bims-metlip 2023-03-12 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2023–03–12
fourteen papers selected by
Sofia Costa, Matterworks

MAW: the reproducible Metabolome Annotation Workflow for untargeted tandem mass spectrometry.
Separation of highly hydrophilic nicotinamide metabolites using a COSMOSIL PBr column.
Small molecule biomarker discovery: Proposed workflow for LC-MS-based clinical research projects.
Lower accuracy of testosterone, cortisol, and free T4 measurements using automated immunoassays in people undergoing hemodialysis.
A sensitive LC-MS/MS method for the quantification of the plant toxins hypoglycin A and methylenecyclopropylglycine and their metabolites in cow's milk and urine and application to farm milk samples from Germany.
Generic Methods for Simultaneous Analysis of Four Direct Oral Anticoagulants in Human Plasma and Urine by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.
Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research.
A simple and rapid HPLC-MS/MS method for therapeutic drug monitoring of amikacin in dried matrix spots.
Quantitative analysis of therapeutic proteins in biological fluids: recent advancement in analytical techniques.
Simple In Vitro 18O Labeling for Improved Mass Spectrometry-Based Drug Metabolites Identification: Deep Drug Metabolism Study.
Direct Chiral Supercritical Fluid Chromatography-Mass Spectrometry Analysis of Monoacylglycerol and Diacylglycerol Isomers for the Study of Lipase-Catalyzed Hydrolysis of Triacylglycerols.
Comparison of the Determination of Some Antihypertensive Drugs in Clinical Human Plasma Samples by Solvent Front Position Extraction and Precipitation Modes.
Sequential isolation of metabolites and lipids from a single sample to achieve multiomics by using TRIzol reagent.
Validation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of N,N-dimethylacetamide and N-monomethylacetamide in paediatric plasma.

J Cheminform. 2023 Mar 04. 15(1): 32

MAW: the reproducible Metabolome Annotation Workflow for untargeted tandem mass spectrometry.

Mahnoor Zulfiqar, Luiz Gadelha, Christoph Steinbeck, Maria Sorokina, Kristian Peters.

  Mapping the chemical space of compounds to chemical structures remains a challenge in metabolomics. Despite the advancements in untargeted liquid chromatography-mass spectrometry (LC-MS) to achieve a high-throughput profile of metabolites from complex biological resources, only a small fraction of these metabolites can be annotated with confidence. Many novel computational methods and tools have been developed to enable chemical structure annotation to known and unknown compounds such as in silico generated spectra and molecular networking. Here, we present an automated and reproducible Metabolome Annotation Workflow (MAW) for untargeted metabolomics data to further facilitate and automate the complex annotation by combining tandem mass spectrometry (MS2) input data pre-processing, spectral and compound database matching with computational classification, and in silico annotation. MAW takes the LC-MS2 spectra as input and generates a list of putative candidates from spectral and compound databases. The databases are integrated via the R package Spectra and the metabolite annotation tool SIRIUS as part of the R segment of the workflow (MAW-R). The final candidate selection is performed using the cheminformatics tool RDKit in the Python segment (MAW-Py). Furthermore, each feature is assigned a chemical structure and can be imported to a chemical structure similarity network. MAW is following the FAIR (Findable, Accessible, Interoperable, Reusable) principles and has been made available as the docker images, maw-r and maw-py. The source code and documentation are available on GitHub ( https://github.com/zmahnoor14/MAW ). The performance of MAW is evaluated on two case studies. MAW can improve candidate ranking by integrating spectral databases with annotation tools like SIRIUS which contributes to an efficient candidate selection procedure. The results from MAW are also reproducible and traceable, compliant with the FAIR guidelines. Taken together, MAW could greatly facilitate automated metabolite characterization in diverse fields such as clinical metabolomics and natural product discovery.

Keywords:  FAIR; Metabolite annotation; Tandem mass spectrometry; Untargeted metabolomics; Workflow

DOI:  https://doi.org/10.1186/s13321-023-00695-y
MethodsX. 2023 ;10 102061

Separation of highly hydrophilic nicotinamide metabolites using a COSMOSIL PBr column.

Makoto Ozaki, Motoshi Shimotsuma, Tsunehisa Hirose.

  Highly hydrophilic compounds such as nicotinamide metabolites are very difficult to separate via high-performance liquid chromatography (HPLC) using octadecyl (C18) columns. In general, for the separation of hydrophilic compounds, hydrophilic interaction liquid chromatography (HILIC) columns are used instead of reversed phase chromatography using C18 columns. However, HILIC columns generally obey complex separation mechanisms because ionic interactions are involved in the retention process, which hinders the optimization of the separation conditions. Additionally, the resulting peak shapes are disturbed when large amounts of aqueous samples are injected. This study demonstrates that COSMOSIL PBr columns, in which both hydrophobic and dispersive interactions occur, show high retention for various hydrophilic compounds under similar separation conditions as those used with C18 columns. Specifically, using a COSMOSIL PBr column, 11 nicotinamide metabolites could be separated under simpler conditions than those used previously with C18 columns, affording better peak shape for each compound. The applicability of the method was evaluated using a tomato sample, from which the nicotinamide metabolites were successfully separated. The results show that the COSMOSIL PBr column is a good alternative to the C18 column for a good separation of all the peaks, including impurity peaks.

Keywords:  HPLC, LC-MS, and LC-MS/MS analysis; Nicotinamide mononucleotide (NMN); Reversed phase chromatography; Separation of nicotinamide metabolites in foods

DOI:  https://doi.org/10.1016/j.mex.2023.102061
J Mass Spectrom Adv Clin Lab. 2023 Apr;28 47-55

Small molecule biomarker discovery: Proposed workflow for LC-MS-based clinical research projects.

S Rischke, L Hahnefeld, B Burla, F Behrens, R Gurke, T J Garrett.

  Mass spectrometry focusing on small endogenous molecules has become an integral part of biomarker discovery in the pursuit of an in-depth understanding of the pathophysiology of various diseases, ultimately enabling the application of personalized medicine. While LC-MS methods allow researchers to gather vast amounts of data from hundreds or thousands of samples, the successful execution of a study as part of clinical research also requires knowledge transfer with clinicians, involvement of data scientists, and interactions with various stakeholders. The initial planning phase of a clinical research project involves specifying the scope and design, and engaging relevant experts from different fields. Enrolling subjects and designing trials rely largely on the overall objective of the study and epidemiological considerations, while proper pre-analytical sample handling has immediate implications on the quality of analytical data. Subsequent LC-MS measurements may be conducted in a targeted, semi-targeted, or non-targeted manner, resulting in datasets of varying size and accuracy. Data processing further enhances the quality of data and is a prerequisite for in-silico analysis. Nowadays, the evaluation of such complex datasets relies on a mix of classical statistics and machine learning applications, in combination with other tools, such as pathway analysis and gene set enrichment. Finally, results must be validated before biomarkers can be used as prognostic or diagnostic decision-making tools. Throughout the study, quality control measures should be employed to enhance the reliability of data and increase confidence in the results. The aim of this graphical review is to provide an overview of the steps to be taken when conducting an LC-MS-based clinical research project to search for small molecule biomarkers.

Keywords:  (U)HPLC (Ultra-), High pressure liquid chromatography; Biomarker Discovery Study; HILIC, Hydrophilic interaction liquid chromatography; HRMS, High resolution mass spectrometry; LC-MS, Liquid chromatography – mass spectrometry; LC-MS-Based Clinical Research; Lipidomics; MRM, Multiple reaction monitoring; Metabolomics; PCA, Principal component analysis; QA, Quality assurance; QC, Quality control; RF, Random Forest; RP, Reversed phase; SVA, Support vector machine

DOI:  https://doi.org/10.1016/j.jmsacl.2023.02.003
Clin Chem Lab Med. 2023 Mar 06.

Lower accuracy of testosterone, cortisol, and free T4 measurements using automated immunoassays in people undergoing hemodialysis.

Heleen I Jansen, Antonius E van Herwaarden, Henk J Huijgen, Marc G Vervloet, Jacquelien J Hillebrand, Anita Boelen, Annemieke C Heijboer.

   OBJECTIVES: Hormone measurements using automated immunoassays (IAs) can be affected by the sample matrix. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) is less affected by these matrix effects. In clinical laboratories, testosterone, cortisol and, free thyroxine (FT4) are often measured using IAs. Renal failure alters serum composition in blood samples from people undergoing hemodialysis (HDp) and have, therefore, a complex serum constitution compared to healthy controls (HC). The goal of this study was to investigate the accuracy of testosterone, cortisol, and FT4 measurements in samples of HDp and to get more insight in the interfering factors.
METHODS: Thirty serum samples from HDp and HC were collected to measure testosterone, cortisol, and FT4 using a well standardized isotope dilution (ID)-LC-MS/MS method and 5 commercially available automated IAs (Alinity, Atellica, Cobas, Lumipulse, UniCel DXI). Method comparisons between LC-MS/MS and IAs were performed using both HDp and HC samples.
RESULTS: Average bias from the LC-MS/MS was for testosterone, cortisol, and FT4 immunoassays respectively up to 92, 7-47 and 16-27% more in HDp than in HC samples and was IA dependent. FT4 IA results were falsely decreased in HDp samples, whereas cortisol and testosterone concentrations in females were predominantly falsely increased. Correlation coefficients between LC-MS/MS and IA results were lower in HDp compared to HC samples.
CONCLUSIONS: Several IAs for testosterone (in women), cortisol, and FT4 are less reliable in the altered serum matrix of samples of HDp than in HC. Medical and laboratory specialists should be aware of these pitfalls in this specific population.

Keywords:  cortisol; free thyroxine; hemodialysis; immunoassay; mass spectrometry; testosterone

DOI:  https://doi.org/10.1515/cclm-2022-1133
Anal Bioanal Chem. 2023 Mar 06.

A sensitive LC-MS/MS method for the quantification of the plant toxins hypoglycin A and methylenecyclopropylglycine and their metabolites in cow's milk and urine and application to farm milk samples from Germany.

Ahmed H El-Khatib, Julika Lamp, Stefan Weigel.

  Hypoglycin A (HGA) and its homologue methylenecyclopropylglycine (MCPrG) are present in ackee and lychee as well as seeds, leaves, and seedlings of some maple (Acer) species. They are toxic to some animal species and humans. The determination of HGA, MCPrG, and their glycine and carnitine metabolites in blood and urine is a useful tool for screening for potential exposure to these toxins. In addition, HGA, MCPrG, and/or their metabolites have been detected in milk. In this work, simple and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) methods without derivatization were developed and validated for the quantification of HGA, MCPrG, and their metabolites in cow's milk and urine. An extraction procedure from milk samples has been developed, whereas a dilute-and-shoot approach was implemented for urine samples. For quantification, the MS/MS analysis was performed in multiple reaction monitoring mode. The methods were validated according to the European Union guidelines using blank raw milk and urine as matrices. The limit of quantification presented here for HGA in milk (1.12 µg/L) is noticeably lower than the lowest published limit of detection (9 µg/L). Acceptable values for recovery (89-106% and 85-104% in milk and urine, respectively) and precision (≤ 20%) were obtained for all the quality control levels. The stability of HGA and MCPrG in frozen milk over a period of 40 weeks has been demonstrated. The method was applied to 68 milk samples from 35 commercial dairy farms and showed the absence of any quantifiable amounts of HGA, MCPrG, and their metabolites.

Keywords:  Methylenecyclopropylacetyl-carnitine; Methylenecyclopropylacetyl-glycine; Methylenecyclopropylformyl-glycine; Sycamore maple (Acer pseudoplatanus)

DOI:  https://doi.org/10.1007/s00216-023-04607-9
Molecules. 2023 Feb 28. pii: 2254. [Epub ahead of print]28(5):

Generic Methods for Simultaneous Analysis of Four Direct Oral Anticoagulants in Human Plasma and Urine by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry.

Jian-Wei Ren, Xin Zheng, Xiao-Hong Han.

  The new direct oral anticoagulants (DOACs) are increasingly used to treat and prevent thromboembolic disorders, and monitoring concentrations may be valuable in some special scenarios to prevent clinical adverse events. This study aimed to develop generic methods for the rapid and simultaneous analysis of four DOACs in human plasma and urine. Protein precipitation and one-step dilution were used to prepare the plasma and urine; the extracts were injected to ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Chromatographic separation was performed on an Acquity™ UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) with gradient elution of 7 min. A triple quadrupole tandem mass spectrometer with an electrospray ionization source was employed to analyze DOACs in a positive ion mode. The methods showed great linearity in the plasma (1~500 ng/mL) and urine (10~10,000 ng/mL) for all analytes (R2 ≥ 0.99). The intra- and inter-day precision and accuracy were within acceptance criteria. The matrix effect and extraction recovery were 86.5~97.5% and 93.5~104.7% in the plasma, while 97.0~101.9% and 85.1~99.5% in the urine. The stability of samples during the routine preparation and storage were within the acceptance criteria of less than ±15%. The methods developed were accurate, reliable, and simple for the rapid and simultaneous measurement of four DOACs in human plasma and urine, and successfully applied to patients and subjects with DOACs therapy for anticoagulant activity assessment.

Keywords:  DOACs; UPLC-MS/MS; human plasma; urine

DOI:  https://doi.org/10.3390/molecules28052254
J Mass Spectrom Adv Clin Lab. 2023 Apr;28 35-46

Pre-analytical sample handling standardization for reliable measurement of metabolites and lipids in LC-MS-based clinical research.

A Sens, S Rischke, L Hahnefeld, E Dorochow, S M G Schäfer, D Thomas, M Köhm, G Geisslinger, F Behrens, R Gurke.

  The emerging disciplines of lipidomics and metabolomics show great potential for the discovery of diagnostic biomarkers, but appropriate pre-analytical sample-handling procedures are critical because several analytes are prone to ex vivo distortions during sample collection. To test how the intermediate storage temperature and storage period of plasma samples from K3EDTA whole-blood collection tubes affect analyte concentrations, we assessed samples from non-fasting healthy volunteers (n = 9) for a broad spectrum of metabolites, including lipids and lipid mediators, using a well-established LC-MS-based platform. We used a fold change-based approach as a relative measure of analyte stability to evaluate 489 analytes, employing a combination of targeted LC-MS/MS and LC-HRMS screening. The concentrations of many analytes were found to be reliable, often justifying less strict sample handling; however, certain analytes were unstable, supporting the need for meticulous processing. We make four data-driven recommendations for sample-handling protocols with varying degrees of stringency, based on the maximum number of analytes and the feasibility of routine clinical implementation. These protocols also enable the simple evaluation of biomarker candidates based on their analyte-specific vulnerability to ex vivo distortions. In summary, pre-analytical sample handling has a major effect on the suitability of certain metabolites as biomarkers, including several lipids and lipid mediators. Our sample-handling recommendations will increase the reliability and quality of samples when such metabolites are necessary for routine clinical diagnosis.

Keywords:  1-AG, 1-arachidonoyl glycerol; 1-LG, 1-linoleoyl glycerol; 2-AG, 2-arachidonoyl glycerol; 2-LG, 2- linoleoyl glycerol; ACN, acetonitrile; AEA, arachidonoyl ethanolamide; BHT, 2,6-di-tert-butyl-4-methylphenol; CAR, carnitine; EC, endocannabinoid; FC, fold change; FT, freezing temperature/storage in ice water; HETE, hydroxyeicosatetraenoate; HRMS, high-resolution mass spectrometry; IRB, Institutional Review Board; IS, internal standard; K3EDTA plasma sampling; K3EDTA, tripotassium ethylenediaminetetraacetic acid; LC, liquid chromatography; LEA, linoleoyl ethanolamide; LLE, liquid–liquid extraction; LLOQ, lowest limit of quantification; LPA, lysophosphatidic acid; LPC O, lysophosphatidylcholine-ether; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPG, lysophosphatidylglycerol; LPI, lysophosphatic inositol; Lipidomics; MS/MS, tandem mass spectrometry; MTBE, methyl tertiary-butyl ether; MeOH, methanol; Metabolomics; OEA, oleoyl ethanolamide; PBS, phosphate-buffered saline; PC, phohsphatidylcholine; PE, phosphotidylethanolamine; PEA, palmitoyl ethanolamide; PI, phosphatidylinositol; Pre-analytics; QC, quality control; REC, Research Ethics Committee; RT, room temperature; Ref, reference sample; SEA, stearoyl ethanolamide; SPE, solid-phase extraction; STD, calibration standard; Sampling protocol; VEA, vaccenic acid ethanolamid; WB, whole blood

DOI:  https://doi.org/10.1016/j.jmsacl.2023.02.002
J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jan 07. pii: S1570-0232(23)00002-8. [Epub ahead of print] 123592

A simple and rapid HPLC-MS/MS method for therapeutic drug monitoring of amikacin in dried matrix spots.

Yan Chen, Yu Wang, Beining Guo, Yaxin Fan, Hailan Wu, Xin Li, Yi Li, Xiaolan Huang, Mengting Chen, Xiaofen Liu, Jing Zhang.

  Individualized treatment of amikacin under the guidance of therapeutic drug monitoring (TDM) is important to reduce the occurrence of toxicity and improve clinical efficacy. In the present study, we developed and validated a simple and high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine the concentration of amikacin in dried matrix spots (DMS) which the matrix is serum. DMS samples were obtained by spotting volumetric blood onto Whatman 903® cards. Samples were punched into 3 mm diameter discs and extracted with 0.2 % formic acid in water. The HILIC column (2.1 mm × 100 mm, 3.0 µm) under gradient elution was applied, and the analysis time was 3 min per injection. The mass spectrometry transitions were m/z 586.3 → 163.0 for amikacin and m/z 591.4 → 163.1 for D5-amikacin. Full validation was conducted for DMS method, and the method was applied for the amikacin TDM and compared with serum method. The linearity was ranged from 0.5 to 100 mg/L. Both within-run and between-run accuracy and precision of DMS ranged from 91.8 % to 109.6 % and 3.6 % to 14.2 %, respectively. The matrix effect was 100.5 %-106.5 % of DMS method. Amikacin remained stable in DMS for at least 6 days at room temperature, 16 days at 4 °C, 86 days at -20 °C and -70 °C. A good agreement between the DMS method and serum method has been shown in Bland-Altman plots and Passing-Bablok regression. All of the results demonstrated that the DMS methods can be a favorable replacement for amikacin TDM.

Keywords:  Amikacin; Dried matrix spot; HPLC-MS/MS; Serum; Therapeutic drug monitoring

DOI:  https://doi.org/10.1016/j.jchromb.2023.123592
Drug Deliv. 2023 Dec;30(1): 2183816

Quantitative analysis of therapeutic proteins in biological fluids: recent advancement in analytical techniques.

Jae Geun Song, Kshitis Chandra Baral, Gyu-Lin Kim, Ji-Won Park, Soo-Hwa Seo, Da-Hyun Kim, Dong Hoon Jung, Nonye Linda Ifekpolugo, Hyo-Kyung Han.

  Pharmaceutical application of therapeutic proteins has been continuously expanded for the treatment of various diseases. Efficient and reliable bioanalytical methods are essential to expedite the identification and successful clinical development of therapeutic proteins. In particular, selective quantitative assays in a high-throughput format are critical for the pharmacokinetic and pharmacodynamic evaluation of protein drugs and to meet the regulatory requirements for new drug approval. However, the inherent complexity of proteins and many interfering substances presented in biological matrices have a great impact on the specificity, sensitivity, accuracy, and robustness of analytical assays, thereby hindering the quantification of proteins. To overcome these issues, various protein assays and sample preparation methods are currently available in a medium- or high-throughput format. While there is no standard or universal approach suitable for all circumstances, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay often becomes a method of choice for the identification and quantitative analysis of therapeutic proteins in complex biological samples, owing to its high sensitivity, specificity, and throughput. Accordingly, its application as an essential analytical tool is continuously expanded in pharmaceutical R&D processes. Proper sample preparation is also important since clean samples can minimize the interference from co-existing substances and improve the specificity and sensitivity of LC-MS/MS assays. A combination of different methods can be utilized to improve bioanalytical performance and ensure more accurate quantification. This review provides an overview of various protein assays and sample preparation methods, with particular emphasis on quantitative protein analysis by LC-MS/MS.

Keywords:  ELISA; LC-MS/MS; Protein drugs; chromatographic separation; protein assays; quantitation

DOI:  https://doi.org/10.1080/10717544.2023.2183816
Int J Mol Sci. 2023 Feb 26. pii: 4569. [Epub ahead of print]24(5):

Simple In Vitro 18O Labeling for Improved Mass Spectrometry-Based Drug Metabolites Identification: Deep Drug Metabolism Study.

Boris Tupertsev, Sergey Osipenko, Albert Kireev, Eugene Nikolaev, Yury Kostyukevich.

  The identification of drug metabolites formed with different in vitro systems by HPLC-MS is a standard step in preclinical research. In vitro systems allow modeling of real metabolic pathways of a drug candidate. Despite the emergence of various software and databases, identification of compounds is still a complex task. Measurement of the accurate mass, correlation of chromatographic retention times and fragmentation spectra are often insufficient for identification of compounds especially in the absence of reference materials. Metabolites can "slip under the nose", since it is often not possible to reliably confirm that a signal belongs to a metabolite and not to other compounds in complex systems. Isotope labeling has proved to be a tool that aids in small molecule identification. The introduction of heavy isotopes is done with isotope exchange reactions or with complicated synthetic schemes. Here, we present an approach based on the biocatalytic insertion of oxygen-18 isotope under the action of liver microsomes enzymes in the presence of 18O2. Using the local anesthetic bupivacaine as an example, more than 20 previously unknown metabolites were reliably discovered and annotated in the absence of the reference materials. In combination with high-resolution mass spectrometry and modern methods of mass spectrometric metabolism data processing, we demonstrated the ability of the proposed approach to increase the degree of confidence in interpretating metabolism data.

Keywords:  18O; drug metabolism; mass spectrometry; metabolites identification

DOI:  https://doi.org/10.3390/ijms24054569
Anal Chem. 2023 Mar 09.

Direct Chiral Supercritical Fluid Chromatography-Mass Spectrometry Analysis of Monoacylglycerol and Diacylglycerol Isomers for the Study of Lipase-Catalyzed Hydrolysis of Triacylglycerols.

Oleksandr Kozlov, Eliška Horáková, Sára Rademacherová, Dávid Maliňák, Rudolf Andrýs, Eliška Prchalová, Miroslav Lísa.

The fast and selective separation method of intact monoacylglycerol (MG) and diacylglycerol (DG) isomers using chiral supercritical fluid chromatography-mass spectrometry (SFC-MS) was developed and employed to study lipase selectivity in the hydrolysis of triacylglycerols (TGs). The synthesis of 28 enantiomerically pure MG and DG isomers was performed in the first stage using the most commonly occurring fatty acids in biological samples such as palmitic, stearic, oleic, linoleic, linolenic, arachidonic, and docosahexaenoic acids. To develop the SFC separation method, different chromatographic conditions such as column chemistry, mobile phase composition and gradient, flow rate, backpressure, and temperature were carefully assessed. Our SFC-MS method used a chiral column based on a tris(3,5-dimethylphenylcarbamate) derivative of amylose and neat methanol as a mobile phase modifier, which provides baseline separation of all the tested enantiomers in 5 min. This method was used to evaluate hydrolysis selectivity of lipases from porcine pancreas (PPL) and Pseudomonas fluorescens (PFL) using nine TGs differing in acyl chain length (14-22 carbon atoms) and number of double bonds (0-6) and three DG regioisomer/enantiomers as hydrolysis intermediate products. PFL exhibited preference of the fatty acyl hydrolysis from the sn-1 position of TG more pronounced for the substrates with long polyunsaturated acyls, while PPL did not show considerable stereoselectivity to TGs. Conversely, PPL preferred hydrolysis from the sn-1 position of prochiral sn-1,3-DG regioisomer, whereas PFL exhibited no preference. Both lipases showed selectivity for the hydrolysis of outer positions of DG enantiomers. The results show complex reaction kinetics of lipase-catalyzed hydrolysis given by different stereoselectivities for substrates.

DOI: https://doi.org/10.1021/acs.analchem.3c00053
Molecules. 2023 Feb 27. pii: 2213. [Epub ahead of print]28(5):

Comparison of the Determination of Some Antihypertensive Drugs in Clinical Human Plasma Samples by Solvent Front Position Extraction and Precipitation Modes.

Kamila Jaglińska, Beata Polak, Anna Klimek-Turek, Emilia Fornal, Anna Stachniuk, Alicja Trzpil, Robert Błaszczyk, Andrzej Wysokiński.

  The determination of the selected antihypertensive drugs in human plasma samples with the novel solvent front position extraction (SFPE) technique is presented. The SFPE procedure combined with LC-MS/MS analysis was used for the first time to prepare a clinical sample containing the drugs mentioned above from different therapeutic groups. The effectiveness of our approach was compared with the precipitation method. The latter technique is usually used to prepare biological samples in routine laboratories. During the experiments, the substances of interest and the internal standard were separated from other matrix components using a prototype horizontal chamber for thin-layer chromatography/high-performance thin-layer chromatography (TLC/HPTLC) with a moving pipette powered by a 3D mechanism, which distributed the solvent on the adsorbent layer. Detection of the six antihypertensive drugs was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Results obtained by SFPE were very satisfactory (linearity R2 ≥ 0.981; %RSD ≤ 6%; LOD and LOQ were in the range of 0.06-9.78 ng/mL and 0.17-29.64 ng/mL, respectively). The recovery was in the range of 79.88-120.36%. Intra-day and inter-day precision had a percentage coefficient of variation (CV) in the range of 1.10-9.74%. The procedure is simple and highly effective. It includes the automation of TLC chromatogram development, which significantly reduced the number of manual operations performed, the time of sample preparation and solvent consumption.

Keywords:  LC-MS/MS analysis; antihypertensive drugs; clinical sample preparation; precipitation; solvent front position extraction

DOI:  https://doi.org/10.3390/molecules28052213
Talanta. 2023 Mar 04. pii: S0039-9140(23)00167-4. [Epub ahead of print]258 124416

Sequential isolation of metabolites and lipids from a single sample to achieve multiomics by using TRIzol reagent.

Cheng-Yen Kao, Chung-Te Chang, Pei-Yun Kuo, Chia-Jen Lin, Huai-Hsuan Chiu, Hsiao-Wei Liao.

  Simultaneous extraction of various types of biomolecule from a single sample can be beneficial for multiomics studies of unique specimens. An efficient and convenient sample preparation approach must be developed that can comprehensively isolate and extract biomolecules from one sample. TRIzol reagent is widely used in biological studies for DNA, RNA, and protein isolation. This study evaluated the feasibility of using TRIzol reagent for the simultaneous isolation of not only DNA, RNA, and proteins but also metabolites and lipids from a single sample. Through the comparison of known metabolites and lipids obtained using the conventional methanol (MeOH) and methyl-tert-butyl ether (MTBE) extraction methods, we determined the presence of metabolites and lipids in the supernatant during TRIzol sequential isolation. Finally, we performed untargeted metabolomics and lipidomics to examine metabolite and lipid alterations associated with the jhp0417 mutation in Helicobacter pylori by using the TRIzol sequential isolation protocol and MeOH and MTBE extraction methods. Metabolites and lipids with significant differences isolated using the TRIzol sequential isolation protocol were consistent with those obtained using the conventional MeOH and MTBE extraction methods. These results indicated that TRIzol reagent can be used to simultaneously isolate metabolites and lipids from a single sample. Thus, TRIzol reagent can be used in biological and clinical research, especially in multiomics studies.

Keywords:  Lipidomics; Metabolomics; Multiomics; Sequential isolation; TRIzol

DOI:  https://doi.org/10.1016/j.talanta.2023.124416
J Sep Sci. 2023 Mar 06. e2201003

Validation of a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of N,N-dimethylacetamide and N-monomethylacetamide in paediatric plasma.

Sebastian P A Rosser, Andrew J McLachlan, Georg Hempel, Jason Chung, Peter J Shaw, Steven J Keogh, Christa E Nath.

  N,N-dimethylacetamide is an excipient used in intravenous busulfan formulations, administered prior to haematopoietic stem cell transplantation. This study aimed to develop and validate a liquid chromatography-tandem mass spectrometry method for simultaneous quantification of N,N-dimethylacetamide and its metabolite N-monomethylacetamide in paediatric plasma for patients receiving busulfan. A 4 μL aliquot of patient plasma was extracted using 196 μL 50% methanol solution and quantified against calibrators prepared in the extraction solvent given negligible matrix effects across three concentrations. 9 [H2 ]-N,N-dimethylacetamide was used as an internal standard. Separation of N,N-dimethylacetamide and N-monomethylacetamide was achieved using a Kinetex® EVO C18 stationary phase (100mm × 2.1mm × 2.6μm) running an isocratic mobile phase of 30% methanol containing 0.1% formic acid at a flow of 0.2 mL/min over 3.0 minutes and an injection volume of 1 μL. N,N-dimethylacetamide and N-monomethylacetamide were linear up to 1200 μg/L and 200 μg/L (lower limit of quantification 1 μg/L for both analytes), respectively. Calibrator accuracy and precision were within ± 10% of the test parameters across four concentration levels. Analytes were stable over 14 days at three different storage conditions. This method was successfully applied to measure N,N-dimethylacetamide and N-monomethylacetamide concentrations in 77 paediatric patients, totalling 1,265 plasma samples. This article is protected by copyright. All rights reserved.

Keywords:  N,N-dimethylacetamide; N-monomethylacetamide; liquid chromatography-tandem mass spectrometry; method validation; pharmacokinetics

DOI:  https://doi.org/10.1002/jssc.202201003