bims-metlip 2023-06-11 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–06–11
twenty papers selected by
Sofia Costa, Matterworks

MetaPro: a web-based metabolomics application for LC-MS data batch inspection and library curation.
Analysis of triacylglycerol and phospholipid sn-positional isomers by liquid chromatographic and mass spectrometric methodologies.
Development and validation of LC-MS/MS methods for the quantification of 101BHG-D01, a novel, long-acting and selective muscarinic receptor antagonist, and its main metabolite M6 in human plasma, urine and feces: Application to a clinical study in healthy Chinese subjects.
Method validation for a greener approach to the quantification of 25-hydroxy vitamin D3 in patient serum using supported liquid extraction and liquid chromatography-tandem mass spectrometry.
Innovative thermally assisted on-line solid phase extraction-reversed phase liquid chromatography applied to targeted nutrimetabolomics in human biofluids.
Estimation of dihydroartemisinin in human plasma using a highly sensitive LTQ Orbitrap mass spectrometer with Xcalibur software.
Identification of nutritional biomarkers through highly sensitive and chemoselective metabolomics.
Trace quantification of GL-V9 and its glucuronide metabolites (5-O-glucuronide GL-V9) in Beagle dog plasma by UPLC-MS/MS and its application to a pharmacokinetic study.
Precision fermentation with mass spectrometry-based spent media analysis.
A Novel Enzymatic Hydrolysis Method for Urine Aldosterone Quantification: A Case for Reassessing Clinical Cut-Offs of Primary Aldosteronism.
OSCA-finder: Redefining the assay of kidney disease diagnostic through metabolomics and deep learning.
Separation of Isobaric Lipids in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions.
Derivatized versus non-derivatized LC-MS/MS techniques for the analysis of estrogens and estrogen-like endocrine disruptors in human plasma.
Metabolomic Analysis of Polymicrobial Wound Infections and an Associated Adhesive Bandage.
High-throughput quantification of microbial-derived organic acids in mucin-rich samples via reverse phase high performance liquid chromatography.
Florfenicol and Florfenicol Amine Quantification in Bull Serum and Seminal Plasma by a Single Validated UHPLC-MS/MS Method.
Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique.
Determination of 1,3-Diphenylguanidine, 1,3-Di-o-tolylguanidine, and 1,2,3-Triphenylguanidine in Human Urine Using Liquid Chromatography-Tandem Mass Spectrometry.
Simultaneous determination of 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid and main plasma aminothiols by HPLC-UV based method.
Validation of an LC-MS/MS method for simultaneous quantification of abiraterone, enzalutamide and darolutamide in human plasma.

Metabolomics. 2023 Jun 08. 19(6): 57

MetaPro: a web-based metabolomics application for LC-MS data batch inspection and library curation.

Shaowei An, Ruimin Wang, Miaoshan Lu, Chao Zhang, Huafen Liu, Jinyin Wang, Cong Xie, Changbin Yu.

   INTRODUCTION: Metabolomics analysis based on liquid chromatography-mass spectrometry (LC-MS) has been a prevalent method in the metabolic field. However, accurately quantifying all the metabolites in large metabolomics sample cohorts is challenging. The analysis efficiency is restricted by the abilities of software in many labs, and the lack of spectra for some metabolites also hinders metabolite identification.
OBJECTIVES: Develop software that performs semi-targeted metabolomics analysis with an optimized workflow to improve quantification accuracy. The software also supports web-based technologies and increases laboratory analysis efficiency. A spectral curation function is provided to promote the prosperity of homemade MS/MS spectral libraries in the metabolomics community.
METHODS: MetaPro is developed based on an industrial-grade web framework and a computation-oriented MS data format to improve analysis efficiency. Algorithms from mainstream metabolomics software are integrated and optimized for more accurate quantification results. A semi-targeted analysis workflow is designed based on the concept of combining artificial judgment and algorithm inference.
RESULTS: MetaPro supports semi-targeted analysis workflow and functions for fast QC inspection and self-made spectral library curation with easy-to-use interfaces. With curated authentic or high-quality spectra, it can improve identification accuracy using different peak identification strategies. It demonstrates practical value in analyzing large amounts of metabolomics samples.
CONCLUSION: We offer MetaPro as a web-based application characterized by fast batch QC inspection and credible spectral curation towards high-throughput metabolomics data. It aims to resolve the analysis difficulty in semi-targeted metabolomics.

Keywords:  Database; Mass spectrometry; Metabolite identification; Metabolomics; Quality assurance; Quality control; Software; Spectral library; Web-based; Workflow

DOI:  https://doi.org/10.1007/s11306-023-02018-6
Mass Spectrom Rev. 2023 Jun 06.

Analysis of triacylglycerol and phospholipid sn-positional isomers by liquid chromatographic and mass spectrometric methodologies.

Mikael Fabritius, Baoru Yang.

  Analysis of triacylglycerol (TG) and phospholipid sn-positional isomers can be divided into two main categories: (a) direct separation by chromatography or other means such as ion mobility mass spectrometry and (b) quantification of regioisomer ratios by structurally informative fragment ions with mass spectrometric methods. Due to long retention times and limited performance, researchers are moving away from direct chromatographic separation of isomers, using mass spectrometry instead. Many established analytical methods are targeting specific isomers of interest instead of untargeted analysis of comprehensive profiles of regioisomers. Challenges remain arising from the large number of isobaric and isomeric lipid species in natural samples, often overlapping chromatographically and sharing structurally informative fragment ions. Further, fragmentation of glycerolipids is influenced by the nature of the attached fatty acids, and the lack of available regiopure standards is still an obstacle for establishing calibration curves required for accurate quantification of regioisomers. Additionally, throughput of many methods is still quite limited. Optimization algorithms and fragmentation models are useful especially for analysis of TG regioisomers, as identification using calibration curves alone without proper separation is difficult with complex samples.

Keywords:  liquid chromatography; mass spectrometry; phospholipid; regioisomer; triacylglycerol

DOI:  https://doi.org/10.1002/mas.21853
J Pharm Biomed Anal. 2023 Sep 05. pii: S0731-7085(23)00267-4. [Epub ahead of print]233 115498

Development and validation of LC-MS/MS methods for the quantification of 101BHG-D01, a novel, long-acting and selective muscarinic receptor antagonist, and its main metabolite M6 in human plasma, urine and feces: Application to a clinical study in healthy Chinese subjects.

Huaye Gao, Minlu Cheng, Haijun Liu, Li Ding.

  101BHG-D01 is a novel, long-acting and selective muscarinic receptor antagonist for the treatment of chronic obstructive pulmonary disease (COPD) and rhinorrhea in rhinitis. To support its clinical study, several liquid chromatography tandem mass spectrometry (LC-MS/MS) methods for the quantification of 101BHG-D01 and its main metabolite M6 in human plasma, urine and feces were developed. The plasma samples were prepared by protein precipitation, and the urine and fecal homogenate samples were pretreated by direct dilution, respectively. The chromatographic separation was performed on an Agilent InfinityLab Poroshell 120 C18 column with 0.1% formic acid and 10.0 mM ammonium acetate buffer solution in water and methanol as the mobile phase. The MS/MS analysis was performed by using multiple reaction monitoring (MRM) under a positive ion electrospray ionization mode. The methods were validated with regards to selectivity, linearity, lower limit of quantitation (LLOQ), accuracy and precision, matrix effect, extraction recovery, dilution integrity, batch size, carryover and stability. The calibration ranges were as follows: 1.00-800 pg/mL for 101BHG-D01 and 1.00-20.0 pg/mL for M6 in plasma; 0.0500-20.0 ng/mL for 101BHG-D01 and M6 in urine; 0.400-400 ng/mL for 101BHG-D01 and 0.100-100 ng/mL for M6 in feces. There was no endogenous or cross interference observed at the retention time of the analytes and internal standard in various biological matrices. Across these matrices, for the lower limit of quantitation quality control (LLOQ QC) samples, the intra- and inter-batch coefficients of variation were within 15.7%. For other QC samples, the intra- and inter-batch coefficients of variation were within 8.9%. The intra- and inter-batch accuracy deviations for all QC samples were within the range of - 6.2-12.0%. No significant matrix effect was observed from the matrices. The extraction recoveries of these methods at different concentrations were consistent and reproducible. The analytes were stable in different matrices under various storage conditions. The other bioanalytical parameters were also fully validated and met the criteria given in the FDA guidance. These methods were successfully applied to a clinical study in healthy Chinese subjects after a single dose administration of 101BHG-D01 inhalation aerosol. After inhalation, 101BHG-D01 was absorbed into plasma rapidly with the time to reach the maximum drug concentration (Tmax) of 5 min and eliminated slowly with a half-life time about 30 h. The cumulative urinary and fecal excretion rates revealed 101BHG-D01 was mainly excreted in feces, rather than urine. The pharmacokinetic results of the study drug laid a foundation for its further clinical development.

Keywords:  101BHG-D01; LC-MS/MS; Long-acting muscarinic antagonist; Metabolite M6; Pharmacokinetics

DOI:  https://doi.org/10.1016/j.jpba.2023.115498
Clin Chem Lab Med. 2023 Jun 09.

Method validation for a greener approach to the quantification of 25-hydroxy vitamin D3 in patient serum using supported liquid extraction and liquid chromatography-tandem mass spectrometry.

Gavin Turrell, Thilini Thrimawithana, Catherine Itsiopoulos, Ronda F Greaves, Rosita Zakaria.



Keywords:  liquid chromatography-tandem mass spectrometry; supported liquid extraction; vitamin D

DOI:  https://doi.org/10.1515/cclm-2023-0444
Anal Chim Acta. 2023 Aug 15. pii: S0003-2670(23)00650-5. [Epub ahead of print]1269 341429

Innovative thermally assisted on-line solid phase extraction-reversed phase liquid chromatography applied to targeted nutrimetabolomics in human biofluids.

Lapo Renai, Luca Marzullo, Giulia Bonaccorso, Serena Orlandini, Fulvio Mattivi, Maria Concetta Bruzzoniti, Massimo Del Bubba.

  In this study, the use of thermal desorption in on-line solid phase extraction coupled with reversed phase liquid chromatography (on-line SPE-LC) was for the first time proposed and demonstrated for the desorption of analytes strongly retained by multiple interaction polymeric sorbents. In detail, this analytical strategy was applied to the on-line SPE-LC targeted analysis of a model set of 34 human gut metabolites characterized by heterogeneous physicochemical properties (i.e., octanol-water partition coefficient in the range -0.3 - 3.4). The novel thermally assisted on-line SPE approach was investigated in comparison to conventional room temperature desorption strategies based on the use of (i) an optimized elution gradient or (ii) organic desorption followed by post-cartridge dilution. The thermally assisted desorption strategy has been shown to be better performing and suitable for the development of a reliable and sensitive method for the analysis of the model group of analytes in urine and serum. In more detail, under the optimized experimental conditions, the proposed method provided negligible matrix effects in both biofluids for almost all target analytes. Moreover, method quantification limits were in the ranges 0.026-7.2 μg L-1 and 0.033-23 μg L-1 for urine and serum, respectively, i.e., comparable to or lower than those reported in methods previously published.

Keywords:  Analyte detachment; Mass spectrometry; Method validation; Nutrimetabolites; Serum; Urine

DOI:  https://doi.org/10.1016/j.aca.2023.341429
Front Pharmacol. 2023 ;14 1157604

Estimation of dihydroartemisinin in human plasma using a highly sensitive LTQ Orbitrap mass spectrometer with Xcalibur software.

Tareq Abu-Izneid, Muhammad Abbas, David G Watson, Yasar Shah, Sayyed I Shah, Fazli Khuda.

  Background: Artemether (ARM), the O-methyl ether prodrug of dihydroartemisinin (DHA), is considered a first-line antimalarial agent. Artemether is extensively metabolized in vivo to its active metabolite DHA, and therefore its determination offers considerable difficulties. In the present study, DHA identification and estimation were accurately performed by the mass spectrometric analysis, using a high-resolution liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) LTQ Orbitrap hybrid mass spectrometer. Methods: The plasma samples were taken from healthy volunteers, and the spiked plasma was extracted by adding 1 mL of a mixture of dichloromethane and tert.-methyl butyl ether (8:2 v/v) to 0.5 mL of plasma. The internal standard solution (artemisinin 500 ng/mL) was added to the plasma samples. After vertexing and centrifugation, the organic layer was separated and transferred into another tube and dried under nitrogen. The residue was reconstituted in 100 μL of acetonitrile and was injected onto the LC-MS system for analysis. Measurement of standards and samples was carried out isocratically on a Surveyor HPLC system combined with an LTQ Orbitrap mass spectrometer using an ACE 5 C18-PFP column. Mobile phase A consisted of 0.1% v/v formic acid in water, Mobile phase B consisted of acetonitrile only, and isocratic elution was carried out with A:B 20:80, v/v. The flow rate was 500 μL/min. The ESI interface was operated in a positive ion mode with a spray voltage of 4.5 kV. Results: Artemether is not a very biologically stable compound and is immediately metabolized to its active metabolite dihydroartemisinin, so no clear peak was observed for artemether. Both artemether and DHA after ionization undergo neutral losses of methanol and water, respectively, in the source of the mass spectrometer. The ions observed were (MH-H2O) m/z 267.15 for DHA and (MH-m/z 283.15 for internal standard artemisinin. The method was validated according to international guidelines. Discussion: The validated method was applied successfully for the determination and quantification of DHA in plasma samples. This method works well for the extraction of drugs, and the Orbitrap system with the help of Xcalibur software accurately and precisely determines the concentration of DHA in spiked as well as volunteer's plasma.

Keywords:  LC-MS; analytical method; antimalarial; artemether; dihydroartemisinin; human plasma; metabolites

DOI:  https://doi.org/10.3389/fphar.2023.1157604
Food Chem. 2023 Jun 01. pii: S0308-8146(23)01099-3. [Epub ahead of print]425 136481

Identification of nutritional biomarkers through highly sensitive and chemoselective metabolomics.

Weifeng Lin, Kiana Mellinghaus, Ana Rodriguez-Mateos, Daniel Globisch.

  The importance of a healthy diet for humans is known for decades. The elucidation of key molecules responsible for the beneficial and adverse dietary effects is slowly developing as the tools are missing. Carbonyl-containing metabolites are a common bioproducts through conversion of diet by the microbiome. In here, we have utilized our recently developed mass spectrometric methodology based on chemoselective conjugation of carbonyl-metabolites. The method has been applied for urine sample analysis from a dietary (poly)phenol intervention study (N = 78 individuals) for the first time. We have identified a series of carbonyl-metabolites of dietary origin and the chemical structure was validated for 30 metabolites. Our sensitive analysis led to the discovery of four unknown dietary markers with high sensitivity and selectivity (AUC > 0.91). Our chemical metabolomics method has been successfully applied for large-scale analysis and provides the basis for targeted metabolomics to identify unknown nutritional and disease-related biomarkers.

Keywords:  Carbonyl metabolites; Chemical biology; Chemical metabolomics; Dietary intervention; Nutritional biomarkers

DOI:  https://doi.org/10.1016/j.foodchem.2023.136481
PLoS One. 2023 ;18(6): e0286467

Trace quantification of GL-V9 and its glucuronide metabolites (5-O-glucuronide GL-V9) in Beagle dog plasma by UPLC-MS/MS and its application to a pharmacokinetic study.

Xuefeng Zhang, Guanlan Liu, Zechun Sang, Qinglong Guo, Yuxin Zhou.

GL-V9, a new synthetic flavonoid derived from wogonin, has shown beneficial biological functions. In this study, accurate and sensitive UPLC-MS/MS methods were developed and validated for the quantification of GL-V9 and its glucuronide metabolite (5-O-glucuronide GL-V9) in Beagle dog plasma. The chromatographic separation was performed on a C8 column (ACE Excel 5 C8 50×3.0 mm) using 0.1% formic acid and acetonitrile were used as mobile phase. Mass detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization (ESI) interface operating in positive ion mode. Quantitative analysis was performed in multiple reaction monitoring (MRM) mode with the transitions of m/z 410.2→126.1 for GL-V9, m/z 586.3→410.0 for 5-O-glucuronide GL-V9 and m/z 180.0→110.3 for phenacetin (internal standard), respectively. The calibration curves for GL-V9 and 5-O-glucuronide GL-V9 showed excellent linearity over the concentration range of 0.5-500 ng/mL with correlation coefficient greater than 0.99. The intra- and inter-day accuracies were within 99.86% to 109.20% for GL-V9 and 92.55% to 106.20% for 5-O-glucuronide GL-V9, respectively. The mean recovery was 88.64% ± 2.70% for GL-V9, and 92.31% ± 6.28% for 5-O-glucuronide GL-V9, respectively. The validated method was successfully applied to the pharmacokinetic study in Beagle dogs after oral and intravenous administration. The oral bioavailability of GL-V9 was approximately 2.47%~4.35% in Beagle dogs and reached steady state on the fifth day after repeated dosing.

DOI: https://doi.org/10.1371/journal.pone.0286467
Biotechnol Bioeng. 2023 Jun 05.

Precision fermentation with mass spectrometry-based spent media analysis.

Hardik Dodia, Avinash Vellore Sunder, Yogen Borkar, Pramod P Wangikar.

  Optimization and monitoring of bioprocesses requires the measurement of several process parameters and quality attributes. Mass spectrometry (MS)-based techniques such as those coupled to gas chromatography (GCMS) and liquid Chromatography (LCMS) enable the simultaneous measurement of hundreds of metabolites with high sensitivity. When applied to spent media, such metabolome analysis can help determine the sequence of substrate uptake and metabolite secretion, consequently facilitating better design of initial media and feeding strategy. Furthermore, the analysis of metabolite diversity and abundance from spent media will aid the determination of metabolic phases of the culture and the identification of metabolites as surrogate markers for product titer and quality. This review covers the recent advances in metabolomics analysis applied to the development and monitoring of bioprocesses. In this regard, we recommend a stepwise workflow and guidelines that a bioprocesses engineer can adopt to develop and optimize a fermentation process using spent media analysis. Finally, we show examples of how the use of MS can revolutionize the design and monitoring of bioprocesses.

Keywords:  bioprocess development; exo-metabolomics; mass spectrometry; productivity markers; spent media analysis

DOI:  https://doi.org/10.1002/bit.28450
Clin Chem. 2023 Jun 05. pii: hvad065. [Epub ahead of print]

A Novel Enzymatic Hydrolysis Method for Urine Aldosterone Quantification: A Case for Reassessing Clinical Cut-Offs of Primary Aldosteronism.

Mehrdad Yazdanpanah, Sarah R Delaney, Daniel R Beriault.

BACKGROUND: Primary aldosteronism (PA) is a common endocrine cause of secondary hypertension. The aldosterone/renin ratio is an important tool for PA screening, and dynamic testing in serum or urine is used to confirm the diagnosis. While LC-MS/MS is considered the gold standard for testing, there is significant interlaboratory variability between the extraction procedures, which can impact diagnostic interpretation. To help overcome this, we present a simple and accurate LC-MS/MS method for the quantification of both serum and urine aldosterone using a novel enzymatic hydrolysis procedure.
METHODS: Serum and urine aldosterone was extracted and measured by LC-MS/MS. Urine-conjugated aldosterone glucuronide was hydrolyzed using a genetically modified glucuronidase enzyme. The assay precision, accuracy, limit of quantification, recovery, and carryover were evaluated and the new assay cut-offs were proposed.
RESULTS: The liquid chromatography method allowed for adequate separation of the aldosterone peak from closely eluting peaks. Significant in vitro aldosterone loss was observed during acid-catalyzed hydrolysis of urine, which was corrected with the addition of the internal standard to the urine before the hydrolysis step. Glucuronidase catalyzed hydrolysis of urine aldosterone glucuronide displays good correlation with the corrected acid-catalyzed hydrolysis. Serum aldosterone showed good agreement with reference values and the consensus range reported for external quality assessment specimens.
CONCLUSION: A simple, fast, and highly accurate method for the detection of serum and urine aldosterone has been developed. The proposed novel enzymatic procedure allows for short hydrolysis time and compensates for urine aldosterone loss during the hydrolysis step.

DOI: https://doi.org/10.1093/clinchem/hvad065
Talanta. 2023 Jun 02. pii: S0039-9140(23)00496-4. [Epub ahead of print]264 124745

OSCA-finder: Redefining the assay of kidney disease diagnostic through metabolomics and deep learning.

Xuanlin Meng, Zhian He, Lizhen Guo, Houwei Lin, Lei Feng.

  Liquid chromatography-mass spectrometry (LC-MS) is a platform for urine and blood sample analysis. However, the high variability in the urine sample reduced the confidence of metabolite identification. Therefore, pre and post-calibration operations are inevitable to ensure an accurate urine biomarker analysis. In this study, the phenomenon of a higher creatinine concentration variable in ureteropelvic junction obstruction (UPJO) patient urine samples than in healthy people was revealed, indicating the urine biomarker discovery of UPJO patients is not adapted to the creatinine calibrate strategy. Therefore, we proposed a pipeline "OSCA-Finder" to reshape the urine biomarker analysis. First, to ensure a more stable peak shape and total ion chromatography, we applied the product of osmotic pressure and injection volume as a calibration principle and integrated it with an online mixer dilution. Therefore, we obtained the most peaks and identified more metabolites in a urine sample with peak area group CV<30%. A data-enhanced strategy was applied to reduce the overfit while training a neural network binary classifier with an accuracy of 99.9%. Finally, seven accurate urine biomarkers combined with a binary classifier were applied to distinguish UPJO patients from healthy people. The results show that the UPJO diagnostic strategy based on urine osmotic pressure calibration has more potential than ordinary strategies.

Keywords:  Biomarker; Data enhancement; LC-MS; OSCA-Finder; UPJO

DOI:  https://doi.org/10.1016/j.talanta.2023.124745
J Am Soc Mass Spectrom. 2023 Jun 05.

Separation of Isobaric Lipids in Imaging Mass Spectrometry Using Gas-Phase Charge Inversion Ion/Ion Reactions.

Jonathan T Specker, Boone M Prentice.

  The diverse array of chemical compounds present in tissue samples results in many isobaric (i.e., same nominal mass) compounds in imaging mass spectrometry experiments. Adequate separation and differentiation of these compounds is necessary to ensure accurate analyte identification and avoid composite images comprising multiple compounds. High-resolution accurate mass (HRAM) measurements are able to resolve these compounds in some instances, but HRAM measurements are not always feasible depending on the instrument platform and the desired experimental time scale. Alternatively, tandem mass spectrometry (MS/MS) can be used to perform gas-phase transformations that improve molecular specificity. While conventional MS/MS methods employ collision induced dissociation (CID) to fragment compounds of interest and then analyze the product masses, gas-phase ion/ion reactions can be used to instead selectively react with desired classes of analytes. Herein, we have used gas-phase charge inversion ion/ion reactions to selectively resolve phosphatidylcholines (PCs) in isobaric lipid mixtures. A 1,4-phenylenedipropionic acid (PDPA) reagent dianion readily reacts with [M + H]+, [M + Na]+, and [M + K]+ ion types to produce demethylated product anions for each PC, [PC - CH3]-. These product anions are no longer isobaric and now differ in mass by 22 Da (protonated versus sodiated) and 16 Da (sodiated versus potassiated), respectively. This reaction has been used to differentiate isobaric lipids in the imaging mass spectrometry analysis of rat brain tissue.

Keywords:  MALDI; imaging mass spectrometry; ion/ion reactions; lipids

DOI:  https://doi.org/10.1021/jasms.3c00081
Ecotoxicol Environ Saf. 2023 Jun 01. pii: S0147-6513(23)00587-0. [Epub ahead of print]260 115083

Derivatized versus non-derivatized LC-MS/MS techniques for the analysis of estrogens and estrogen-like endocrine disruptors in human plasma.

J Vitku, L Horackova, L Kolatorova, M Duskova, T Skodova, M Simkova.

  Bisphenols, parabens, alkylphenols and triclosan are anthropogenic substances with a phenolic group that have been introduced to the environment in recent decades. As they possess hormone-like effects, they have been termed endocrine disruptors (EDs), and can interfere with steroid pathways in organisms. To evaluate the potential impact of EDs on steroid biosynthesis and metabolism, sensitive and robust methods enabling the concurrent measurement of EDs and steroids in plasma are needed. Of crucial importance is the analysis of unconjugated EDs, which possess biological activity. The aim of the study was to develop and validate LC-MS/MS methods with and without a derivatization step for the analysis of unconjugated steroids (estrone-E1, estradiol-E2, estriol-E3, aldosterone-ALDO) and different groups of EDs (bisphenols, parabens, nonylphenol-NP and triclosan-TCS), and compare these methods on a set of 24 human plasma samples using Passing-Bablok regression analysis. Both methods were validated according to FDA and EMA guidelines. The method with dansyl chloride derivatization allowed 17 compounds to be measured: estrogens (E1, E2, E3), bisphenols (bisphenol A-BPA, BPS, BPF, BPAF, BPAP, BPZ, BPP), parabens (methylparaben-MP, ethylparaben-EP, propylparaben-PP, butylparaben-BP, benzylparaben-BenzylP), TCS and NP, with lower limits of quantification (LLOQs) between 4 and 125 pg/mL. The method without derivatization enabled 15 compounds to be analyzed: estrogens (E1, E2, E3), ALDO, bisphenols (BPA, BPS, BPF, BPAF, BPAP, BPZ), parabens (MP, EP, PP, BP, BenzylP) with LLOQs between 2 and 63 pg/mL, and NP and BPP in semiquantitative mode. Adding 6 mM ammonium fluoride post column into mobile phases in the method without derivatization achieved similar or even better LLOQs than the method with the derivatization step. The uniqueness of the methods lies in the simultaneous determination of different classes of unconjugated (bioactive) fraction of EDs together with selected steroids (estrogens + ALDO in the method without derivatization), which provides a useful tool for evaluating the relationships between EDs and steroid metabolism.

Keywords:  Aldosterone; Bisphenol; Estrogen; Nonylphenol; Paraben; Triclosan

DOI:  https://doi.org/10.1016/j.ecoenv.2023.115083
J Am Soc Mass Spectrom. 2023 Jun 08.

Metabolomic Analysis of Polymicrobial Wound Infections and an Associated Adhesive Bandage.

Monica Ness, Avery L Holmes, Chaoyi Wu, Ekram Hossain, Carolyn B Ibberson, Laura-Isobel McCall.

Concerns about ion suppression, spectral contamination, or interference have led to avoidance of polymers in mass spectrometry (MS)-based metabolomics. This avoidance, however, has left many biochemical fields underexplored, including wounds, which are often treated with adhesive bandages. Here, we found that despite previous concerns, the addition of an adhesive bandage can still result in biologically informative MS data. Initially, a test LC-MS analysis was performed on a mixture of known chemical standards and a polymer bandage extract. Results demonstrated successful removal of many polymer-associated features through a data processing step. Furthermore, the bandage presence did not interfere with metabolite annotation. This method was then implemented in the context of murine surgical wound infections covered with an adhesive bandage and inoculated with Staphylococcus aureus, Pseudomonas aeruginosa, or a 1:1 mix of these pathogens. Metabolites were extracted and analyzed by LC-MS. On the bandage side, we observed a greater impact of infection on the metabolome. Distance analysis showed significant differences between all conditions and demonstrated that coinfected samples were more similar to S. aureus-infected samples compared to P. aeruginosa-infected samples. We also found that coinfection was not merely a summative effect of each monoinfection. Overall, these results represent an expansion of LC-MS-based metabolomics to a novel, previously under-investigated class of samples, leading to actionable biological information.

DOI: https://doi.org/10.1021/jasms.3c00066
J Med Microbiol. 2023 Jun;72(6):

High-throughput quantification of microbial-derived organic acids in mucin-rich samples via reverse phase high performance liquid chromatography.

Alex R Villarreal, Sarah K Lucas, Joshua R Fletcher, Ryan C Hunter.

  Organic acids (short chain fatty acids, amino acids, etc.) are common metabolic byproducts of commensal bacteria of the gut and oral cavity in addition to microbiota associated with chronic infections of the airways, skin, and soft tissues. A ubiquitous characteristic of these body sites in which mucus-rich secretions often accumulate in excess, is the presence of mucins; high molecular weight (HMW), glycosylated proteins that decorate the surfaces of non-keratinized epithelia. Owing to their size, mucins complicate quantification of microbial-derived metabolites as these large glycoproteins preclude use of 1D and 2D gel approaches and can obstruct analytical chromatography columns. Standard approaches for quantification of organic acids in mucin-rich samples typically rely on laborious extractions or outsourcing to laboratories specializing in targeted metabolomics. Here we report a high-throughput sample preparation process that reduces mucin abundance and an accompanying isocratic reverse phase high performance liquid chromatography (HPLC) method that enables quantification of microbial-derived organic acids. This approach allows for accurate quantification of compounds of interest (0.01 mM - 100 mM) with minimal sample preparation, a moderate HPLC method run time, and preservation of both guard and analytical column integrity. This approach paves the way for further analyses of microbial-derived metabolites in complex clinical samples.

Keywords:  amino acids; glycoproteins; high performance liquid chromatography; mucin; organic acids; reverse phase liquid chromatography; targeted metabolomics

DOI:  https://doi.org/10.1099/jmm.0.001708
Vet Med Int. 2023 ;2023 6692920

Florfenicol and Florfenicol Amine Quantification in Bull Serum and Seminal Plasma by a Single Validated UHPLC-MS/MS Method.

Anisa Bardhi, Juan E Romano, Giampiero Pagliuca, Alice Caneschi, Andrea Barbarossa.

Florfenicol is a broad-spectrum antibiotic belonging to the amphenicols class that inhibits protein synthesis by binding to bacteria's ribosomal subunits. This drug is commonly used in veterinary medicine to treat bacterial infectious diseases in cattle, swine, poultry, and fish. The proposed method uses a quick protein precipitation with acetonitrile for the extraction of florfenicol and florfenicol amine in serum and seminal plasma, followed by analysis in UHPLC-MS/MS for their simultaneous quantification. A BEH C18 reversed-phase column was chosen for analyte separation, allowing to obtaining sharp and symmetrical peak shapes in a chromatographic run of just 3.5 min under programmed conditions. Two specific transitions were observed for each analyte, and florfenicol-d3 was used as the internal standard. The approach was fully validated in each matrix over ranges suitable for field concentrations of florfenicol and florfenicol amine, showing good linearity during each day of testing (R2 always >0.99). Excellent accuracy and precision were demonstrated, for both analytes, by calculated bias always within ±15% and CV% always below 15% at all QC levels tested. The satisfactory outcomes obtained during recovery, matrix effect, and process efficiency investigations in serum and seminal plasma confirmed the strength of the method for the quantification of target compounds. To our knowledge, this is the first LC-MS/MS-validated approach for the quantification of florfenicol and florfenicol amine in serum and seminal plasma and was successfully applied for the determination of their concentration-time profiles in bulls. This paves the way to understanding the pharmacokinetics of this antibiotic and its active metabolite in bull's seminal plasma, which will enable the design of more appropriate treatment protocols.

DOI: https://doi.org/10.1155/2023/6692920
Fa Yi Xue Za Zhi. 2023 Apr 25. pii: 1004-5619(2023)02-0151-10. [Epub ahead of print]39(2): 151-160

Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique.

Jiao-Jiao Ji, Duo-Qi Xu, Ping Xiang, Hui Yan, Min Shen.

   OBJECTIVES: To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments.
METHODS: Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring (MRM) mode.
RESULTS: The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges (r>0.99), the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm.
CONCLUSIONS: The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

Keywords:  drug-facilitated sexual assault; forensic medicine; liquid chromatography-tandem mass spectrometry (LC-MS/MS); micro-segmentation; psychoactive substance; single hair; toxicological analysis; zolpidem

DOI:  https://doi.org/10.12116/j.issn.1004-5619.2022.321101
Environ Sci Technol. 2023 Jun 08.

Determination of 1,3-Diphenylguanidine, 1,3-Di-o-tolylguanidine, and 1,2,3-Triphenylguanidine in Human Urine Using Liquid Chromatography-Tandem Mass Spectrometry.

Zhong-Min Li, Kurunthachalam Kannan.

  1,3-Diphenylguanidine (DPG), 1,3-di-o-tolylguanidine (DTG), and 1,2,3-triphenylguanidine (TPG) are rubber additives widely present in the indoor environment. Nevertheless, little is known about their human exposure. We developed a method for the quantification of DPG, DTG, and TPG in human urine, using high-performance liquid chromatography-tandem mass spectrometry. The quantitative analysis of target analytes at parts-per-trillion levels in urine was optimized using hydrophilic-lipophilic balanced solid-phase extraction and isotopic dilution. The method limits of detection and quantification were in the range of 0.002-0.02 and 0.005-0.05 ng/mL, respectively. The recoveries of all analytes in human urine fortified at 1, 5, 10, and 20 ng/mL concentrations were in the range of 75.3-111%, with standard deviations of 0.7-4%. The repeated measurement of similarly fortified human urine yielded intra-day and inter-day variations of 0.47-3.90 and 0.66-3.76%, respectively. The validated method was applied in the measurement of DPG, DTG, and TPG in real human urine samples, which revealed the occurrence of DPG in children's urine samples (n = 15) with a detection frequency of 73% and at a median concentration of 0.05 ng/mL. DPG was found in 20% of adults' urine samples (n = 20).

Keywords:  1,2,3-triphenylguanidine; 1,3-di-o-tolylguanidine; 1,3-diphenylguanidine; human exposure; rubber additive; urine

DOI:  https://doi.org/10.1021/acs.est.3c00412
Sci Rep. 2023 06 07. 13(1): 9294

Simultaneous determination of 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid and main plasma aminothiols by HPLC-UV based method.

Justyna Piechocka, Monika Wyszczelska-Rokiel, Rafał Głowacki.

The report presents the first method for simultaneous determination of plasma 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA), an adduct of cysteine (Cys) and active form of vitamin B6 pyridoxal 5'-phosphate (PLP), as well as total low molecular-weight thiols content, including Cys, homocysteine (Hcy), cysteinyl-glycine (Cys-Gly), and glutathione (GSH). The assay is based on high performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) and involves disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP), derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) followed by sample deproteinization with perchloric acid (PCA). The chromatographic separation of obtained stable UV-absorbing derivatives is achieved on ZORBAX SB-C18 (150 × 4.6 mm, 5.0 µm) column using gradient elution with eluent consisted of 0.1 mol/L trichloroacetic acid (TCA), pH 1.7 and acetonitrile (ACN), delivered at a flow rate 1 mL/min. Under these conditions, the analytes are separated within 14 min at room temperature, and quantified by monitoring at 355 nm. Regarding HPPTCA, the assay linearity was demonstrated within a 1-100 µmol/L in plasma and the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). The accuracy ranged from 92.74 to 105.57% and 95.43 to 115.73%, while precision varied from 2.48 to 6.99% and 0.84 to 6.98% for intra- and inter-day measurements, respectively. The utility of the assay was proved by application to plasma samples delivered by apparently healthy donors (n = 18) in which the HPPTCA concentration ranged from 19.2 to 65.6 µmol/L. The HPLC-UV assay provides complementary tool for routine clinical analysis, facilitating further studies on the role of aminothiols and HPPTCA in living systems.

DOI: https://doi.org/10.1038/s41598-023-36548-9
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 May 26. pii: S1570-0232(23)00162-9. [Epub ahead of print]1225 123752

Validation of an LC-MS/MS method for simultaneous quantification of abiraterone, enzalutamide and darolutamide in human plasma.

Stefan A J Buck, Peter de Bruijn, Inge M Ghobadi-Moghaddam-Helmantel, Mei H Lam, Ronald de Wit, Stijn L W Koolen, Ron H J Mathijssen.

  Currently, several oral androgen receptor signalling inhibitors are available for the treatment of advanced prostate cancer. Quantification of plasma concentrations of these drugs is highly relevant for various purposes, such as Therapeutic Drug Monitoring (TDM) in oncology. Here, we report a liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for the simultaneous quantification of abiraterone, enzalutamide, and darolutamide. The validation was performed according to the requirements of the U.S. Food and Drug Administration and European Medicine Agency. We also demonstrate the clinical applicability of the quantification of enzalutamide and darolutamide in patients with metastatic castration-resistant prostate cancer.

Keywords:  Abiraterone; Darolutamide; Enzalutamide; Human plasma; LC-MS/MS

DOI:  https://doi.org/10.1016/j.jchromb.2023.123752