bims-metlip 2024-11-10 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2024–11–10
33 papers selected by
Sofia Costa, Matterworks

Exploring electromembrane extraction coupled to fast LC-MS/MS as a high-throughput platform for determination of 12 polar endogenous metabolites in human plasma.
Expanding Spatial Metabolomics Coverage with Lithium-Doped Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging.
Quantitative analysis of methylmalonic acid in human plasma by LC-MS/MS after derivatization.
Multiparametric LC-MS/MS method for simultaneous determination of eleven antifungal drugs and metabolites in human plasma.
An accurate measurement method for serum homocysteine measurement by ID-LC/MS/MS and the application of external quality assessment.
Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids.
Validation of an LC-MS/MS Method for Analysis of 58 Drugs of Abuse in Oral Fluid and Method Comparison with an Established LC-HRMS Method.
Evaluation of in-house-built pipette-tip micro-solid-phase extraction devices for sample preparation in the analysis of amino compounds by liquid chromatography-tandem mass spectrometry.
Measurement of tepotinib by UPLC‒MS/MS and its interaction with naringenin in rats.
A broad-spectrum peptide screening method using an optimized solid-phase extraction and liquid chromatography-high-field asymmetric ion mobility spectrometry-mass spectrometry for doping control in equine urine.
Rapid and Simultaneous Determination of 13 Sulfonamides in Soil by Matrix Solid-Phase Dispersion With High-Performance Liquid Chromatography-Tandem Mass Spectrometry.
Multiplatform metabolomic interlaboratory study of a whole human stool candidate reference material from omnivore and vegan donors.
Quantifying the extended energy metabolome of industrially important microorganisms (Saccharomyces cerevisiae) using ultra-performance liquid chromatography with mass spectrometry.
Liquid chromatography-mass spectrometric method for the simultaneous analysis of branched-chain amino acids and their ketoacids from dried blood spot as secondary analytes for the detection of maple syrup urine disease.
Human Biomonitoring of Serum Polybrominated Diphenyl Ethers by Supported Liquid Extraction and Gas Chromatography Coupled With Tandem Mass Spectrometry.
Enhanced Lipidomics Analysis of Breast Cancer Cells Using Three-phase Liquid Extraction and Ultra High-performance Liquid Chromatography Coupled With Quadrupole Time-of-Flight Tandem Mass Spectrometry.
Deducing formation routes of oxylipins by quantitative multiple heart-cutting achiral-chiral 2D-LC-MS.
An accurate and precise liquid chromatography-tandem mass spectrometry method for the determination of six phosphatidylethanol homologues in whole blood with phospholipid interferences minimized.
Evaluating quackery formulations: LC-MS/MS based method for detecting glucocorticoid content.
Separation of nucleobases, nucleosides, nucleotides and oligonucleotides by hydrophilic interaction liquid chromatography (HILIC): A state-of-the-art review.
Custom GC×GC configuration for the selective isolation or removal of compounds from complex samples.
Development and validation of an LC-MS/MS method for highly concentrated tacrolimus and cyclosporine samples prepared from pharmaceutical products to assess drug loss from feeding tubes.
A Rapid and Simple HPLC-MS/MS Method for the Quantitative Determination of Colistin for Therapeutic Drug Monitoring in Clinical Practice.
Development and validation of an LC‒MS/MS method for the determination of cyclocreatine phosphate and its related endogenous biomolecules in rat heart tissues.
Investigation of the Cholesterol Biosynthesis by Heart-Cut Liquid Chromatography and Mass Spectrometric Detection.
Elucidation of endogenous and exogenous chemicals in maternal serum using high-resolution mass spectrometry.
Recent developments in the analytical approaches of acyl-CoAs to assess their role in mitochondrial fatty acid oxidation disorders.
Membrane-based preparation for mass spectrometry imaging of cultures of bacteria.
Detection of IDH mutation in glioma by desorption electrospray ionization (DESI) tandem mass spectrometry.
A Fast-Forward Dilute-and-Shoot Multielement Method for Analysis of 33 Elements in Human Whole Blood, Serum, and Urine by Inductively Coupled Plasma Mass Spectrometry: A Streamlined Approach for Clinical Diagnostic and Biomonitoring.
A liquid chromatography - Tandem mass spectrometry method for determination of ocrelizumab in serum of patients with multiple sclerosis.
Supercritical Fluid Chromatography in Bioanalysis-A Review.
Maximizing sensitivity and selectivity in LC × LC-HRMS for pesticide analysis via exploitation of per-aqueous liquid chromatography.

J Chromatogr A. 2024 Nov 22. pii: S0021-9673(24)00825-2. [Epub ahead of print]1737 465451

Exploring electromembrane extraction coupled to fast LC-MS/MS as a high-throughput platform for determination of 12 polar endogenous metabolites in human plasma.

Anne Oldeide Hay, Frederik André Hansen.

  High efficiency in the analytical workflow, including fast sample preparation and LC-MS/MS analysis, is an advantage when analyzing a high number of samples. It can however be a challenge when determining polar analytes in complex, biological samples, and one must expect to make a compromise between a simple sample preparation followed by a long chromatographic separation, or vice versa, to limit matrix effects. In this proof-of-concept work, a one-step 96-well (parallel extraction) electromembrane extraction (EME) method was coupled to flow injection-MS/MS of 0.7 min per sample, allowing a very high-throughput analysis of 12 polar, endogenous metabolites from unprecipitated plasma of limited dilution. The throughput of the EME method matched the subsequent analysis. Recoveries ranged from 6 to 93 %, and repeatability and linearity were 2-15 % and R2 ≥ 0.9949, respectively, for all but two compounds. Matrix effects were approximately 50 % after EME and varied <11 % between 6 plasma donors, which represented a major improvement relative to a simple protein precipitation where signals were entirely suppressed. The work demonstrates a potential for EME coupled to flow injection-MS/MS to serve as a high-throughput platform for bioanalysis, not just of polar analytes, but also hydrophobic drugs both basic and acidic.

Keywords:  Acetyl choline; Electromembrane extraction; Flow injection - mass spectrometry; High throughput; Polar endogenous metabolites

DOI:  https://doi.org/10.1016/j.chroma.2024.465451
Anal Chem. 2024 Nov 06.

Expanding Spatial Metabolomics Coverage with Lithium-Doped Nanospray Desorption Electrospray Ionization Mass Spectrometry Imaging.

Kiera Nguyen, Gillian Carleton, Julian J Lum, Kyle D Duncan.

Spatial metabolomics has emerged as a powerful tool capable of revealing metabolic gradients throughout complex heterogeneous tissues. While mass spectrometry imaging (MSI) technologies designed to generate spatial metabolomic data have improved significantly over time, metabolite coverage is still a significant limitation. It is possible to achieve deeper metabolite coverage by imaging in positive and negative polarities or imaging several serial sections with different targeted biomolecular classes. However, this significantly increases the number of tissue samples required for biological studies and reduces the capacity for larger sample cohorts. Herein, we introduce lithium-doped nanospray desorption electrospray ionization (nano-DESI) as a simple and robust method to increase spatial metabolomics coverage, which is achieved through enhancements to ionization efficiencies in positive ion mode for metabolites and lipids lacking basic moieties, and improved structurally diagnostic tandem mass spectra for [M + Li]+ adducts. Specifically, signal intensities were found to be enhanced by 10-1000× for 96 compounds including small molecule metabolites, fatty acids, neutral lipids (e.g., diacylglycerols, DAG), and phospholipids when lithium was added to the ESI solvent. In addition, proof-of-principle results reveal that lithium-doped nano-DESI MSI was able to comprehensively visualize metabolites and lipids in the prostaglandin (PG) biosynthetic pathway with PG isomeric resolution in an ovarian tumor section. These data show colocalization of fatty acid (FA) 20:4 containing DAGs, FA 20:4 monoacylglycerols (MAGs), and FA 20:4 with PGE2 and disparate localizations of PGD2. Overall, this study describes a simple and powerful approach to more comprehensively probe the spatial metabolome with MSI.

DOI: https://doi.org/10.1021/acs.analchem.4c03553
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Oct 31. pii: S1570-0232(24)00368-4. [Epub ahead of print]1248 124359

Quantitative analysis of methylmalonic acid in human plasma by LC-MS/MS after derivatization.

Danhong Shen, Wei Chen, Jindi Liu, Yanhua Liu, Hongjun Shi.

  Methylmalonic acid (MMA) is a reverse biomarker of vitamin B12 that is increasingly utilized in clinical practice. However, its low sensitivity and susceptibility to strong interference from isomer present chromatographic challenges. We have developed a rapid derivatization method for plasma MMA at room temperature, converting it to the corresponding 2,2,2-trifluoroethylamide derivative using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and 2,2,2-trifluoroethylamine hydrochloride (TFEA). Amidization was completed within 10 min, followed by protein precipitation extraction of the amides with trichloroacetic acid for Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. This technique notably enhanced the signal-to-noise ratio of MMA in chromatography. The derivatized MMA exhibited excellent linearity within a concentration range of 42.4-2711.9 nmol/L, with a correlation coefficient (R2) of 0.9990. The intraday and interday precision of replicate measurements ranged from 2.4 % to 4.4 % and 2.6 % to 2.8 %, respectively, while the recovery fell between 97.9 % and 100.1 %.

Keywords:  Derivatization; Liquid chromatography-tandem mass; Methylmalonic acid; Vitamin B12

DOI:  https://doi.org/10.1016/j.jchromb.2024.124359
J Pharm Biomed Anal. 2024 Nov 01. pii: S0731-7085(24)00599-5. [Epub ahead of print]253 116557

Multiparametric LC-MS/MS method for simultaneous determination of eleven antifungal drugs and metabolites in human plasma.

Jean-Joseph Bendjilali-Sabiani, Céline Constans, Olivier Mathieu, Yoann Cazaubon.

  A multiparametric liquid chromatography-tandem mass spectrometry method has been developed for the simultaneous quantification of 11 antifungal drugs and their metabolites in human plasma. This method addresses the critical need for therapeutic drug monitoring in the treatment of invasive fungal infections, which are increasingly prevalent among immunocompromised patients and those in intensive care units. The method quantifies flucytosin, fluconazole, itraconazole, hydroxy-itraconazole, posaconazole, isavuconazole, voriconazole, voriconazole-N-oxide, anidulafungin, caspofungin, and micafungin. Key challenges in method development included optimising mass spectrometer settings, chromatographic conditions, and sample preparation techniques to ensure accurate, sensitive, and specific detection. Validation of this method was conducted in accordance with the guidelines set by the USA Food and drug administration and the European Medicines Agency covering linearity, precision, accuracy, selectivity, matrix effect, and stability. The method exhibited robust performance with intra- and inter-assay precision under 10 % and average accuracy for intra- and inter-assay comparison of -2.35 % and 0.80 %, respectively. Limits of detection (0.002 to 0.110 mg/L) and a quantification range between 0.005 and 200 mg/L make this method suitable for clinical TDM applications. The ability to simultaneously analyse eleven antifungals and their metabolites within a single 5-minute run enhances its utility in clinical settings, particularly for critically ill patients who may experience significant pharmacokinetic variations. The method requires only 100 µL of plasma, demonstrating good analytical performances rendering it a valuable tool for optimising antifungal therapy and improving patient outcomes in ICU management.

Keywords:  Antifungal; Assay method; Mass spectrometry; Multiparametric analysis; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jpba.2024.116557
Clin Chim Acta. 2024 Nov 01. pii: S0009-8981(24)02274-5. [Epub ahead of print] 120021

An accurate measurement method for serum homocysteine measurement by ID-LC/MS/MS and the application of external quality assessment.

Mo Wang, Jie Shi, Yichuan Song, Shunli Zhang, Rui Zhang.

   BACKGROUND: Serum homocysteine (Hcy) measurement accuracy is essential for detecting and diagnosing cardio-cerebrovascular illnesses. Although many different methods have been developed to determine the concentration of Hcy, those different assays showed nonequivalent results. This study aimed to develop an accurate and precise isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC/MS/MS) method for serum homocysteine (Hcy) and assign values for reference materials used in external quality assessment (EQA) programs.
METHODS: Different concentrations of homocysteine calibration solutions were prepared with d4-Hcy as the internal standard. This method employed DTT to reduce protein-bound Hcy, followed by protein precipitation, and gradient elution on a Supelcosil LC-CN column for chromatographic separation, and multiple reaction monitor (MRM) mode with electrospray ionization (ESI) for mass spectrometric detection. After optimized ID-LC/MS/MS parameters, imprecision, trueness, the limit of quantification (LoQ), the limit of detection (LoD), and measurement uncertainty were evaluated to check the methodological performance. The established method was employed in assigning values to EQA samples, which were sent to 63 clinical laboratories in Beijing to measure Hcy.
RESULTS: At 10.69,15.99, and 37.80 μmol/L levels, the present method demonstrated analytical imprecision of 0.57 %, 0.65 %, and 0.57 %, and recoveries of 99.67 % to 100.21 %, respectively. The bias between the target values of GBW (Guojia Biaozhun Wuzhi) were - 0.79 % to 0.62 %. The LoQ and LoD for Hcy were 0.36 nmol/L and 0.27 nmol/L. The method had an uncertainty (U 95 %) of 1.34 % to 1.48 %. For the three levels of EQA samples, the percentage of laboratories meeting the trueness evaluation criteria (±10 %) was 81.67 %, 83.33 %, and 71.67 % respectively.
CONCLUSIONS: With optimal method precision and trueness, the ID-LC/MS/MS method to measure serum Hcy can be used for value assignment of EQA samples, which can provide reliable data for monitoring the accuracy of clinical laboratory for Hcy measurement.

Keywords:  External quality assessment; Homocysteine; Isotope Dilution Liquid Chromatography-tandem Mass Spectrometry

DOI:  https://doi.org/10.1016/j.cca.2024.120021
J Lipid Res. 2024 Oct 25. pii: S0022-2275(24)00182-2. [Epub ahead of print] 100677

Quantifying acyl-chain diversity in isobaric compound lipids containing monomethyl branched-chain fatty acids.

C R Green, M J Kolar, G H McGregor, A T Nelson, M Wallace, C M Metallo.

  Compound lipids comprise a diverse group of metabolites present in living systems, and metabolic- and environmentally-driven structural distinctions across this family is increasingly linked to biological function. However, methods for deconvoluting these often isobaric lipid species are lacking or require specialized instrumentation. Notably, acyl-chain diversity within cells may be influenced by nutritional states, metabolic dysregulation, or genetic alterations. Therefore, a reliable, validated method of quantifying structurally similar even-, odd-, and branched-chain acyl groups within intact compound lipids will be invaluable for gaining molecular insights into their biological functions. Here we demonstrate the chromatographic resolution of isobaric lipids containing distinct combinations of straight-chain and branched-chain acyl groups via ultra-high-pressure liquid chromatography (UHPLC)-mass spectrometry (MS) using a C30 liquid chromatography column. Using metabolically-engineered adipocytes lacking branched-keto acid dehydrogenase A (Bckdha), we validate this approach through a combination of fatty acid supplementation and metabolic tracing using monomethyl branched-chain fatty acids and valine. We observe resolution of numerous isobaric triacylglycerols and other compound lipids, demonstrating the resolving utility of this method. This approach adds to the toolbox for laboratories to quantify and characterize acyl chain diversity across the lipidome.

Keywords:  BCKDH; Branched-chain fatty acids; C30 chromatography; stable isotope tracing

DOI:  https://doi.org/10.1016/j.jlr.2024.100677
J Anal Toxicol. 2024 Nov 04. pii: bkae087. [Epub ahead of print]

Validation of an LC-MS/MS Method for Analysis of 58 Drugs of Abuse in Oral Fluid and Method Comparison with an Established LC-HRMS Method.

Yufang Zheng, Magnus Axelsson, Moa Andresen Bergström.

  Liquid chromatography-mass spectrometry (LC-MS) methods for detection of multiple drugs of abuse (DoA) in oral fluid (OF) samples are being implemented in many clinical routine laboratories. Therefore, there is a need to develop new multi-analyte methods with simple sample pre-treatment and short analysis times. The purpose of this work was to validate a method detecting 58 DoA to be used with two different OF sampling kits, the saliva collection system (SCS) from Greiner Bio-One and Quantisal from Immunalysis, using the same sample pretreatment and analytical method. A set of 110 samples collected with the SCS kit was further compared to an LC-HRMS (high resolution mass spectrometry) method in another laboratory. The method was successfully validated, with precision and accuracy of ≤15% and z-scores of <2 for external controls. Using a sensitive LC-MS/MS instrument, the detection limits were <1 µg/L in neat oral fluid. In the comparative study between the LC-MS/MS and LC-HRMS methods using SCS samples, a good agreement was observed. Discrepancies were limited to lower concentration ranges, attributable to differences in cut-off thresholds between the methods. This work contributes to the development of LC-MS multi-analyte methods for OF samples, which are suitable for clinical routine laboratories.

Keywords:  Clinical toxicology; Drugs of Abuse; High Resolution Mass Spectrometry; Oral Fluid

DOI:  https://doi.org/10.1093/jat/bkae087
J Chromatogr A. 2024 Oct 28. pii: S0021-9673(24)00854-9. [Epub ahead of print]1738 465480

Evaluation of in-house-built pipette-tip micro-solid-phase extraction devices for sample preparation in the analysis of amino compounds by liquid chromatography-tandem mass spectrometry.

Larissa Silva Maciel, Michaela Hříbková, Koit Herodes.

  Amino compounds are of significant interest in dietary, clinical, and quality control fields. Efficient extraction is crucial for comprehensive metabolomics, especially for amino acids and biogenic amines, but traditional solid-phase extraction (SPE) methods are costly and require large solvent volumes. Miniaturized SPE techniques, like pipette-tip micro-solid-phase extraction (PT-µ-SPE), offer promising alternatives by improving throughput and reducing solvent and sorbent usage. This study presents PT-µ-SPE for the screening and quantification of amino compounds in bee products, particularly honey. The method involves derivatization with diethyl ethoxymethylenemalonate (DEEMM) and analysis using liquid chromatography-triple quadrupole mass spectrometry. Silica-based SCX sorbents were effective for a broad range of amino compounds, while WCX sorbents were better for aliphatic amines. The method's extraction efficiency was assessed across sample loading, washing, and elution solution, with recovery rates of 70 - 120% in oat bran, sea buckthorn leaves, and honey extracts. Matrix effects were observed for four amino compounds in honey. Limits of detection (LoD) and quantification (LoQ) ranged from 0.37 to 57 µg L⁻¹ and 1.13 to 174 µg L⁻¹, respectively. Covering 48 amino compounds under different PT-µ-SPE conditions, this method has been applied to several samples, demonstrating accuracy, environmental sustainability, cost-effectiveness, portability, and versatility in amino compound analysis.

Keywords:  Amino compounds; Derivatization; Honey; Pipette-tip micro-solid-phase extraction; Sample preparation

DOI:  https://doi.org/10.1016/j.chroma.2024.465480
BMC Chem. 2024 Nov 04. 18(1): 215

Measurement of tepotinib by UPLC‒MS/MS and its interaction with naringenin in rats.

Zhe Chen, Chaojie Chen, Ya-Nan Liu, Xinhao Xu, Shunbin Luo.

  We established a method based on ultra performance liquid chromatography tandem mass spectrometry (UPLC‒MS/MS) to quantitatively measure tepotinib, which was validated as acceptable and used in the evaluation of food-drug interactions between tepotinib and naringenin in rats. We used pemigatinib as the internal standard (IS), and acetonitrile and 0.1% formic acid aqueous solution constituted the mobile phase. To extract the target analyte, acetonitrile was used for protein precipitation (PPT). For UPLC‒MS/MS, we performed liquid chromatography using a C18 column, and mass spectrometry was performed in positive multiple reaction monitoring (MRM) mode. Excellent linearity was shown in the range of 0.1-500 ng/mL, and the coefficient of correlation was > 0.99. Notably, the lower limit of quantification (LLOQ) for tepotinib was determined to be 0.1 ng/mL. The intra- and inter-day accuracy of tepotinib ranged from - 1.7 to 7.3%, while the precision was ≤ 8.4%, at three concentrations except LLOQ. The recovery of each substance was ≥ 81.2%, and the matrix effects were within 90.5-98.6%. The stabilities of all analytes under different conditions met all requirements for quantitation in plasma samples. The relevant parameters, such as LLOQ, were evaluated in accordance with the principles of the Food and Drug Administration (FDA) biological verification method. Food-drug interaction study had shown that the plasma concentration of tepotinib could be significantly increased, accompanied by a decrease in clearance rate when administered with 50 mg/kg naringenin. The results showed that naringenin could increase the plasma concentration and decrease the clearance rate of tepotinib when naringenin and tepotinib were administered at the same time.

Keywords:  Drug‒drug interaction; Naringenin; Pharmacokinetics; Tepotinib; UPLC‒MS/MS

DOI:  https://doi.org/10.1186/s13065-024-01293-1
Anal Methods. 2024 Nov 06.

A broad-spectrum peptide screening method using an optimized solid-phase extraction and liquid chromatography-high-field asymmetric ion mobility spectrometry-mass spectrometry for doping control in equine urine.

Kohei Ohnuma, Misato Hirano-Kodaira, Michiko Bannai, Yoshibumi Shimizu, Masayuki Yamada, Kenji Kinoshita, Gary Ngai-Wa Leung, Hideaki Ishii.

The abuse of prohibited peptide-based drugs with a broad spectrum of chemical characteristics poses a significant concern for the horseracing industry. Recently, there has been a notable increase in positive cases of small-peptide drugs reported in equine and canine sports. In addition to small peptides, large peptides (over 2 kDa) with structural diversity have also entered the market in increasing numbers as drugs for humans and livestock. However, the simultaneous analysis of both small- and large-peptide-based drugs is still challenging. In this study, a screening method was developed to cover 74 analytes, including peptides, their catabolites, and/or their mimetics, with molecular weights ranging from 0.3 kDa to greater than 5 kDa. The simultaneous extraction of both small and large peptides was achieved using a weak cation-exchange solid-phase extraction cartridge with a mixture of different pore sizes (suitable for large peptides), followed by analysis using liquid chromatography high-field asymmetric ion mobility spectrometry tandem mass spectrometry (LC-FAIMS-MS/MS). For method validation, the limits of detection (LoDs), reproducibility, recovery, matrix effect, selectivity, and carryover were evaluated. Remarkably, the LoDs of ∼80% of the analytes were less than or equal to 50 pg ml-1, with the lowest LoD (1 pg ml-1) being observed for selected peptides in horse urine. These results indicate a substantial advancement in achieving comprehensive coverage for both small and large peptides with high sensitivity for the purpose of doping control in horseracing and equestrian sports.

DOI: https://doi.org/10.1039/d4ay01477d
J Sep Sci. 2024 Nov;47(21): e70015

Rapid and Simultaneous Determination of 13 Sulfonamides in Soil by Matrix Solid-Phase Dispersion With High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

JinPeng Yuan, Chen Liu, HaiLong Jiang, ZhaoTing Zhou, Meng Xie, YouMin Sun.

  Sulfonamides, the most frequent antibacterial agent, are widely used due to their low cost and excellent antibacterial effect. With the emergence of the environment, the potential hazard to the ecological environment has attracted the great attention of humans. Based on matrix solid-phase dispersion coupled with high-performance liquid chromatography-tandem mass spectrometry, an accurate, fast, and sensitive analytical method was developed for the determination of sulfonamides (SAs) in soil. Several influencing factors including the type of dispersants, the ratio of sample-to-sorbent, eluents, and solvent volume were investigated, and the matrix effects were evaluated. Under optimized conditions, the calibration curves exhibited excellent linearity with correlation coefficients (r) exceeding 0.996. The limit of detection (based on signal-to-noise ratio [S/N] = 3) and limit of quantification (based on S/N = 10) were 0.024-0.058 and 0.079-0.195 µg/kg, respectively. The recoveries ranging from 70.12% to 123.63% were obtained with a relative standard deviation of less than 15% at three levels (20, 40, and 200 µg/kg). The developed method was successfully applied to analyze 13 SAs at trace levels in a real soil sample. This proposed method would be an alternative and suitable for routine application in the future owing to its rapidity, sensitivity, and affordability.

Keywords:  high‐performance liquid chromatography‐tandem mass spectrometry; matrix solid‐phase dispersion; sample preparation; soil; sulfonamides

DOI:  https://doi.org/10.1002/jssc.70015
Metabolomics. 2024 Nov 04. 20(6): 125

Multiplatform metabolomic interlaboratory study of a whole human stool candidate reference material from omnivore and vegan donors.

Abraham Kuri Cruz, Marina Amaral Alves, Thorkell Andresson, Amanda L Bayless, Kent J Bloodsworth, John A Bowden, Kevin Bullock, Meagan C Burnet, Fausto Carnevale Neto, Angelina Choy, Clary B Clish, Sneha P Couvillion, Raquel Cumeras, Lucas Dailey, Guido Dallmann, W Clay Davis, Amy A Deik, Alex M Dickens, Danijel Djukovic, Pieter C Dorrestein, Josie G Eder, Oliver Fiehn, Roberto Flores, Helen Gika, Kehau A Hagiwara, Tuan Hai Pham, James J Harynuk, Juan J Aristizabal-Henao, David W Hoyt, Focant Jean-François, Matilda Kråkström, Amit Kumar, Jennifer E Kyle, Santosh Lamichhane, Yuan Li, Seo Lin Nam, Rupasri Mandal, A Paulina de la Mata, Michael J Meehan, Thomas Meikopoulos, Thomas O Metz, Thomai Mouskeftara, Nathalie Munoz, G A Nagana Gowda, Matej Orešic, Morgan Panitchpakdi, Stefanuto Pierre-Hugues, Daniel Raftery, Blake Rushing, Tracey Schock, Harold Seifried, Stephanie Servetas, Tong Shen, Susan Sumner, Kieran S Tarazona Carrillo, Dejong Thibaut, Jesse B Trejo, Lieven Van Meulebroek, Lynn Vanhaecke, Christina Virgiliou, Kelly C Weldon, David S Wishart, Lu Zhang, Jiamin Zheng, Sandra Da Silva.

   INTRODUCTION: Human metabolomics has made significant strides in understanding metabolic changes and their implications for human health, with promising applications in diagnostics and treatment, particularly regarding the gut microbiome. However, progress is hampered by issues with data comparability and reproducibility across studies, limiting the translation of these discoveries into practical applications.
OBJECTIVES: This study aims to evaluate the fit-for-purpose of a suite of human stool samples as potential candidate reference materials (RMs) and assess the state of the field regarding harmonizing gut metabolomics measurements.
METHODS: An interlaboratory study was conducted with 18 participating institutions. The study allowed for the use of preferred analytical techniques, including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR).
RESULTS: Different laboratories used various methods and analytical platforms to identify the metabolites present in human stool RM samples. The study found a 40% to 70% recurrence in the reported top 20 most abundant metabolites across the four materials. In the full annotation list, the percentage of metabolites reported multiple times after nomenclature standardization was 36% (LC-MS), 58% (GC-MS) and 76% (NMR). Out of 9,300 unique metabolites, only 37 were reported across all three measurement techniques.
CONCLUSION: This collaborative exercise emphasized the broad chemical survey possible with multi-technique approaches. Community engagement is essential for the evaluation and characterization of common materials designed to facilitate comparability and ensure data quality underscoring the value of determining current practices, challenges, and progress of a field through interlaboratory studies.

Keywords:  Fecal matter; Gut metabolomics; Human stool; Lipidomics; Metabolomics; Multiplatform analysis; Reference materials

DOI:  https://doi.org/10.1007/s11306-024-02185-0
J Chromatogr B Analyt Technol Biomed Life Sci. 2024 Oct 24. pii: S1570-0232(24)00351-9. [Epub ahead of print]1248 124342

Quantifying the extended energy metabolome of industrially important microorganisms (Saccharomyces cerevisiae) using ultra-performance liquid chromatography with mass spectrometry.

Jordan I Oliver, Antony N Davies, Richard Dinsdale.

  This study has developed a new targeted methodology for the separation, detection, and quantification of metabolites from the wider energy metabolome of industrially important microorganisms such as Saccharomyces cerevisiae in a single analytical sample. This has been achieved using UHPLC-MS technology in HILIC mode. Absolute concentrations of metabolites nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide reduced (NADH), nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide phosphate reduced (NADPH), flavin adenine dinucleotide (FAD), adenosine-monophosphate (AMP), adenosine-diphosphate (ADP), and adenosine-triphosphate (ATP) were determined in a single extraction and analytical methodology. This study demonstrated the development of a rapid, statistically robust, and reproducible methodology through regression calibrations of standard samples from 0.1 to 100 µMol providing a correlation value of r2 = >0.98 for all metabolites. Sample preparation, extraction and analytical methodologies used showed high accuracy, sensitivity, and recovery. With an LOD and LOQ for the targeted analysis of metabolites from the wider energy metabolism in a single sample and analytical run with the lowest LOD of 0.055 nMol (±0.002) and lowest LOQ of 0.167 nMol (±0.006). This method was then applied to Saccharomyces cerevisiae cell culture to evaluate the methodology in industrially used microbial cultures. Results obtained have been statistically determined to be robust and reproducible through recovery analysis using deuterated and isotopically labelled internal standards AMP-15N, ADP-15N and ATP-d14.

Keywords:  Energy metabolome; Metabolomics; Saccharomyces cerevisiae; Targeted metabolomics; UHPLC-MS

DOI:  https://doi.org/10.1016/j.jchromb.2024.124342
J Mass Spectrom Adv Clin Lab. 2024 Nov;34 8-20

Liquid chromatography-mass spectrometric method for the simultaneous analysis of branched-chain amino acids and their ketoacids from dried blood spot as secondary analytes for the detection of maple syrup urine disease.

Arya Raveendran, Ashutosh Gupta, Leslie E Lewis, Krishnananda Prabhu, Sudheer Moorkoth.

   Background: Maple syrup urine disease (MSUD) is an aminoacidopathy caused by a defective branched-chain alpha-ketoacid dehydrogenase complex, leading to the accumulation of branched-chain amino acids (BCAAs) and their respective keto acids (BCKAs). A comprehensive test was developed to measure BCAAs and BCKAs using LC-MS from dried blood spot (DBS) samples for the diagnosis and prevention of MSUD in newborns and infants.
Methods: Analytes were extracted from DBS using a methanol:0.1 % v/v formic acid solution (75:25) containing internal standards and analyzed on a Luna PFP column (150 mm × 4.6 mm, 3 µm) at a flow rate of 0.3 mL/min. The method was validated for linearity, accuracy, precision, recovery, carry-over, matrix effect, hematocrit, blood volume, and punch position effects. Biomarker stability in the matrix and stock solution was assessed. Correlation with the plasma method was determined using Pearson's correlation coefficient and Bland-Altman analysis. The method established reference ranges for the Udupi district population in South India.
Results: The method demonstrated linearity (r2 > 0.99), with a lower limit of detection at 2 µM (BCAA) and 1 µM (BCKA), and acceptable recovery of QC samples. Hematocrit, blood volume, punch position, and storage condition effects were within acceptable limits. Correlation and Bland-Altman analysis showed strong interconvertibility between plasma and DBS assays. Reference ranges for leucine, isoleucine, valine, KIC, KIV, and KMV were established.
Conclusion: The developed DBS method, requiring no derivatization and involving simple sample preparation with short run times, is a cost-effective and reliable approach for the confirmatory diagnosis of MSUD.

Keywords:  Amino acids; Branched chain keto acids; Dried blood spot; LC-MS; MSUD

DOI:  https://doi.org/10.1016/j.jmsacl.2024.10.001
J Sep Sci. 2024 Nov;47(21): e70010

Human Biomonitoring of Serum Polybrominated Diphenyl Ethers by Supported Liquid Extraction and Gas Chromatography Coupled With Tandem Mass Spectrometry.

Miao Liu, Xiangyu Jia, Rongfei Peng, Zhijun Bai, Jun Yuan, Lei Tan.

  This study aims to develop and validate a robust analytical method for the quantification of polybrominated diphenyl ethers (PBDEs) in human serum using gas chromatography-tandem mass spectrometry. We compared procedural blanks, recoveries, and operational convenience of liquid-liquid extraction and supported liquid extraction for the determination of serum PBDEs. We evaluated different extraction solvents for their effect on PBDE recoveries. Supported liquid extraction was selected for method validation due to its operational convenience. The method demonstrated satisfactory linearity, sensitivity, and reproducibility, with the range of 0.10-5.00 µg/L for most PBDE congeners and 0.20-10.0 µg/L for PBDE-154 and PBDE-183, with limits of detection ranging from 2 to 48 ng/L, and with matrix effects ranging from 94% to 113%. Quality control assessments indicated that recoveries ranged from 85% to 110% and relative standard deviations of less than 11%. The proposed method was applied to biomonitoring of 111 healthy adults, revealing detectable levels of PBDEs in over 90% of the samples. BDE-47 and BDE-183 were the most prevalent, with mean concentrations of 4.13 and 22.1 ng/L, respectively. Detection frequencies ranged from 0.90% for BDE-17 and BDE-85 to 25.2% for BDE-47. Males had higher mean concentrations of BDE-183 than females.

Keywords:  human biomonitoring; liquid–liquid extraction; polybrominated diphenyl ethers; sample preparation; supported liquid extraction

DOI:  https://doi.org/10.1002/jssc.70010
J Sep Sci. 2024 Nov;47(21): e70014

Enhanced Lipidomics Analysis of Breast Cancer Cells Using Three-phase Liquid Extraction and Ultra High-performance Liquid Chromatography Coupled With Quadrupole Time-of-Flight Tandem Mass Spectrometry.

Binhong He, Fengying Ye, Jieqing Feng, Ting Zhou.

  Lipid extraction of complex biological samples is essential for high-quality data in liquid chromatography-mass spectrometry (LC-MS)-based lipidomics. This study introduces a three-phase liquid extraction (3PLE)-ultra-high-performance LC coupled with quadrupole time-of-flight tandem MS method. This method was successfully applied to lipidomics analysis of breast cancer cells, including highly metastatic MDA-MB-231 and slightly metastatic MCF7 cells. The 3PLE method employed an n-hexane/methyl tert-butyl ether/acetonitrile/water solvent system that formed one aqueous and two organic phases. Neutral and polar lipids were enriched in the upper and middle organic phases, respectively, and combined for detection, thereby reducing analysis time. Compared with the Bligh and Dyer method, 3PLE achieved higher sensitivity and detected more features, with over a 50% increase in the relative abundance of nearly 50% of the differential lipids. In total, 21 differential lipids were identified in the MDA-MB-231 group and 22 in the MCF7 group compared to normal breast epithelial cells (MCF10A). Pathway analysis suggested that lipid changes in breast cancer cells were associated with glycerophospholipid metabolism, arachidonic acid metabolism, sphingolipid metabolism, and linoleic acid metabolism. The study presents a highly efficient lipidomics method, providing a scientific foundation for understanding breast cancer pathogenesis and aiding in diagnosis.

Keywords:  breast cancer cells | lipidomics | liquid chromatography | mass spectrometry | three‐phase liquid extraction

DOI:  https://doi.org/10.1002/jssc.70014
J Lipid Res. 2024 Nov 04. pii: S0022-2275(24)00199-8. [Epub ahead of print] 100694

Deducing formation routes of oxylipins by quantitative multiple heart-cutting achiral-chiral 2D-LC-MS.

Nadja Kampschulte, Rebecca Kirchhoff, Ariane Löwen, Nils Helge Schebb.

  Several oxylipins are regulators of inflammation. They are formed by enzymes such as lipoxygenases or cyclooxygenases, but also stereorandomly by autoxidation. Reversed-phase liquid chromatography-tandem-mass-spectrometry (LC-MS/MS) methods for oxylipin quantification do not separate enantiomers. Here, we combine sensitive and selective oxylipin analysis with chiral separation using two-dimensional (2D)-LC-MS/MS. By multiple heart-cutting, the oxylipin peaks are transferred onto a chiral column. 45 enantiomeric pairs of (di-)hydroxy-fatty acids are separated with full gradient elution within 1.80min, yielding lower limits of quantification <1pg on column. Concentrations as well as enantiomeric fractions of oxylipins can be determined, even at low concentrations or at high enantiomeric excess of one isomer. The developed achiral-chiral multiple heart-cutting 2D-LC-MS/MS method offers unprecedented selectivity, enabling a better understanding of the formation route of these lipid mediators. This is demonstrated by distinguishing the formation of hydroxy-fatty acids by (acetylated) cyclooxygenase-2 and radical-mediated autoxidation. Applying the method to human M2-like-macrophages, we show that the so-called specialized pro-resolving mediators (SPM) 5,15-DiHEPE and 7,17-DiHDHA as well as 5,15-DiHETE were present as (S,S)-enantiomers, supporting their enzymatic formation. In contrast, at least eight isomers (including protectin DX but not neutroprotectin D1) of 10,17-DiHDHA are present in immune cells, indicating formation by autoxidation. In human plasma of healthy subjects, none of these dihydroxy-fatty acids are not present. However, we demonstrate that all four isomers quickly form via autoxidation if the samples are stored improperly. Thus, dihydroxy-FA should only be reported as SPM, such as resolvin D5 or resolvin E4, if an enantioselective analysis has been carried out.

Keywords:  arachidonic acid; autoxidation; cyclooxygenase; enantioselective analysis; enzymatic oxidation; lipid oxidation; lipidomics; lipoxygenase; resolvins; sample storage

DOI:  https://doi.org/10.1016/j.jlr.2024.100694
J Chromatogr A. 2023 Oct 13. pii: S0021-9673(23)00676-3. [Epub ahead of print]1711 464451

An accurate and precise liquid chromatography-tandem mass spectrometry method for the determination of six phosphatidylethanol homologues in whole blood with phospholipid interferences minimized.

Marisa Maria, Nuno R Neng, Thomas Berg.

  Alcohol consumption is associated with a wide risk of different diseases, injury and death, and has significant social and economic consequences worldwide. Phosphatidylethanol (PEth) is a group of promising direct alcohol biomarkers, with a significantly longer half-life in blood than ethanol, which can be measured to predict different drinking patterns, such as heavy- and social drinking. This study aimed to develop and validate an accurate and precise LC-MS/MS method for the determination of six PEth homologues in whole blood with minimal interference from unwanted phospholipids. Different organic solvent mixtures for liquid-liquid extraction were investigated to obtain satisfactory recovery of PEth homologues and removal of the lyso-phospholipids and other early eluting phospholipids. The mixture of heptane/2-propanol (80:20, v:v) gave lower phospholipid background and better signal/noise values for the PEth peaks. An LC-MS/MS TQ-S system from Waters was used for the instrumental analysis. The main part of unwanted phospholipids were separated from the PEth homologues on an Acquity BEH C18 column (50 × 2.1 mm ID, 1.7 µm particles) using a buffer-free mobile phase of 0.025 % ammonia in Type 1 water, pH 10.7, as solvent A and methanol as solvent B. Validation and quantification of 22 authentic blood samples showed that the developed LC-MS/MS method is sensitive, precise and accurate for the determination of the six PEth homologues in whole blood. Lower limit of quantification was 10 nM for all compounds. No matrix effects were observed, possibly due to the successful strategies incorporated to avoid the influence of unwanted phospholipids.

Keywords:  Alcohol consumption; LC-MS/MS; Liquid-liquid extraction (LLE); PEth 16:0/18:1; Phosphatidylethanol; Phospholipid removal

DOI:  https://doi.org/10.1016/j.chroma.2023.464451
Toxicol Rep. 2024 Dec;13 101763

Evaluating quackery formulations: LC-MS/MS based method for detecting glucocorticoid content.

Hafiza Monaza Batool, Muhammad Irfan Jalees, Madeeha Batool.

  Glucocorticoids are widely used as highly effective drugs for treating inflammatory diseases. In this study, a method was developed and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously determine four glucocorticoids, including betamethasone, dexamethasone, hydrocortisone, and prednisolone in unauthorized or unregulated medicinal powders often associated with quackery formulations. Commercially available standards were used for method development and glucocorticoid detection. Glucocorticoids were extracted from the samples with methanol, which were then chromatographically separated using two mobile phases (0.1 % formic acid in water and 0.1 % formic acid in acetonitrile) in an isocratic flow on an Agilent Poroshel 120 C18 column (2.1 mm x 75 mm x 2.7 m). The validated analytical measuring range (AMR) of betamethasone and dexamethasone was 7.8-500 ng/mL, whereas, for hydrocortisone and prednisolone, AMR was 7.8-1000 ng/mL. The method showed an excellent coefficient of determination (r2) >0.990 for betamethasone, hydrocortisone, and prednisolone, while for dexamethasone 0.986. Accuracy and precision (intra/inter days) of these glucocorticoids showed a bias of 6-15 % (<20 %) and a coefficient of variation (CV) of <15 %. For each dilution factor, the integrity of samples was maintained after dilution. The developed method is sensitive and valuable for detecting, quantifying, and confirming the selected glucocorticoids in various quackery formulation powders commonly used in Pakistani setups.

Keywords:  Glucocorticoids; Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Method development; Quackery formulations; Validation

DOI:  https://doi.org/10.1016/j.toxrep.2024.101763
J Chromatogr A. 2024 Oct 22. pii: S0021-9673(24)00841-0. [Epub ahead of print]1738 465467

Separation of nucleobases, nucleosides, nucleotides and oligonucleotides by hydrophilic interaction liquid chromatography (HILIC): A state-of-the-art review.

Yong Guo.

  The polar nature of nucleobases, nucleosides and nucleotides makes hydrophilic interaction chromatography (HILIC) a good choice of technology for separation. Both naturally occurring and modified nucleosides and nucleotides have been successfully separated in HILIC. A wide range of stationary phases with different retention and selectivity are suitable for the separation of nucleobases, nucleosides and nucleotides; and a sufficient knowledge base is also available to guide method development. Although oligonucleotides are significantly different from nucleotides in terms of polarity and charges, HILIC has been shown to be a viable alternative to ion-pairing reversed-phase liquid chromatography (IP-RPLC). Only a few polar stationary phases have been shown to provide satisfactory performance; however, the requirements for the mobile phase composition including organic solvent, mobile phase pH and salt concentration are sufficiently understood. This review provides a comprehensive evaluation of the chromatographic conditions with a historical perspective on adopting and developing HILIC for the separation of nucleobases, nucleosides, nucleotides and oligonucleotides. The areas for more research and potential directions for future development activities are identified and discussed.

Keywords:  HILIC; Nucleobase; Nucleoside; Nucleotide; Oligonucleotide; Retention; Selectivity

DOI:  https://doi.org/10.1016/j.chroma.2024.465467
J Chromatogr A. 2024 Oct 29. pii: S0021-9673(24)00858-6. [Epub ahead of print]1738 465484

Custom GC×GC configuration for the selective isolation or removal of compounds from complex samples.

Matyas Ripszam, Tobias Bruderer, Federico Maria Vivaldi, Serena Reale, Fabio Di Francesco.

  We developed a novel approach to selectively isolate or remove nearly any compound from complex mixtures of volatile organic compounds. This was achieved by customizing a GC×GC system with a Deans switch, a passive splitter, and a custom-made adapter for sample recollection. The new setup was evaluated with 106 standard chemicals covering a wide range of volatility (boiling points: 56 - 343 ⁰C) and polarity (log P: 0.2 - 9.4). The method was used to remove two notorious malodorous compounds from spoiled wine samples. We found that the recovery can be maximized if a custom-made adapter is attached directly on the flame ionization detector port (average recovery rate of 76 ± 7 % for the standards). Eventually, we could selectively isolate or remove chemicals with peaks separated by a minimum distance of 50 ms for the second column throughout the whole chromatographic run. The developed system is expected to mainly be used in the field of flavor and fragrance analysis (i.e., selection of flavors and odorants of interest or removal of off-flavor or malodorous compounds). At present, we can reasonably collect about 100 ng of each single compound and are currently working on sample enrichment to improve our method to isolate sufficient amounts for further chemical analysis (e.g. high sensitivity nuclear magnetic resonance or chemical ionization tandem mass spectrometry).

Keywords:  Chemical isolation; Comprehensive two-dimensional GC; Flavor and fragrances; Preparative GC; Thermal desorption; Volatile organic compounds

DOI:  https://doi.org/10.1016/j.chroma.2024.465484
J Mass Spectrom Adv Clin Lab. 2024 Nov;34 28-33

Development and validation of an LC-MS/MS method for highly concentrated tacrolimus and cyclosporine samples prepared from pharmaceutical products to assess drug loss from feeding tubes.

Yi Xiao, Mari Ishak Gabra, Edward Leung.

   Introduction: Tacrolimus and cyclosporine are common immunosuppressants utilized post-organ transplantation to manage allograft rejection. Both have narrow therapeutic indices and are frequently measured to support dose adjustments. Although nasogastric tubes are commonly used to provide nutritional support and serve as a route for immunosuppressant administration, they were never validated for such purposes.
Objective: To develop and validate a liquid chromatography - tandem mass spectrometry (LC-MS/MS) method for highly concentrated tacrolimus and cyclosporine samples prepared from pharmaceutical products to support the validation of feeding tube administration of these immunosuppressants.
Methods: The method involved stepwise dilutions with dimethyl sulfoxide before analysis using online sample preparation and LC-MS/MS. It was validated in a CLIA-certified clinical laboratory that measures immunosuppressants by LC-MS/MS and is designed to support clinical studies evaluating drug loss from feeding tubes.
Results: The method was linear between 6.8 µg/mL and 75 µg/mL for tacrolimus, and between 0.9 mg/mL and 10 mg/mL for cyclosporine, with r2 > 0.99 and total precision <5 % at all QC levels. The method demonstrated good recovery using cyclosporine Certified Reference Material, tacrolimus European Pharmacopeia Reference Standard, and prepared pharmaceutical products. Minimal matrix effects were observed.
Conclusion: An analytical method was developed and validated for in vitro studies with simulated administration of tacrolimus or cyclosporine to assess loss during drug administration using feeding tubes.

Keywords:  Immunosuppressants; LC-MS/MS; NG tubes

DOI:  https://doi.org/10.1016/j.jmsacl.2024.10.002
Drug Des Devel Ther. 2024 ;18 4877-4887

A Rapid and Simple HPLC-MS/MS Method for the Quantitative Determination of Colistin for Therapeutic Drug Monitoring in Clinical Practice.

Na Zhang, Yiran Xu, Beibei Liang, Jinru Zeng, Rui Wang, Yun Cai.

  Colistin is the last-line option for the treatment of multidrug-resistant gram-negative bacterial infections with narrow therapeutic window. It is essential to ensure its efficacy and safety by therapeutic drug monitoring (TDM). Quantitative determination of colistin is difficult due to its complex ingredients. Previous determination methods demand intricate sample pre-treatment which are not only time-consuming but also costly, and is difficult to apply in clinical practice. Therefore, in order to carry out quantitative determination of colistin accurately and quickly, we establish a rapid high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) with simple sample pre-treatment process. The sample was purified by acetonitrile to remove the plasma protein. Then purified colistin was effectively separated from terfenadine, an internal standard (IS) using Phenomenex Kinetex C18 column (50.0×2.1mm, 5µm) with acetonitrile and water mobile phase at a flow rate of 0.5 mL/min and 40°C column temperature. Colistin and IS were monitored in positive ion mode. Our method expressed good linearity in 50.0~6000 ng/mL of colistin B and 28.31~3397.51 ng/mL of colistin A in plasma. Methodology validations, including selectivity, precision, accuracy, recovery, stability, matrix effect, and dilution integrity met acceptance criteria of Bioanalytical Method Validation (M10) of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH).

Keywords:  HPLC-MS/MS; colistin; human plasma; protein precipitation; rapid and simple detection

DOI:  https://doi.org/10.2147/DDDT.S479329
BMC Chem. 2024 Nov 04. 18(1): 214

Development and validation of an LC‒MS/MS method for the determination of cyclocreatine phosphate and its related endogenous biomolecules in rat heart tissues.

Ibrahim F Abo-Elmagd, Amr M Mahmoud, Medhat A Al-Ghobashy, Marianne Nebsen, Mostafa A Rabie, Ahmed F Mohamed, Lamiaa A Ahmed, Nesrine S El Sayed, Reem K Arafa, Robert Todd, Salwa A Elgebaly.

  The cardioprotective drug cyclocreatine phosphate has been awarded Food and Drug Administration-orphan drug designation for the prevention of ischemic injury to enhance cardiac graft recovery and survival in heart transplantation. Cyclocreatine phosphate is the water-soluble derivative of cyclocreatine. Estimating the levels of Cyclocreatine phosphate, Adenosine triphosphate, Creatine Phosphate, Creatine and Cyclocreatine helps us in understanding the energy state as well as evaluating the heart cells' function. The quantification of endogenous compounds imposes a challenging task for analysts because of the absence of a true blank matrix, whose use is required according to international guidelines. Recently, the International Council for Harmonization issued a new guideline that contains guidance on the validation of methods used to quantify endogenous components, such as the background subtraction approach that was employed in our current study. Specifically, we developed and validated a sensitive, reliable and accurate liquid chromatography-tandem mass spectrometry assay to determine simultaneously the levels of mentioned endogenous compounds in rat heart tissue. Tissue samples were prepared by protein precipitation extraction using water: methanol (1:1). Using Ultra Performance Liquid Chromatography, Chromatographic separation was achieved with ZORBAX Eclipse Plus C18 4.6 × 100 mm,3.5 μm column and conditions as following: ammonium acetate (pH 8.5): acetonitrile, 70:30 mobile phase, 0.7 mL/min flow rate and 25 °C temperature. Electrospray ionization mass detector with Multiple reaction monitoring mode was then employed, using both positive and negative modes, Analysis was carried out using 5.00-2000.00 ng/mL linear concentration range within 2 min for each analyte. According to Food and Drug Administration guidelines for bioanalytical methods, validation was carried out. We investigated the matrix effect, recovery efficiency and process efficiency for the analyte in neat solvent, postextraction matrix and tissue. The results stated mean percentage recoveries higher than 99%, accuracy 93.32-111.99%, and Relative Standard Deviation (RSD) below 15% within the concentration range of our study which indicated that target analytes' stability in their real matrix is sufficient under the employed experimental conditions.

Keywords:  Background subtraction; Cardioprotective; Cyclocreatine phosphate; Endogenous biomolecules; LC-MS/MS

DOI:  https://doi.org/10.1186/s13065-024-01304-1
J Chromatogr A. 2024 Oct 26. pii: S0021-9673(24)00849-5. [Epub ahead of print]1738 465475

Investigation of the Cholesterol Biosynthesis by Heart-Cut Liquid Chromatography and Mass Spectrometric Detection.

Pia Wittenhofer, Laura Kiesewetter, Oliver J Schmitz, Sven W Meckelmann.

  The biosynthesis and homeostasis of cholesterol are essential for cellular function. Cholesterol is a major lipid with multiple roles in membrane stability, signaling, or as a precursor for other molecules. Because of the structural similarity of the sterols involved in the biosynthesis, their accurate identification and quantification is still challenging. Moreover, the huge difference in the concentration of cholesterol and its precursors can cause interferences during the detection. To overcome these problems, a heart-cut liquid chromatographic method was developed by evaluating 38 different columns to achieve optimal separation. The method efficiently separates all sterol biosynthesis intermediates, with detection limits in the low nmol/L-range and an upper limit of quantification of 9 mmol/L for cholesterol by using triple quadrupole mass spectrometric detection. Investigation of lung carcinoma cells treated with statins demonstrated the capability to detect a biological response, showing inhibition of sterol synthesis. This technique offers a robust tool for studying cholesterol biosynthesis and its role in disease.

Keywords:  2D-LC; Cancer; Lipidomics; Statins; Sterols

DOI:  https://doi.org/10.1016/j.chroma.2024.465475
Ecotoxicol Environ Saf. 2024 Nov 01. pii: S0147-6513(24)01332-0. [Epub ahead of print]286 117256

Elucidation of endogenous and exogenous chemicals in maternal serum using high-resolution mass spectrometry.

Akifumi Eguchi, Kenichi Sakurai, Midori Yamamoto, Chisato Mori.

  The increasing exposure to environmental chemicals calls for comprehensive non-targeted analysis to detect unrecognized substances in human samples. We examined human serum samples to classify compounds as endogenous or exogenous using public databases and to explore the relationships between exposure markers and metabolic patterns. Serum samples from 84 pregnant women at 32 weeks gestation were analyzed using LC-QToFMS. Using the PubChemLite for Exposomics database, we annotated and classified 106 compounds (51 endogenous, 55 exogenous). The compound patterns were analyzed using three dimensional reduction methods: Principal Component Analysis (PCA), regularized Generalized Canonical Correlation Analysis (rGCCA), and Uniform Manifold Approximation and Projection (UMAP). OPTICS clustering applied to these methods revealed two distinct clusters, with 89 % of significant compounds overlapping between clusters. The detected exogenous compounds included dietary substances, phthalates, nitrogenous compounds, and parabens. Pathway enrichment analysis showed that chemical exposure was linked to changes in amino acid metabolism, protein and mineral transport, and energy metabolism. While we found associations between exposure and metabolite changes, we could not establish causality. Our approach of analyzing both exogenous and endogenous chemicals from the same dataset using PubChemLite database presents a new method for exposome research, despite limitations in sample size and peak annotation accuracy. These findings contribute to understanding multiple chemical exposures and their metabolic effects in human biomonitoring.

Keywords:  Clustering; Database; Liquid chromatography-high resolution mass spectrometry; Non-targeted analysis

DOI:  https://doi.org/10.1016/j.ecoenv.2024.117256
Mol Genet Metab. 2023 Oct 20. pii: S1096-7192(23)00341-4. [Epub ahead of print] 107711

Recent developments in the analytical approaches of acyl-CoAs to assess their role in mitochondrial fatty acid oxidation disorders.

Madhulika Singh, Hyung L Elfrink, Amy C Harms, Thomas Hankemeier.

  Fatty acid oxidation disorders (FAOD) are inborn errors of metabolism that occur due to deficiency of specific enzyme activities and transporter proteins involved in the mitochondrial metabolism of fatty acids, causing a deficiency in ATP production. The identification of suitable biomarkers plays a crucial role in predicting the future risk of disease and monitoring responses to therapies. Acyl-CoAs are directly involved in the steps of fatty acid oxidation and are the primary biomarkers associated with FAOD. However, acyl-CoAs are not used as diagnostic biomarkers in hospitals and clinics as they are present intracellularly with low endogenous levels. Additionally, the analytical method development of acyl-CoAs is quite challenging due to diverse physicochemical properties and instability. Hence, secondary biomarkers such as acylcarnitines are used for the identification of FAOD. In this review, the focus is on the analytical techniques that have evolved over the years for the identification and quantitation of acyl-CoAs. Among these techniques, liquid chromatography-mass spectrometry clearly has an advantage in terms of sensitivity and selectivity. Stable isotope labeling by essential nutrients in cell culture (SILEC) enables the generation of labeled internal standards. Each acyl-CoA species has a distinct pattern of instability and degradation, and the use of appropriately matched internal standards can compensate for such issues. Although significant progress has been made in measuring acyl-CoAs, more efforts are needed for bringing these technical advancements to hospitals and clinics. This review also highlights the difficulties involved in the routine use of acyl-CoAs as a diagnostic biomarker and some of the measures that can be adopted by clinics and hospitals for overcoming these limitations.

Keywords:  Acyl-CoA; Biomarkers; Fatty acid oxidation disorders; LC-MS; Newborn screening; SILEC

DOI:  https://doi.org/10.1016/j.ymgme.2023.107711
Anal Bioanal Chem. 2024 Nov 04.

Membrane-based preparation for mass spectrometry imaging of cultures of bacteria.

Farès Slimani, Laurence Hotel, Aurélie Deveau, Bertrand Aigle, Patrick Chaimbault, Vincent Carré.

  The study of the dialogue between microorganisms at the molecular level is becoming essential to understand their relationship (antagonist, neutral, or beneficial interactions) and its impact on the organization of the microbial community. Mass spectrometry imaging (MSI) with matrix-assisted laser desorption/ionization (MALDI) is a technique that reveals the spatial distribution of molecules on a sample surface that may be involved in interactions between organisms. An experimental limitation to perform MALDI MSI is a flat sample surface, which in many cases could not be achieved for bacterial colonies such as filamentous bacteria (e.g., Streptomyces). In addition, sample heterogeneity affects sample dryness and MALDI matrix deposition prior to MSI. To avoid such problems, we introduce an additional step in the sample preparation. A polymeric membrane is interposed between the microorganisms and the agar-based culture medium, allowing the removal of bacterial colonies prior to MSI of the homogeneous culture medium. A proof of concept was evaluated on Streptomyces ambofaciens (a soil bacterium) cultures on solid media. As the mycelium was removed at the same time as the polymeric membrane, the metabolites released into the medium were spatially resolved by MALDI MSI. In addition, extraction of the recovered mycelium from the membrane confirmed the identification of the metabolites by ESI MS/MS analysis. This approach allows both the spatial distribution of metabolites produced by microorganisms in an agar medium to be studied under well-controlled sample preparation and their structure to be elucidated. This capability is illustrated using desferrioxamine E, a siderophore produced by S. ambofaciens.

Keywords:  Bacteria; MALDI; Mass spectrometry imaging; Membrane-based; Metabolomic; Preparation method

DOI:  https://doi.org/10.1007/s00216-024-05622-0
Sci Rep. 2024 11 06. 14(1): 26865

Detection of IDH mutation in glioma by desorption electrospray ionization (DESI) tandem mass spectrometry.

Mahdiyeh Shahi, Steven Pringle, Michael Morris, Diogo Moniz Garcia, Alfredo Quiñones-Hinojosa, R Graham Cooks.

  Desorption electrospray ionization (DESI) tandem mass spectrometry (MS) is used to assess mutation status of isocitrate dehydrogenase (IDH) in human gliomas. Due to the diffuse nature of gliomas, total gross resection is not normally achieved during surgery, leading to tumor recurrence. The mutation status of IDH has clinical significance due to better prognosis in IDH-mutant patients. The mutant IDH converts alpha-ketoglutaric acid (α-KG) into 2-hydroxyglutarate (2HG), which accumulates abnormally in cells. Immunohistochemical staining (IHC) and genetic testing, the gold standards, are incompatible with intraoperative applications but DESI tandem mass spectrometry (MS/MS) can be used to assess the mutation status of IDH enzyme from tissue intraoperatively. Here, on off-line evaluation is made of the performance of two different types of mass spectrometers in characterization of IDH mutation status. The intensity of 2HG is measured against glutamate (Glu), an intrinsic reference molecule, in both tandem MS measurements. In both cases using DESI clear separation between IDH-mutant (mut) and IDH-wildtype (wt) samples (p < 0.0001) is observed, despite the short analysis time. Due to the higher detection sensitivity, multiple reaction monitoring experiments using a triple quadrupole show slightly better performance compared to product ion MS/MS performed on a simple linear ion trap. Both DESI-MS platforms are capable of providing information on IDH mutation status, which might in future be used at the time of surgery to support decision-making on resection regions, especially at tumor margins.

Keywords:  2-hydroxyglutarate; Ambient ionization; Brain cancer; Molecular diagnostics; Multiple reaction monitoring (MRM); Oncometabolite

DOI:  https://doi.org/10.1038/s41598-024-77044-y
J Anal Methods Chem. 2024 ;2024 9944995

A Fast-Forward Dilute-and-Shoot Multielement Method for Analysis of 33 Elements in Human Whole Blood, Serum, and Urine by Inductively Coupled Plasma Mass Spectrometry: A Streamlined Approach for Clinical Diagnostic and Biomonitoring.

Sandra Huber, Jörg Michel, Maurice Reijnen, Maria Averina, Bjørn Bolann, Jon Øyvind Odland, Solrunn Hansen, Jan Brox.

The analysis of toxic and essential elements in human matrices is used in clinical diagnostics and for biomonitoring of different populations to study related health outcomes. This work aimed to develop fast and reliable methods for the analysis of a broad range of elements in liquid human matrices, such as whole blood, serum, and urine, with a similar setup for the three matrices and different analysis needs. An easy and fast-forward dilute-and-shoot method for 33 elements (i.e., Ag, Al, As, B, Ba, Be, Bi, Cd, Ce, Co, Cr, Cu, Hg, I, Li, Mn, Mo, Ni, Pb, Pd, Pt, Sb, Se, Sn, Sr, Te, Th, Tl, U, V, W, Zn, and Zr) was developed. 200 µL of either sample material was diluted with an alkaline reagent to a volume of 4 mL in total. Sample dilution and preparation of matrix-matched calibration standards were performed in 48-well plates by an automated liquid handler. Diluted samples were analyzed by inductively coupled plasma mass spectrometry on a Perkin Elmer NexIon 300D ICP-MS instrument equipped with an ESI-FAST SC2DX autosampler in kinetic energy discrimination mode with helium as cell gas at either 4.8 mL or 5.7 mL and 1600 W RF generator power. The method validation results showed good accuracy for fresh human samples from an external quality assessment scheme with measured concentrations within the assigned concentration ranges. Good precision and reproducibility for most elements were demonstrated with variation coefficients below or far below 8% and 15% for whole blood, 8% and 10% for serum, and 10% and 10% for urine, respectively. The developed reagent and instrumental setup were applicable to all three matrices. This minimizes the risk of human errors when switching between analyses of the different sample matrices and allows a rapid and easy analysis of whole blood, serum, and urine within one day if needed. The method demonstrated robustness over time, withstanding minor changes in the preparation of working solutions and samples, instrumental analysis, and setup. Analysis of human real samples showed the method's applicability for 33 toxic and essential elements in whole blood, serum, and urine and at concentrations relevant to clinical diagnostics as well as biomonitoring.

DOI: https://doi.org/10.1155/2024/9944995
Talanta. 2024 Oct 28. pii: S0039-9140(24)01490-5. [Epub ahead of print]283 127111

A liquid chromatography - Tandem mass spectrometry method for determination of ocrelizumab in serum of patients with multiple sclerosis.

Patrik Matlak, Hana Brozmanova, Pavel Sistik, Denisa Moskorova, Ivana Kacirova, Pavel Hradilek, Milan Grundmann.

  Ocrelizumab is a second generation recombinant humanized IgG1 monoclonal antibody used for the treatment of multiple sclerosis that selectively target B cells expressing the CD20 antigen. This study aimed to develop and validate a UPLC-MS/MS method for quantification of ocrelizumab in human serum, which can be used in clinical applications for therapeutic drug monitoring. The analysis of ocrelizumab was performed using a bottom-up approach on a liquid chromatography coupled to tandem mass spectrometry detection. The method involved immunoglobulin precipitation with cold methanol followed by peptide digestion with trypsin. The resulting specific peptides were separated on an Acquity UPLC BEH C18 column at 55 °C using gradient elution. The method was validated according to European Medicines Agency (EMEA) guidelines and demonstrated intra- and inter-assay precision with coefficients of variation ranging from 1.6 % to 6.1 % and accuracies between 90.2 % and 107.2 %. Stability testing, including autosampler, long-term and freeze-thaw stability, showed no more than 15 % variation. The method was successfully applied to 169 patient samples, revealing ocrelizumab concentrations ranging from 0.5 to 21.8 mg/L in patients on 6-month dosing regimen and 20.5-65.0 mg/L in 16 patients receiving an initial two-week dose of 300 mg. The newly developed UPLC-MS/MS method met all criteria for accuracy, precision and stability, confirming its suitability for clinical use in monitoring ocrelizumab levels in multiple sclerosis patients.

Keywords:  Antibodies; Liquid chromatography; Mass spectrometry; Multiple sclerosis; Ocrelizumab; Serum

DOI:  https://doi.org/10.1016/j.talanta.2024.127111
J Sep Sci. 2024 Nov;47(21): e70003

Supercritical Fluid Chromatography in Bioanalysis-A Review.

Laura Toribio, María Teresa Martín, José Bernal.

  In the last decade, the instrumentation improvements in supercritical fluid chromatography (SFC) and the hyphenation to mass spectrometry (MS), have increased the SFC acceptance between scientists, becoming today a valuable tool in analytical chemistry. The unique selectivity, short analysis times, low consumption of organic solvents, and the greener mobile phase, have contributed to expanding its applicability which has led to an increase in the number of publications especially in the bioanalysis area. This work reviews the advantages and main applications of SFC in bioanalysis during the last 5 years. Fundamental aspects concerning mobile phase composition, stationary phase, hyphenation to MS as well as matrix effect have been discussed. Finally, the most relevant applications have been summarized.

Keywords:  Metabolomics; doping analysis; forensic analysis; lipidomics

DOI:  https://doi.org/10.1002/jssc.70003
J Chromatogr A. 2024 Oct 05. pii: S0021-9673(24)00777-5. [Epub ahead of print]1738 465403

Maximizing sensitivity and selectivity in LC × LC-HRMS for pesticide analysis via exploitation of per-aqueous liquid chromatography.

Elena Bandini, Stephanie A Schuster, Turaj Rahmani, Frédéric Lynen.

  Accurate monitoring of pesticide residues at minimal concentrations is imperative for adherence to stringent regulatory standards in numerous countries. This study presents an innovative methodology employing comprehensive two-dimensional liquid chromatography coupled with high-resolution mass spectrometry (LC × LC-HRMS). The approach ensures high sensitivity and selectivity in detecting targeted compounds. A pivotal component of this methodology is the utilization of per-aqueous liquid chromatography (PALC) as the first dimension, facilitating the use of water-based mobile phases and addressing solvent mismatch issues. The second dimension employs reversed-phase liquid chromatography (RPLC), enhancing the separation of compounds. PALC proves instrumental in refocusing and enables the practical application of narrow-diameter columns (1.5 mm I.D.). This column design permits a direct split-free connection of the LC × LC to an electrospray-based mass spectrometer (ESI-MS), contributing to heightened sensitivity. The MS acquisition is performed in a targeted single-ion monitoring mode, ensuring reliable quantification and identification of the pesticide compounds. A comprehensive evaluation of key performance metrics, including signal-to-noise ratio, limit of detection, and response linearity, is conducted. The methodology achieves a limit of detection below the ng mL-1 and exhibits response linearity within the concentration range of 1-100 ng mL-1. The robustness of the approach is further demonstrated through intra-day and inter-day repeatability validations. Furthermore, the platform is finally tested on a surface water sample. This study not only introduces an advanced analytical methodology for pesticide multi-residue analysis but also underscores the significance of PALC in enhancing sensitivity by facilitating the use of smaller-diameter columns and water-based mobile phases, along with the role of RPLC in enhancing separation. The proposed approach showcases promising results in achieving detection limits that match the stringent regulatory standards and reliable quantification for effective pesticide residue monitoring.

Keywords:  Comprehensive liquid chromatography; High-resolution mass spectrometry; Per-aqueous liquid chromatography; Pesticides analysis; Sensitivity

DOI:  https://doi.org/10.1016/j.chroma.2024.465403