hydroxyl-Containing Metabolites in Biological Tissues.

Lingzhi Wang, Qingce Zang, Ying Zhu, Jialin Liu, Xinzhu Li, Xinyi Tu, Xin Li, Zeper Abliz, Ruiping Zhang.

On-tissue chemical derivatization combined with mass spectrometry imaging (MSI) can effectively visualize low-abundance and poorly ionizable molecules in biological tissues. Owing to the lack of an effective chemical reaction environment on the tissue surface, the development of direct one-step derivatization reactions is challenging. Herein, we present a two-step reaction involving on-tissue chemical oxidation followed by derivatization combined with airflow-assisted desorption electrospray ionization-MSI, enabling the visualization of primary and secondary hydroxyl-containing metabolites (PSHMs) within the tissue sections. This method indirectly achieved on-tissue derivatization by combining two reactions. Hydroxyl was converted to carbonyl using chemical oxidants, and subsequently, carbonyl was derived using Girard's P reagent. Using this methodology, 169 PSHMs, including hydroxy fatty acids (OH-FAs), fatty alcohols (FOHs), and sterol lipids, were detected and imaged in the tissues of rat brain, kidney, and liver. Moreover, we found that the abundant PSHMs, fatty aldehydes, and oxo fatty acids were significantly dysregulated in the liver and kidney tissues of type 2 diabetic rats; in particular, OH-FAs and FOHs were remarkably up-regulated in the diabetic rat liver tissues. The aberrations of these oxidative metabolites provide insights into the understanding of the molecular pathological mechanism of diabetes. This study demonstrates a novel, two-step reaction strategy for on-tissue derivatization with the analysis of previously inaccessible molecules using MSI.

DOI: https://doi.org/10.1021/acs.analchem.2c04316
Anal Chim Acta. 2023 Jan 25. pii: S0003-2670(22)01251-X. [Epub ahead of print]1239 340680

Rapid quantification of seven major neonicotinoids and neonicotinoid-like compounds and their key metabolites in human urine.

Sonja A Wrobel, Daniel Bury, Vladimir N Belov, Jan M Klenk, Bernhard Hauer, Heiko Hayen, Anderson J Martino-Andrade, Holger M Koch, Thomas Brüning, Heiko U Käfferlein.

  Neonicotinoids and neonicotinoid-like compounds (NNIs) are frequently used insecticides worldwide and exposure scenarios can vary widely between countries and continents. We have developed a specific and robust analytical method based on liquid chromatography-electrospray tandem mass spectrometry coupled to online-SPE (online-SPE-LC-ESI-MS-MS) to analyze the seven most important NNIs from a global perspective together with nine of their key metabolites in human urine. The method also includes the neonicotinoid-like flupyradifurone (FLUP), an important future substitute for classical neonicotinoids, and two of its major human metabolites, 5-hydroxy- and N-desfluoroethyl-FLUP. Validation of the method was carried out using pooled urine samples from low-dose human metabolism studies and spiked urine samples with a wide range of creatinine concentrations. Depending on the analyte, the limits of quantitation were between 0.06 and 2.1 µg L-1, the inter-day and intra-day imprecisions ≤6%, and the mean relative recoveries between 89% and 112%. The method enabled us to successfully quantify NNIs and their metabolites at current environmental exposures in 34 individuals of the German general population and 43 pregnant women from Brazil with no known occupational exposures to NNIs.

Keywords:  Biomarker; Human biomonitoring; Insecticides; Neonicotinoids; Online-SPE-LC-MS-MS; Standard synthesis; Urine

DOI:  https://doi.org/10.1016/j.aca.2022.340680
Foods. 2022 Dec 28. pii: 163. [Epub ahead of print]12(1):

Comparison of Workflows for Milk Lipid Analysis: Phospholipids.

Cheng Li, Zhiqian Liu, Leah Marett, Jennie Pryce, Simone Rochfort.

  Milk is a rich source of lipids, with the major components being triglycerides (TAG) and phospholipids (mainly phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI)). Liquid chromatography-mass spectrometry (LC-MS) is the predominant technique for lipid identification and quantification across all biological samples. While fatty acid (FA) composition of the major lipid classes of milk can be readily determined using tandem MS, elucidating the regio-distribution and double bond position of the FA remains difficult. Various workflows have been reported on the quantification of lipid species in biological samples in the past 20 years, but no standard or consensus methods are currently available for the quantification of milk phospholipids. This study will examine the influence of several common factors in lipid analysis workflow (including lipid extraction protocols, LC stationary phases, mobile phase buffers, gradient elution programmes, mass analyser resolution and isotope correction) on the quantification outcome of bovine milk phospholipids. The pros and cons of the current LC-MS methods as well as the critical problems to be solved will also be discussed.

Keywords:  liquid chromatography-mass spectrometry; milk; phospholipids; quantification

DOI:  https://doi.org/10.3390/foods12010163
Anal Chem. 2023 Jan 11.

Segmented Flow Strategies for Integrating Liquid Chromatography-Mass Spectrometry with Nuclear Magnetic Resonance for Lipidomics.

Jiajun Lei, Rohit Mahar, Mario C Chang, James Collins, Matthew E Merritt, Timothy J Garrett, Richard A Yost.

Building an accurate lipid inventory relies on coordinated information from orthogonal analytical capabilities. Integrating the familiar workflow of liquid chromatography (LC), high-resolution mass spectrometry (HRMS), and tandem mass spectrometry (MS/MS) with proton nuclear magnetic resonance spectroscopy (1H NMR) would be ideal for building that inventory. For absolute lipid structural elucidation, LC-HRMS/MS can provide lower-level structural information with superior sensitivity, while 1H NMR can provide invaluable higher-order structural information for the disambiguation of isomers with absolute chemical specificity. Digitization of the LC eluent followed by splitting the microfractions into two flow paths in a defined ratio for HRMS/MS and NMR would be the ideal strategy to permit correlation of the MS and NMR data as a function of chromatographic retention time. Here, we report an active segmentation platform to transform analytical flow rate LC eluent into parallel microliter segmented flow queues for high confidence correlation of the MS, MS/MS, and NMR data. The practical details in implementing this strategy to achieve an integrated LC-MS-NMR platform are presented, including the development of an active segmentation technology using a four-port two-way valve to transform the LC eluent into parallel segmented flows for online MS analysis followed by offline segment-specific 1H NMR and optimization of the detector response toward segmented flow. To demonstrate the practicality of this novel platform, it was tested using lipid mixture samples.

DOI: https://doi.org/10.1021/acs.analchem.2c03974
Anal Chim Acta. 2023 Jan 25. pii: S0003-2670(22)01219-3. [Epub ahead of print]1239 340648

Development of a matrix-based candidate reference material for human glycated albumin using isotope-dilution liquid chromatography/tandem mass spectrometry.

Rui Zhang, Rui Zhao, Wenhua Ren, Mo Wang, Gaowei Fan, Jie Shi, Yichuan Song, Xiaojie Zhou, Qingtao Wang.

  Glycated albumin (GA) in human serum is tested clinically as a short-term indicator for glucose monitoring. Here, we evaluated a candidate serum reference material (RM) at three different GA concentrations to help standardize serum GA measurements. Both albumin and GA were quantitatively determined using isotope-dilution liquid chromatography/tandem mass spectrometry with lysine-4,4,5,5-D4·2HCl (D4-lysine) and Nε-l3C6-(l-deoxy-d-fructose-1-yl)-l-lysine (13C6-DOF-lysine) as internal standards and lysine and synthetic DOF-lysine as calibration standards. The method was evaluated with the RM, JCCRM611-1, from the Reference Material Institute for Clinical Chemistry Standards. The homogeneity and stability of the candidate RMs were examined using a commercial biochemical analyzer. Fifteen units were randomly selected, and statistical analysis showed no inhomogeneity. The candidate RMs were stable for at least 6 months at -80 °C. The coefficients of variation (CVs) for the JCCRM611-1 RM ranged from 3.2% to 2.3%, and the biases ranged from 4.12% to -1.84%. GA was tested at low, medium, and high concentrations, which were quantified as 249.53 ± 13.29, 408.02 ± 11.70, and 637.22 ± 17.03 mmol/mol, respectively. The overall CVs ranged from 0.99% to 2.51%. The candidate RMs can potentially be used to develop a traceability chain to improve the accuracy of GA measurements.

Keywords:  Glycated albumin; Human serum; Liquid chromatography; Lysine; Reference material; Tandem mass spectrometry

DOI:  https://doi.org/10.1016/j.aca.2022.340648
Anal Chem. 2023 Jan 10. 95(1): 83-99

NMR Metabolomics Methods for Investigating Disease.

G A Nagana Gowda, Daniel Raftery.

DOI: https://doi.org/10.1021/acs.analchem.2c04606
Foods. 2022 Dec 21. pii: 23. [Epub ahead of print]12(1):

Comprehensive Chemical Characterization of Chia (Salvia hispanica L.) Seed Oil with a Focus on Minor Lipid Components.

Marianna Oteri, Giovanni Bartolomeo, Francesca Rigano, Juan Aspromonte, Emanuela Trovato, Giorgia Purcaro, Paola Dugo, Luigi Mondello, Marco Beccaria.

  A comprehensive chemical characterization of different lipid components, namely fatty acid composition after derivatization in fatty acid methyl esters (FAMEs), triacylglycerols (TAGs), phospholipids (PLs), free fatty acids (FFAs), sterols, carotenoids, tocopherols, and polyphenols in Chia seed oil, obtained by Soxhlet extraction, was reported. Reversed phase liquid chromatography (RP-LC) coupled to UV and mass spectrometry (MS) detectors was employed for carotenoids, polyphenols, and TAGs determination; normal phase-LC in combination with fluorescence detector (FLD) was used for tocopherols analysis; PL and FFA fractions were investigated after a rapid solid phase extraction followed by RP-LC-MS and NanoLC coupled to electron ionization (EI) MS, respectively. Furthermore, gas chromatography (GC)-flame ionization (FID) and MS detectors were used for FAMEs and sterols analysis. Results demonstrated a significant content of bioactive compounds, such as the antioxidant tocopherols (22.88 µg mL-1), and a very high content of essential fatty acids (81.39%), namely α-linolenic (62.16%) and linoleic (19.23%) acids. In addition, for the best of authors knowledge, FFA profile, as well as some carotenoid classes has been elucidated for the first time. The importance of free fatty acids in vegetable matrices is related to the fact that they can be readily involved in metabolic processes or biosynthetic pathways of the plant itself. For a fast and reliable determination of this chemical class, a very innovative and sensitive NanoLC-EI-MS analytical determination was applied.

Keywords:  NanoLC-EI-MS; chia seed oil; chromatographic techniques; essential fatty acids; lipidomics; mass spectrometry; minor lipid compounds; nutritional indices; ω3 fatty acids

DOI:  https://doi.org/10.3390/foods12010023
Clin Biochem. 2023 Jan 07. pii: S0009-9120(23)00004-8. [Epub ahead of print]113 52-58

Establishment of Korean Pediatric Reference Intervals for Estradiol using Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Eun Jeong Won, Ahram Yi, Young Jin Ko, Serim Kim, Seong-Ho Kang, Geon Park, Sook-Jin Jang.

   BACKGROUND: Ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) is a reliable and accurate method for measuring steroid hormone levels. There is an increasing need for sensitive and precise methods to measure estradiol in pediatric patients. Here, we established reference intervals for estradiol in healthy children using a UHPLC-MS/MS-based method for the first time in South Korea.
METHODS: Serum estradiol was measured using a Sciex Triple QuadTM 6500 + UHPLC-MS/MS (Sciex, Framingham, MA, USA). Reference intervals for estradiol were established according to the CLSI document EP28-A3c:2008. The reference intervals were validated using serum samples from 634 pediatric patients, including neonates, children, and adolescents. Among them, 389 specimens were used in analysis of the specimen acceptance time. Statistical analysis was performed using MedCalc (MedCalc, Ostend, Belgium) and Analyse-it (Analyse-it Software Ltd., Leeds, United Kingdom) software.
RESULTS: Reference intervals for boys (n = 297) were <16.6, <7.3, <19.0, <30.5, 7.6-96.5, and 10.6-134.4 pmol/L among those aged <1, 1-5, 6-9, 10-11, 12-14, and 15-17 years, respectively. Reference intervals for girls (n = 337) were <114.7, <24.2, <34.8, 8.0-177.0, 10.4-480.5, and 9.1-486.7 pmol/L among those aged <1, 1-5, 6-9, 10-11, 12-14, and 15-17 years, respectively. Overall, there was no effect of specimen acceptance time on estradiol measurements in boys or girls, except for that in the group aged 10-11 years.
CONCLUSIONS: The reference intervals for healthy children were validated using a UHPLC-MS/MS-based method. The highly analytical sensitive UHPLC-MS/MS method may be useful for estradiol determination in pediatric patients.

Keywords:  Estradiol; High-performance liquid chromatography-tandem mass spectrometry (UHPLC–MS/MS); Reference intervals

DOI:  https://doi.org/10.1016/j.clinbiochem.2023.01.004
Zhongguo Yi Xue Ke Xue Yuan Xue Bao. 2022 Dec;44(6): 941-949

[A Method for Quantitative Determination of Amino Acids and Organic Acids in Trace Random Urine and Preliminary Application in Research on Vitamin B12 Nutritional Status and Related Metabolism].

Mei-Yan Song, Xiao-Lan Huang, Fang Wang, Min Zhang, La-la Mi, Zhi-Zhen Liu.

  Objective To develop a method for the quantification of amino acids and organic acids in trace urine by high performance liquid chromatography-tandem mass spectrometry.Methods Random urine samples(10 μl each)were precipitated by acetonitrile and underwent derivatization with 3 mol/L HCl in n-butanol.The analytes were separated by ACE Excel 2 AQ column(50×2.1 mm,2 μm).Electrospray ionization in positive ion mode was carried out and the analytes were detected in multiple reaction monitoring mode.According to existing guidelines,the method was systematically evaluated in terms of sensitivity,specificity,accuracy,precision,recovery,matrix effect,and stability.Then,the established method was employed to detect 19 target compounds in urine samples from 70 healthy children,27 children with suspected vitamin B12 deficiency,and 3 children with cblC type methylmalonic acidemia.Results The lower limit of quantification of the method for the 19 compounds ranged from 0.01 μmol/L to 1.00 μmol/L,and the calibration curves were linear,R2>0.990.The method showed good accuracy with relative error less than ±15% and the intra-day and intra-day precision less than 15%.The run time was 8 min.No obvious matrix effect was detected except for arginine,and the recovery ranged from 80.20% to 114.97%.The samples were stable after 8 h at room temperature and 3 freeze-thaw cycles.The measured values of the compounds in the urine of healthy children were within the children's reference intervals published by Labcorp.The levels of methylmalonic acid(P=0.030)and homocysteine(P<0.001)in the urine samples of children with suspected vitamin B12 deficiency were higher than those in healthy children.The levels of methylmalonic acid,methylcitric acid,and homocysteine in the urine samples of children with cblC type methylmalonic acidemia were 5.14-76.52 times higher than the median levels of healthy children. Conclusions The method established in this study has small sample demand and short run time,which can accurately quantify the levels of amino acids and metabolites in the urine of children.Moreover,it can provide data support for related studies about the metabolic characteristics of urine amino acids and their metabolites in children with vitamin B12 deficiency.

Keywords:  amino acid; liquid chromatography-tandem mass spectrometry; organic acid; random urine; vitamin B12

DOI:  https://doi.org/10.3881/j.issn.1000-503X.14968
Se Pu. 2023 Jan;41(1): 58-65

[Determination of organophosphate diesters in facility vegetable soils using ultra-high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry].

Mengfei Liu, Mei Wang, Ang Zhao, Lin Zhu, Chun Wang, Chao Wei, Wei Liu, Jianzhong Xu.

  Organophosphate diesters (Di-OPEs) are biotic or abiotic degradation products of organophosphate esters (OPEs). Current analytical methods focus on detecting Di-OPEs in human urine. Human exposure to Di-OPEs in environmental matrices has not been systematically studied. Soil plays an important role in the environmental migration and transformation of organic pollutants. Previous studies found that OPEs are ubiquitous in soil. However, few studies reported OPEs metabolite pollution in soil, especially in facility vegetable soil. In this study, an ultra-high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry (UPHLC-Orbitrap HRMS) method was developed for the determination of five Di-OPEs (bis(2-chloroethyl) phosphate (BCEP), bis(1,3-dichloro-2-propyl) phosphate (BDCP), di-n-butyl phosphate (DnBP), diphenyl phosphate (DPhP), and bis(2-ethylhexyl) phosphate (DEHP)) in the facility vegetable soil. The pretreatment process and chromatographic and mass spectrometric conditions were optimized in the present study. Comparative study of the purification effects of different solid-phase extraction columns showed that Oasis WAX cartridge had best purification efficiency for the five Di-OPEs. The cartridge was first activated using 3 mL methanol, 3 mL methanol containing 5% (v/v) ammonia, and 3 mL 0.1 mol/L sodium acetate-acetic acid buffer solution. Then, the cartridge was rinsed with 3 mL of 30% (v/v) methanol aqueous solution, and finally eluted using 8 mL methanol containing 5% (v/v) ammonia. The effects of mobile phase (with respect to solvent composition and flow rate) and column temperature on the shape and intensity of chromatographic peaks were studied. The optimized UHPLC conditions were as follows: chromatographic column, Thermo Accucore RP-MS; column temperature, 30 ℃; mobile phase, 0.2 mmol/L ammonium acetate aqueous solution and methanol; flow rate, 0.2 mL/min. In the UHPLC-Orbitrap HRMS experiment, the five Di-OPEs were analyzed in full MS mode with negative ionization. Instrumental parameters, such as sheath gas and auxiliary gas, were optimized to determine the MS conditions. The optimized Orbitrap HRMS conditions were as follows: heating electrospray ionization source (HESI), full MS mode with negative ionization; scan range, m/z 100-500; ion transfer tube temperature, 320 ℃; automatic gain control of target particle count, 1×106; sheath gas flow rate, 8.58 L/min; auxiliary gas flow rate, 17.40 L/min; spray voltage, 3.2 kV; and S-lens voltage, 50 V. The limits of detection and quantification were 0.001-0.047 ng/g and 0.004-0.156 ng/g, respectively. The correlation coefficients of the calibration curve were 0.9985-0.9999. At three spiked levels, 5.0, 25.0, and 50.0 ng/g, the recoveries of the five Di-OPEs ranged from 56.9% to 133.0% with relative standard deviations of 4.4%-18.9%. The established method was applied to the analysis of the five Di-OPEs in 16 facility vegetable soils. The detection frequencies of the five Di-OPEs exceeded 60% in all soil samples, indicating that the Di-OPEs were ubiquitous in the facility vegetable soil. The contents of the five Di-OPEs in the facility vegetable soil samples ranged from 2.53-6.94 ng/g. DnBP (1.37-3.20 ng/g) and DPhP (0.47-2.44 ng/g) were the predominant congeners in the facility vegetable soil samples, accounting for 23.4%-68.8% and 16.3%-35.9% of the five Di-OPEs, respectively. The developed method is simple, sensitive, and reproducible and can be used effectively for the determination of Di-OPEs in soil. The results of this study will be helpful for understanding the environmental behavior of Di-OPEs and their human exposure in facility vegetable soils.

Keywords:  facility vegetable soils; organophosphate diesters (Di-OPEs); ultra-high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry (UHPLC-Orbitrap HRMS)

DOI:  https://doi.org/10.3724/SP.J.1123.2022.05002
Se Pu. 2023 Jan;41(1): 94-103

[Determination of five amanita peptide toxins in poisonous mushrooms by ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometry].

Liying He, Xiaoqin Tang, Jian Zhao, Qianzhan Yang, Li Li.

  Food poisoning by toxic mushrooms occurs frequently worldwide. It is one of the most common food poisoning events and the main cause of death. Amanita peptide toxins are the most common lethal toxins in poisonous mushrooms. Presently, a novel method based on ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometry (UPLC-Q/Orbitrap HRMS) was developed for the determination of five amanitapeptide toxins (α-amanitin, β-amanitin, γ-amanitin, phalloidin, and phallacidin). Because the isotope summit of α-amanitin affects the detection of β-amanitin, it cannot be distinguished by low resolution mass spectrometry. Therefore, experimental conditions including chromatography and mass spectrometry were explored in detail. The five peptide toxins were extracted from poisonous mushrooms with pure water and filtered through a 0.22 μm teflon microporous membrane. The procedure was rapid, simple, and environmentally friendly. Chromatographic separation was performed on a strong polarity HSS T3 column (100 mm×2.0 mm, 2.1 μm) with gradient elution using acetonitrile and 5 mmol/L ammonium acetate containing 0.1% (v/v) formic acid as mobile phases at a flow rate of 0.3 mL/min. The column temperature was set to 40 ℃. The analytes were ionized using a heating electrospray ionization source and collected in positive ion mode. Full scanning/data-dependent secondary mass spectrometry (Full mass-ddMS2) mode was used for qualitative analysis of the targets within 10 min. The target ion selective scan (Targeted-SIM) mode was used for quantification by external standard calibration. The measured and theoretical values of the exact mass and the MS2 fragment ions of the five compounds were within an error of 5×10-6. Method validation was performed according to the criteria recommended by the Chinese National Standard. All the compounds showed an excellent linear relationship in the range of 1.0-20.0 μg/L. The correlation coefficients (r) ranged from 0.9974 to 0.9989. The limit of detection was 0.006 mg/kg for all five compounds. Recoveries ranged from 81.8% to 102.4%. There was no matrix effect in the blank mushroom sample for the five compounds, and the relative standard deviations ranged from 3.2% to 8.3%. This method provides abundant compound characteristic mass information, such as retention time, exact mass, fragment ions, and other information. The data can be used to identify suspected compounds based on the extracted ion flow diagram and isotope distribution information. Comparison between the actual exact mass and the theoretical exact mass, combined with the fragment ions enables identification of the structures of unknown compounds and collision methods, which can be confirmed in the absence of standard materials. In this study, the isomer of γ-amanitin was identified as amaninamide. The novel method is simple, accurate, specific, and sensitive. The method permits the rapid qualitative and quantitative detection of compound in public health emergency settings and will provide reliable technical support for the rapid screening of such toxic compounds and the structural locking of unknown toxins in the future.

Keywords:  amatoxins; phalloidin; poisonous mushroom; ultra performance liquid chromatography-quadrupole electrostatic field orbitrap high resolution mass spectrometry (UPLC-Q/Orbitrap HRMS)

DOI:  https://doi.org/10.3724/SP.J.1123.2022.03010
Anal Chim Acta. 2023 Jan 25. pii: S0003-2670(22)01262-4. [Epub ahead of print]1239 340691

A strategy for screening and identification of new amino acid-conjugated bile acids with high coverage by liquid chromatography-mass spectrometry.

Yan-Zhen Wang, Peng-Cheng Mei, Pei-Rong Bai, Na An, Jin-Gang He, Jie Wang, Quan-Fei Zhu, Yu-Qi Feng.

  Bile acids (BAs) are a class of vital gut microbiota-host cometabolites, and they play an important role in maintaining gut microbiota-host metabolic homeostasis. Very recently, a new mechanism of BA anabolic metabolism mediated by gut microbiota (BA-amino acid conjugation) has been revealed, which provides a perspective for the research on BA metabolism and gut metabolome. In this study, we established a polarity-switching multiple reaction monitoring mass spectrometry-based screening method to mine amino acid-conjugated bile acids (AA-BAs) derived from host-gut microbiota co-metabolism. In addition, a retention time-based annotation strategy was further proposed to identify the AA-BA isomers and epimers. Using the developed methods, we successfully screened 118 AA-BA conjugates from mouse and human feces, 28 of them were confirmed by standards, and 62 putatively identified based on their predicted retention times. Moreover, we observed that the levels of most AA-BAs were significantly downregulated in the feces of chronic sleep deprivation mice, suggesting that the AA-BA metabolism was closely related to the physiological state of the host.

Keywords:  Amino acid conjugation; Bile acids; Gut microbiota-host cometabolites; Multiple reaction monitoring; Relative retention time; Sleep deprivation

DOI:  https://doi.org/10.1016/j.aca.2022.340691
Sci Rep. 2023 Jan 13. 13(1): 696

Examination of human osteoarchaeological remains as a feasible source of polar and apolar metabolites to study past conditions.

Diego Badillo-Sanchez, Maria Serrano Ruber, Anna M Davies-Barrett, Jatinderpal K Sandhu, Donald J L Jones, Martin Hansen, Sarah A Inskip.

Metabolomics is a modern tool that aids in our understanding of the molecular changes in organisms. Archaeological science is a branch of archaeology that explores different archaeological materials using modern analytical tools. Human osteoarchaeological material are a frequent finding in archaeological contexts and have the potential to offer information about previous human populations, which can be illuminating about our current condition. Using a set of samples comprising different skeletal elements and bone structures, here we explore for the first time the possibility of extracting metabolites from osteoarchaeological material. Here, a protocol for extraction and measurement of extracted polar and less-polar/apolar metabolites by ultra-high performance liquid chromatography hyphenated to high resolution mass spectrometry is presented to measure the molecules separated after a reversed phase and hydrophilic interaction liquid chromatography column. Molecular information was obtained, showing that osteoarchaeological material is a viable source of molecular information for metabolomic studies.

DOI: https://doi.org/10.1038/s41598-023-27401-0
Molecules. 2022 Dec 28. pii: 248. [Epub ahead of print]28(1):

Simultaneous Measurement of Cortisol, Cortisone, Dexamethasone and Additional Exogenous Corticosteroids by Rapid and Sensitive LC-MS/MS Analysis.

Federico Ponzetto, Mirko Parasiliti-Caprino, Fabio Settanni, Antonello Nonnato, Giulio Mengozzi, Ezio Ghigo, Roberta Giordano.

  The simultaneous measurement of dexamethasone and cortisol has proven the ability to increase the diagnostic performance of the overnight dexamethasone-suppression test. Furthermore, the therapeutic drug monitoring of administered corticosteroid drugs could represent a crucial tool for investigating unexpected variations of steroid hormones' circulating levels. In this work, an LC-MS/MS method for the quantification of cortisol, cortisone, dexamethasone and six additional exogenous corticosteroids in the serum/plasma matrix was developed and validated in compliance with the ISO/IEC requirements. To assess the efficiency of the validated method, serum samples of 75 patients undergoing the dexamethasone-suppression test and 21 plasma samples of patients under immunosuppressive treatment after kidney transplant were analyzed. In all dexamethasone-suppression test samples, it was possible to measure the circulating levels of cortisol, cortisone and dexamethasone. Concentrations of the latter were for all tested patients above the proposed cutoff for the dexamethasone-suppression test's results, and the cortisol concentrations showed good correlation with the ones measured by routine immunometric analysis, therefore confirming the screening outcome for all enrolled patients. Prednisone was detected and quantified in all enrolled patients, confirming the use of such a corticosteroid for immunosuppressive therapy. Thanks to these two applications, we proved the overall performance of the developed LC-MS/MS method for four target analytes. The future implementation of such an analytical tool in the clinical biochemistry laboratory's routine will guarantee a single and versatile tool for simultaneously monitoring dexamethasone-suppression-test results and corticosteroid drugs' administration.

Keywords:  Cushing’s syndrome; LC–MS/MS; cortisol; cortisone; dexamethasone; diagnosis; hypercortisolism; serum; steroids

DOI:  https://doi.org/10.3390/molecules28010248
Molecules. 2022 Dec 26. pii: 184. [Epub ahead of print]28(1):

The Development of an Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Method for Biogenic Amines in Fish Samples.

Tong Li, Ruiguo Wang, Peilong Wang.

  Biogenic amines (BAs) are a group of substances that are formed from amino acids by decarboxylation or amination and transamination of aldehydes and ketones. They may have either an aliphatic, aromatic, or heterocyclic structure. Their quantity determines their effects and optimum amounts are essential for physiological functions, but excess BAs causes various toxic effects throughout the human body. In our study, to rapidly determine 14 BAs (histamine, tyramine, dopamine, tryptamine, serotonin, putrescine, spermine, spermidine, octopamine, benzylamine, 1-Phenylethanamine, cadaverine, 2-Phenethylamine, and agmatine) in real fish samples, an ultra-performance liquid chromatography-tandem mass spectrometry method was established. The fish sample was extracted by acetonitrile with 0.1% formic acid and stable biogenic amine derivatives could be obtained by benzoyl chloride derivatization with a shorter reaction time. The method showed good linearity with a linear range of 3-4 orders of magnitude and regression coefficients ranging from 0.9961 to 0.9999. The calculated LODs ranged from 0.1 to 20 nM and the LOQs ranged from 0.3 to 60 nM. Satisfactory recovery was obtained from 84.6% to 119.3%. The proposed method was employed to determine the concentration levels of biogenic amine derivatives in different fish. The results indicated that this method was suitable for the analysis of biogenic amines.

Keywords:  UPLC–MS/MS; benzoyl chloride; biogenic amines; derivatization

DOI:  https://doi.org/10.3390/molecules28010184
Anal Chem. 2023 Jan 10. 95(1): 128-133

Tandem Mass Spectrometry in the Analysis of Petroleum-Based Compounds.

Ruth O Anyaeche, Jaskiran Kaur, Wanru Li, Hilkka Kenttämaa.

DOI: https://doi.org/10.1021/acs.analchem.2c04583
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Dec 21. pii: S1570-0232(22)00483-4. [Epub ahead of print]1215 123578

Sample preparation and chromatographic methods for the determination of protein-bound uremic retention solutes in human biological samples: An overview.

Sara R Fernandes, Andreia N Meireles, Sara S Marques, Luís Silva, Luisa Barreiros, Benedita Sampaio-Maia, Manuel Miró, Marcela A Segundo.

  Protein-bound uremic retention solutes, such as indole-3-acetic acid, indoxyl sulfate, p-cresol and p-cresol sulfate, are associated with the development of several pathologies, namely renal, cardiovascular, and bone toxicities, due to their potential accumulation in the human body, thus requiring analytical methods for monitoring and evaluation. The present review addresses conventional and advanced sample treatment procedures for sample handling and the chromatographic analytical methods developed for quantification of these compounds in different biological fluids, with particular focus on plasma, serum, and urine. The sample preparation and chromatographic methods coupled to different detection systems are critically discussed, focusing on the different steps involved for sample treatment, namely elimination of interfering compounds present in the sample matrix, and the evaluation of their environmental impact through the AGREEprep tool. There is a clear trend for the application of liquid-chromatography coupled to tandem mass spectrometry, which requires protein precipitation, solid-phase extraction and/or dilution prior to analysis of biological samples. Furthermore, from a sustainability point of view, miniaturized methods resorting to microplate devices are highly recommended.

Keywords:  Biological samples; Indole-3-acetic acid; Indoxyl sulfate; Protein-bound compounds; p-Cresol; p-Cresol sulfate

DOI:  https://doi.org/10.1016/j.jchromb.2022.123578
Int J Environ Res Public Health. 2022 Dec 24. pii: 297. [Epub ahead of print]20(1):

Application of High-Performance Liquid Chromatography Combined with Fluorescence Detector and Dispersive Liquid-Liquid Microextraction to Quantification of Selected Bisphenols in Human Amniotic Fluid Samples.

Szymon Szubartowski, Tomasz Tuzimski.

  Bisphenol A (BPA) is a widely produced chemical worldwide found in numerous everyday products. Its endocrine-disrupting properties and omnipresence have aroused concern and led to several restrictions on its use. These restrictions and growing public awareness about the toxicity of BPA have resulted in market products labeled "BPA-free", with BPAs often being replaced by other bisphenols. This is why constant biomonitoring of bisphenol levels in various body fluids and tissues is essential. In this study, we propose the use of simple, cost-effective high-performance liquid chromatography coupled with the fluorescence detector (HPLC-FLD) method for the determination of simultaneously selected bisphenols in amniotic fluid. For the sample preparation, a fast, simple, and "green" dispersive liquid-liquid microextraction (DLLME) method was used, achieving mean recovery values in the range of 80.9-115.9% with relative standard deviations below 12% for all analytes. Limits of quantification (LOQs) determined in the amniotic fluid matrix ranged from 6.17 to 22.72 ng/mL and were obtained from a calibration curve constructed using least-squares linear regression analysis for all cases. The presented sample preparation procedure can be easily adopted for LC-MS analysis.

Keywords:  amniotic fluid; bisphenol A (BPA); bisphenol A analogues; bisphenol A diglicydyl ether (BADGE); dispersive liquid–liquid microextraction (DLLME); fluorescence detector (FLD); high-performance liquid chromatography (HPLC)

DOI:  https://doi.org/10.3390/ijerph20010297
J Pharm Biomed Anal. 2022 Dec 31. pii: S0731-7085(22)00643-4. [Epub ahead of print]225 115222

Screening method for the simultaneous determination of allantoin and uric acid from dried blood spots.

Michal Kopčil, Roman Kanďár.

  Uric acid and its oxidation product allantoin are excellent biomarkers of oxidative stress in humans. Currently, there are high requirements not only for tests monitoring oxidative stress but also for screening laboratory tests in general. The highest demand is imposed on the simplest sampling, easy transport of the sample, and the shortest possible analysis time. The possible solution how to fulfil the requirements is sampling by dried blood spot technique with subsequent HPLC-MS/MS analysis. A fast, sensitive, and reliable HPLC-MS/MS method for the simultaneous determination of uric acid and allantoin from dried blood spots using stable isotopically labelled analogs as internal standards was developed. The separation took place in the reversed phase within 3 min, with protein precipitation and extraction in a one-step procedure. The analytical parameters of the method were satisfactory with an excellent linear range. The presented method was used to determine allantoin and uric acid levels in dried blood spot samples from 100 healthy volunteer donors. The median uric acid concentration in the cohort was 239.3 µmol/L and the median allantoin concentration was 5.6 µmol/L. The presented analytical protocol and method are suitable for screening and monitoring allantoin and uric acid levels as biomarkers of oxidative stress in clinical practice.

Keywords:  Allantoin; Dried blood spot; Liquid chromatography; Oxidative stress; Tandem mass spectrometry; Uric acid

DOI:  https://doi.org/10.1016/j.jpba.2022.115222
J Anim Sci. 2023 Jan 11. pii: skad017. [Epub ahead of print]

Technical note: Simultaneous determination of amino thiols in pig tissue by ultra-high performance liquid chromatography with fluorescence detection.

Marko Rudar, Alexandra Gachman, Melissa Boersma.

  Sulfur amino acid nutrition and metabolism is linked to animal disease. While validated methods for the determination of amino thiol levels in plasma or serum are available, there is a dearth of validated methods for their measurement in tissue. A robust and reproducible ultra-high performance liquid chromatography method has been validated for the simultaneous determination of concentrations of cysteine (Cys), cysteinylglycine (CysGly), homocysteine (Hcys), γ-glutamylcysteine (γ-GluCys), and glutathione (GSH) in pig tissue. Tissue was homogenized and deproteinized with trichloroacetic acid. Amino thiols in the acid-soluble fraction of the tissue homogenate are reduced with tris-(2-carboxyethyl)-phosphine hydrochloride and derivatized with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). Amino thiols were resolved under reversed-phase gradient conditions on a Waters Acquity BEH C18 column (1.7 µm, 2.1 × 100 mm) within 4.5 min and detected with fluorescence. The peak area ratio of analyte to 2-mercaptopropionylglycine internal standard, added to external calibration standards and samples, was used to develop linear calibration curves. Linear calibrations were performed over the range of 15 to 1500 nmol/g for Cys, CysGly, Hcys, and γ-GluCys and 150 to 15000 nmol/g for GSH. Linearity, lower limit of detection, lower limit of quantitation, accuracy, precision, sample stability, and carryover were evaluated. We demonstrate excellent linearity for all analytes within their respective concentration range (r 2 > 0.99) and excellent recovery of amino thiols from spiked samples (mean ± standard deviation across tissues; Cys, 100.0 ± 2.2%; CysGly, 95.4 ± 5.1%; Hcys, 96.6 ± 2.0%; γ-GluCys, 102.2 ± 2.7%; GSH, 100.6 ± 3.3%). The intra-day and inter-day precisions did not exceed 5% and 10%, respectively. Repeated freezing and thawing of tissue homogenate did not affect measured amino thiol concentrations, ABD-labeled amino thiols were stable for one week after derivatization, and there was no sample carryover across consecutive injections. We confirm the identity of each ABD-labeled amino thiol with Orbitrap mass spectrometry. Finally, we apply the method to the determination of amino thiol concentrations in liver and jejunum tissues in newly weaned pigs and show that, despite elevated Cys and maintained GSH concentrations in liver, both γ-GluCys and GSH decline in jejunum of weaned pigs.

Keywords:  Cysteine; fluorescence; glutathione; mass spectrometry; pig; ultra-high performance liquid chromatography

DOI:  https://doi.org/10.1093/jas/skad017
Molecules. 2022 Dec 22. pii: 96. [Epub ahead of print]28(1):

A Comparative Study on the Reduction Modes for Quinone to Determine Ubiquinone by HPLC with Luminol Chemiluminescence Detection Based on the Redox Reaction.

Naoya Kishikawa, Mahmoud El-Maghrabey, Miharu Tobo, Naotaka Kuroda.

  Ubiquinone (UQ) is considered one of the important biologically active molecules in the human body. Ubiquinone determination in human plasma is important for the investigation of its bioavailability, and also its plasma level is considered an indicator of many illnesses. We have previously developed sensitive and selective chemiluminescence (CL) method for the determination of UQ in human plasma based on its redox cycle with dithiothreitol (DTT) and luminol. However, this method requires an additional pump to deliver DTT as a post-column reagent and has the problems of high DTT consumption and broadening of the UQ peak due to online mixing with DTT. Herein, an HPLC (high-performance liquid chromatography) system equipped with two types of online reduction systems (electrolytic flow cell or platinum catalyst-packed reduction column) that play the role of DTT was constructed to reduce reagent consumption and simplify the system. The newly proposed two methods were carefully optimized and validated, and the analytical performance for UQ determination was compared with that of the conventional DTT method. Among the tested systems, the electrolytic reduction system showed ten times higher sensitivity than the DTT method, with a limit of detection of 3.1 nM. In addition, it showed a better chromatographic performance and the best peak shape with a number of theoretical plates exceeding 6500. Consequently, it was applied to the determination of UQ in healthy human plasma, and it showed good recovery (≥97.9%) and reliable precision (≤6.8%) without any interference from plasma components.

Keywords:  chemiluminescence; electrolytic reduction; human plasma; redox cycle; reduction column; ubiquinone

DOI:  https://doi.org/10.3390/molecules28010096
J Pharmacol Toxicol Methods. 2023 Jan 07. pii: S1056-8719(23)00001-1. [Epub ahead of print] 107250

Simultaneous quantification of thalidomide, lenalidomide and pomadomide in plasma by LC-MS/MS.

Bin Wang, Wanting Qiang, Jia Yi, Shouhong Gao, Bosu Meng, Yuhui Mu, Wang Bolong, Zhipeng Wang, Xia Tao.

   OBJECTIVE: To develop a new method for quantitatively analyzing three immunomodulators (thalidomide, lenalidomide and pomadomide) by liquid chromatography tandem mass spectrometry (LC-MS/MS).
METHODS: Using thalidomide-d4 as internal standard, the three analytes were separated on Agilent Zorbax SB-C18(2.1 mm × 100 mm, 3.5 μm, Agilent, USA) column and monitored in multiple reactions monitoring mode in Agilent G6460A triple quadrupole mass spectrometer operating in positive ionization mode. The sample was pretreated by protein precipitation using methanol at 3-fold volume to sample. The mobile phase was comprised of 0.1% formic acid in water (phase A) and acetonitrile (phase B) and was delivered in gradient elution program. The flow rate was 0.3 mL/min, and the injection volume was 5 μL.
RESULTS: The accuracy and stability of the method are within ±15.0%, and the precision is no >15.0%. The recoveries were 85.04% ~ 119.07%, and the matrix effect was 73.68% ~ 116.75%. Specificity, linearity, LLOQ, carry-over and dilution were all in line with the requirements of pharmacopeia and guidelines. The peak concentrations of thalidomide, lenalidomide shows huge inter-individual differences.
CONCLUSIONS: This newly developed method was sensitive, simple, and robust and can be used in therapeutic drug monitoring of three immunomodulators in multiple myeloma patients.

Keywords:  LC-MS/MS; Lenalidomide; Multiple myeloma; Pomadomide; Thalidomide

DOI:  https://doi.org/10.1016/j.vascn.2023.107250
Se Pu. 2023 Jan;41(1): 76-86

[Simultaneous determination of 28 fragrance components in cosmetics by gas chromatography-tandem mass spectrometry].

Dunming Xu, Yifeng Wu, Yifan Wang, Fangfang Chen, Shudi Zhang, Guoyin Lai.

  With the increasing number of cosmetic products, their flavor and fragrance components are receiving greater and greater attention. Establishing an analytical method of determining these components in cosmetics is one of the most effective measures to eliminate consumers' concerns. In this study, a method for the simultaneous determination of 28 fragrance residues in cosmetics by gas chromatography-tandem mass spectrometry (GC-MS/MS) was developed. The samples were extracted using methanol and those containing more oil and grease were purified using a neutral alumina solid-phase extraction column, whereas those with more complex compositions were purified by QuEChERS. The analytes in the samples were measured by GC-MS/MS, characterized using their retention times and characteristic ion pairs, and quantified with an external standard. The respective limits of detection (LODs, S/N=3) and quantification (LOQs, S/N>10) of the compounds were in the ranges 2-20 and 5-50 μg/kg. The linearities of the concentration curves of the 28 substances were good in the ranges 1-100, 2-200, 4-200, and 10-1000 μg/L, and the correlation coefficients of the quantitative ion pairs were >0.999. Twenty-eight fragrances were added to blank samples at spiked levels of 50-500 μg/kg, and the recoveries ranged from 71.3% to 120.4%, with RSDs of 1.5%-14.6%. The method could be applied in the determination of fragrances in cosmetics because it was simple, sensitive, and stable and could effectively exclude the interferences of complex matrices. The method was used to determine the fragrance components in 16 cosmetic products, and some fragrance components were detected in 12 samples. Increased attention should be paid to the safeties of fragrances and flavors used in cosmetics.

Keywords:  cosmetics; fragrances; gas chromatography-tandem mass spectrometry (GC-MS/MS)

DOI:  https://doi.org/10.3724/SP.J.1123.2022.03043
Anal Bioanal Chem. 2023 Jan 11.

Selective Schiff base formation via gas-phase ion/ion reactions to enable differentiation of isobaric lipids in imaging mass spectrometry.

Xizheng Diao, Nicholas R Ellin, Boone M Prentice.

  The separation and identification of lipids in complex mixtures are critical to deciphering their cellular functions. Failure to resolve isobaric compounds (e.g., via high mass resolution or tandem mass spectrometry) can result in incorrect identifications in mass spectrometry experiments. In imaging mass spectrometry, unresolved peaks can also result in composite images of multiple compounds, giving inaccurate depictions of molecular distributions. Gas-phase ion/ion reactions can be used to selectively react with specific chemical functional groups on a target analyte, thereby extracting it from a complex mixture and shifting its m/z value to an unobstructed region of the mass range. Herein, we use selective Schiff base formation via a novel charge inversion ion/ion reaction to purify phosphatidylserines from other isobaric (i.e., same nominal mass) lipids and reveal their singular distributions in imaging mass spectrometry. The selective Schiff base formation between singly deprotonated phosphatidylserine (PS) lipid anions and doubly charged N,N,N',N'-tetramethyl-N,N'-bis(6-oxohexyl)hexane-1,6-diaminium (TMODA) cations is performed using a modified commercial dual source hybrid Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. This process is demonstrated using the isobaric lipids [PS 40:6 - H]- (m/z 834.528) and [SHexCer d38:1 - H]- (m/z 834.576), which produces [PS 40:6 + TMODA - H - H2O]+ (m/z 1186.879), and [SHexCer d38:1 + TMODA - H]+ (m/z 1204.938) product ions following the gas-phase charge inversion reaction. These product ions differ by roughly 18 Da in mass and are easily separated by low mass resolution analysis, while the isobaric precursor ions require roughly 45,000 mass resolving power (full-width at half maximum) to separate. Imaging mass spectrometry using targeted gas-phase ion/ion reactions shows distinct spatial distributions for the separated lipid product ions relative to the composite images of the unseparated precursor ions.

Keywords:  Charge inversion; Gas-phase chemistry; Imaging mass spectrometry; Ion/ion reactions; Lipids; Schiff base formation

DOI:  https://doi.org/10.1007/s00216-023-04523-y
Anal Chim Acta. 2023 Jan 25. pii: S0003-2670(22)01248-X. [Epub ahead of print]1239 340677

Online coupling of matrix solid-phase dispersion to direct analysis in real time mass spectrometry for high-throughput analysis of regulated chemicals in consumer products.

Xianshuang Meng, Shanshan Sun, Hua Bai, Qiang Ma.

  The current work is the first study on online coupling of matrix solid-phase dispersion (MSPD) to direct analysis in real time mass spectrometry (DART-MS) bridging with solid-phase analytical derivatization (SPAD) based on a graphene oxide nanosheets (GONs)-coated cotton swab. Proof-of-concept demonstrations were explored for high-throughput analysis of a diversity of regulated chemicals in consumer products such as textiles, toys, and cosmetics. On-demand sorbent combinations were blended with samples, packed into MSPD columns, and mounted on a homemade 3D-printed rack module for automated sample feeding. To achieve good synergy between MSPD and DART-MS, a cotton swab with a conical tip deposited with GONs was attached to the bottom of the MSPD column. The swabs serve as a solid-phase microextraction probe for convenient enrichment of the eluted analytes from MSPD, thermal desorption of the enriched analytes by DART, and sensitive detection by a hybrid quadrupole-Orbitrap mass spectrometer. Furthermore, the utility of an on-swab SPAD strategy was demonstrated for the detection of formaldehyde by use of the derivatizing reagent of dansyl hydrazine, contributing to improved ionization efficiency without compromising the overall coherence of the analytical workflow. The MSPD-DART-MS methodology was systematically optimized and validated, obtaining acceptable recovery (71.7-110.3%), repeatability (11.8-19.3%), and sensitivity (limits of detection and quantitation in the ranges of 6.2-19.5 and 23.7-75.9 μg/kg) for 32 target analytes. The developed protocol streamlined sample extraction, clean-up, desorption, ionization, and detection, highlighting the appealing potential for high-throughput analysis of samples with complex matrices.

Keywords:  Consumer products; Direct analysis in real time mass spectrometry; Matrix solid-phase dispersion; Solid-phase analytical derivatization

DOI:  https://doi.org/10.1016/j.aca.2022.340677
Anal Chem. 2023 Jan 09.

Development of a 3D-Printed Ionization Source for Single-Cell Analysis.

Qinlei Liu, Sandra Martínez-Jarquín, Wenjie Ge, Renato Zenobi.

Understanding the physiologies and pathologies of diseases requires a thorough understanding of metabolic heterogeneity in cells. This technical note presents a 3D printing technology for manufacturing an ionization source that is specially adapted for mass spectrometry-based single-cell analysis. This all-in-one 3D-printed electrospray ionization source integrates the sample introduction, metabolite extraction, and ionization into one device, simplifying the process of single-cell analysis and improving the reproducibility of the measurement. We successfully used it for high-throughput analysis of three types of cancer cells (around 17 cells/min) and used the t-distributed stochastic neighbor embedding algorithm to distinguish different cell types based on detected metabolites. By simply adjusting the printing parameters of the 3D-printed ionization source, it can be applied to cells with different sizes. The proposed 3D-printed ionization source promises to open new possibilities for single-cell analysis.

DOI: https://doi.org/10.1021/acs.analchem.2c04279
Int J Mol Sci. 2022 Dec 21. pii: 100. [Epub ahead of print]24(1):

Development of the Chromatographic Method for Simultaneous Determination of Azaperone and Azaperol in Animal Kidneys and Livers.

Izabella Kośka, Paweł Kubalczyk.

  A precise and accurate method for the simultaneous determination of azaperone and azaperol in meat tissues has been developed. This paper describes the first method to be so fast, simple, and useful, especially for many laboratories that do not have sophisticated equipment. This method is based on LC separation and UV-Vis detection. During the sample preparation, the meat tissue was homogenized in acetonitrile at a ratio of 1:4 (tissue weight:acetonitrile volume). The homogenate was centrifuged, the supernatant was evaporated in a lyophilizator, and then the evaporation residue was dissolved in 20 µL of ethanol. For deproteinization, 15 µL of perchloric acid was added, and the sample prepared in this way was injected into a chromatographic column and analyzed using reversed-phased HPLC. The mobile phase consisted of 0.05 mol/L phosphate buffer pH 3.00 (component A) and acetonitrile (component B). UV detection was conducted at 245 nm. The experimentally determined LOQs were 0.25 µg/kg for azaperone and 0.12 µg/kg for azaperol. For both analytes, the calibration curves showed linearity in the tested concentration range from 50 to 300 µg/kg of tissue. The accuracy of the presented method did not exceed 15%, and the recovery was in the range of 85-115%. A validated analytical procedure was implemented for the analysis of various animal tissues for their content of azaperone and azaperol.

Keywords:  animal kidneys; animal livers; azaperol; azaperone; high-performance liquid chromatography; sedatives

DOI:  https://doi.org/10.3390/ijms24010100