bims-metlip 2021-09-19 papers

bims-metlip

Biomed News

on Methods and protocols in metabolomics and lipidomics

Issue of 2021–09–19
nineteen papers selected by
Sofia Costa, Icahn School of Medicine at Mount Sinai

Accurate quantification of endogenous N-acylethanolamides by chemical isotope labeling coupled with liquid chromatography-tandem mass spectrometry.
Optimization of a liquid chromatography-ion mobility-high resolution mass spectrometry platform for untargeted lipidomics and application to HepaRG cell extracts.
Thermal degradation of metabolites in urine using multiple isotope-labelled internal standards for off-line GC metabolomics - effects of injector and oven temperatures.
Pharmacokinetic and metabolic analysis of an Alzheimer's disease therapeutic in rat serum via microfluidic CZE-MS.
Metabolome Exploration by High-Resolution Mass Spectrometry Methodologies of Two New Yeast Species: Starmerella reginensis and Starmerella kourouensis.
Bifunctionalized isotopologues as calibrants for standard-free quantitation of drug metabolites in plasma by liquid chromatography coupled with radiometry and mass spectrometry.
Development and validation of a LC-MS/MS method for in vivo quantification of meta-iodobenzylguanidine (mIBG).
Optimization and validation of capillary electrophoresis- and gas chromatography-tandem mass spectrometry methods for the analysis of intermediate metabolites in glycolysis and pentose phosphate pathways within biological samples.
Acetonitrile adduct analysis of underivatised amino acids offers improved sensitivity for hydrophilic interaction liquid chromatography tandem mass-spectrometry.
A generic sample preparation method for the multiplex analysis of seven therapeutic monoclonal antibodies in human plasma or serum with liquid chromatography-tandem mass spectrometry.
Optimizing oxytocin LC-MS/MS sensitivity by choosing the right column.
A simplified LC-MS/MS method for the quantification of the cardiovascular disease biomarker trimethylamine-N-oxide and its precursors.
Synthesis and characterization of 13C labeled carnosine derivatives for isotope dilution mass spectrometry measurements in biological matrices.
Analysis of aristolochic acid I in mouse serum and tissues by using magnetic solid-phase extraction and UHPLC-MS/MS.
A highly sensitive, simple, and fast gas chromatography-mass spectrometry method for the quantification of serum short-chain fatty acids and their potential features in central obesity.
A relative quantitation method for measuring DNA methylation and hydroxymethylation using guanine as an internal standard.
Using random forest to detect multiple inherited metabolic diseases simultaneously based on GC-MS urinary metabolomics.
Stable isotope tracing to assess tumor metabolism in vivo.
Recent advances in metabolic engineering-integration of in silico design and experimental analysis of metabolic pathways.

Anal Chim Acta. 2021 Sep 22. pii: S0003-2670(21)00665-6. [Epub ahead of print]1179 338839

Accurate quantification of endogenous N-acylethanolamides by chemical isotope labeling coupled with liquid chromatography-tandem mass spectrometry.

Yan-Qing Chen, Hong Shen, Ru-Jie Yang, Jian-Bo Wan.

  N-acylethanolamides (NAEs) are a class of naturally occurring lipid molecules with pleiotropic activities ranging from energy homeostasis to analgesic functioning. However, the comprehensive quantitation of endogenous NAEs is challenged by the sub-trace level (nM) in complex biological samples and the limited availability of stable isotope labeled internal standards (SIL-IS). Herein, a sensitive method was developed to accurately determine 20 NAEs in biological samples by chemical isotope labeling strategy coupled with liquid chromatography - tandem mass spectrometry (LC-MS/MS). A pair of efficient derivatization reagents, acetyl chloride-d0 (ACC-d0) and acetyl chloride-d3 (ACC-d3), were used to label NAEs in biological samples and NAE standard mixture, respectively. The heavily labeled NAE derivatives of the standard substances were used as one-to-one internal standards to minimize the matrix effects and potential ion suppression in MS analysis. Although no chemical moiety with high ionization capability was introduced, the detection sensitivity of the derivatized NAEs were substantially enhanced, as evidenced by 6- to 170-fold increase in LOQs, compared to non-derivatized NAEs. The derivatized NAEs provided the stable and abundant specific product ions in MS/MS spectrum, which were used as the quantitation ions for multiple reaction monitoring (MRM) analysis. The validated LC-MS/MS method was also successfully applied to determine NAEs in serum samples and liver tissues from control and alcohol-fed mice, which shown its practicability in the analysis of endogenous NAE in biological samples. Collectively, the proposed method offers a sensitive and accurate quantification of endogenous NAEs, which may facilitate the understanding of NAE metabolisms and their functions in the physiological and pathological processes.

Keywords:  Acetyl chloride; Chemical isotope labeling; LC-MS/MS; N-acylethanolamides

DOI:  https://doi.org/10.1016/j.aca.2021.338839
Talanta. 2021 Dec 01. pii: S0039-9140(21)00729-3. [Epub ahead of print]235 122808

Optimization of a liquid chromatography-ion mobility-high resolution mass spectrometry platform for untargeted lipidomics and application to HepaRG cell extracts.

Katyeny Manuela da Silva, Elias Iturrospe, Joris Heyrman, Jeremy P Koelmel, Matthias Cuykx, Tamara Vanhaecke, Adrian Covaci, Alexander L N van Nuijs.

  Analytical methods to evaluate the lipidome of biological samples need to provide high data quality to ensure comprehensive profiling and reliable structural elucidation. In this perspective, liquid chromatography-high resolution mass spectrometry (LC-HRMS) is the state-of-the-art technique for lipidomic analysis of biological samples. There are thousands of lipids in most biological samples, and therefore separation methods before introduction to the mass spectrometer is key for relative quantitation and identification. Chromatographic methods differ across laboratories, without any consensus on the best methodologies. Therefore, we designed an experiment to determine the optimal LC methodology, and assessed the value of ion mobility for an additional dimension of separation. To apply an untargeted method for hypothesis generation focused on lipidomics, LC-HRMS parameters were optimized based on the measurement of 50 panel lipids covering key human metabolic pathways. Reversed-phase liquid chromatography columns were compared based on a quality scoring system considering the signal-to-noise ratio, peak shape, and retention factor. Furthermore, drift tube ion mobility spectrometry (DTIMS) was implemented to increase peak capacity and confidence during annotation by providing collision cross section (CCS) values for the analytes under investigation. However, hyphenating DTIMS to LC-HRMS may result in a reduced sensitivity due to impaired duty cycles. To increase the signal intensity, a Box-Behnken design (BBD) was used to optimize four key factors, i.e. drift entrance voltage, drift exit voltage, rear funnel entrance, and rear funnel exit voltages. Application of a maximized desirability function provided voltages for the above-mentioned parameters resulting in higher signal intensity compared to each combination of parameters used during the BBD. In addition, the influence of single pulse and Hadamard 4-bit multiplexed modes on signal intensity was explored and different trap filling and release times of ions were evaluated. The optimized LC-DTIM-HRMS platform was applied to extracts from HepaRG cells and resulted in 3912 high-quality features (<30% median relative standard deviation; n = 6, t = 24 h). From these features, 436 lipid species could be annotated (i.e., matching based on accurate mass <5 ppm, isotopic pattern, in-silico MS/MS fragmentation, and in-silico CCS database matching <3%). The application of LC-DTIM-HRMS for untargeted analysis workflows is growing and the platform optimization, as described here, can be used to guide the method development and CCS database comparison for high confidence lipid annotation.

Keywords:  Collision cross section; Hadamard multiplexing ion mobility; High-resolution mass spectrometry; Human HepaRG cells; Liver intracellular extracts; Untargeted lipidomics

DOI:  https://doi.org/10.1016/j.talanta.2021.122808
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Sep 04. pii: S1570-0232(21)00383-4. [Epub ahead of print]1181 122902

Thermal degradation of metabolites in urine using multiple isotope-labelled internal standards for off-line GC metabolomics - effects of injector and oven temperatures.

Emmanouil D Tsochatzis, Caroline Nebel, Marianne Danielsen, Ulrik K Sundekilde, Trine Kastrup Dalsgaard.

  Thermal processes are widely used in small molecule chemical analysis and metabolomics for derivatization, vaporization, chromatography, and ionization, especially in gas chromatography mass spectrometry (GC/MS). An optimized derivatization protocol has been successfully applied using multiple isotope labelled analytical internal standards of selected deuterated and 13C selected compounds, covering a range of different groups of metabolites for non-automated GC metabolomics (off-line). Moreover, the study was also realized in a pooled urine sample, following metabolic profiling. A study of thermal degradation of metabolites due to GC inlet and oven programs (fast, slow) was performed, where the results indicated that both GC oven programs (fast and slow) negatively affected the thermal stability of the metabolites, while the fast-ramp GC program also suppressed MS signals. However, the use of multiple internal standards can overcome this drawback. The application of extended temperature ramp GC program presented identical behaviour on metabolite stability and better chromatographic separation combined with much lower signal suppression, compared to a short temperature ramp program. No effects were observed for organic acids, fatty acids, sugars and sugar alcohols, while significant differences were observed for amino acids. GC metabolomics is a strong tool that can facilitate analysis, but special attention is required for sampling handling and heating, before and during the GC analysis. The use and application of multiple multi-group internal standards is highly recommended.

Keywords:  Derivatization; GC-QToF MS; Internal standards; Metabolomics; Temperature ramping; Thermal degradation

DOI:  https://doi.org/10.1016/j.jchromb.2021.122902
Biomed Chromatogr. 2021 Sep 13. e5243

Pharmacokinetic and metabolic analysis of an Alzheimer's disease therapeutic in rat serum via microfluidic CZE-MS.

Z D Kelley, M A Lovell, B C Lynn.

  Sensitive, high-throughput methods for pharmacokinetic (PK) profiling are essential for potential therapeutics during critical stages of clinical trials. The application of a microfluidic capillary zone electrophoresis mass spectrometry (CZE-MS) method for PK profiling allows for rapid, sensitive, and in-depth analysis of multiple samples within a short timeframe. Here, a CZE-MS approach for PK analysis was compared to a traditional UHPLC-MS approach when analyzing serum extracts from rats treated with a potential Alzheimer's disease (AD) therapeutic, BNC-1. Resulting PK data generated from both methods displayed statistical similarity. Additionally, the separation efficiency attributed to the use of the CZE-MS method provided substantial metabolic regulation data that was not apparent in the UHPLC-MS method. Additionally, the coupling of the CZE-MS method to the data processing software, MZmine2, was used to monitor changes in metabolism and observe putative BNC-1-derived metabolites. The ability for fast analyses without sacrificing sensitivity or metabolic information, suggests that this CZE-MS method is an ideal method for metabolomics-inclusive, high-throughput PK profiling.

Keywords:  Microfluidics; capillary zone electrophoresis; metabolomics; pharmacokinetics

DOI:  https://doi.org/10.1002/bmc.5243
J Agric Food Chem. 2021 Sep 16.

Metabolome Exploration by High-Resolution Mass Spectrometry Methodologies of Two New Yeast Species: Starmerella reginensis and Starmerella kourouensis.

Olivier Perruchon, Isabelle Schmitz-Afonso, Cécile Grondin, Jean-Luc Legras, Carlos Afonso, Abdelhakim Elomri.

  Nature is harnessed since ancient times to fulfill human needs, and yeast culture has been mastered for bakery, brewery, or the preparation of beverages. In this context, the two recently discovered yeast species Starmerella reginensis and Starmerella kourouensis, belonging to a genus related to fermentative activities in the literature, were explored via untargeted metabolomics approaches. Ultrahigh-performance liquid chromatography hyphenated with tandem mass spectrometry and a deep investigation of molecular networks and spectral data allowed the annotation of, respectively, 439 and 513 metabolites for S. reginensis and S. kourouensis, with approximatively 30% compound annotations and 40% chemical class annotations for both yeast strains. These analyses and Fourier transform ion cyclotron resonance mass spectrometry accurate metabolic profiles unveiled a rich content of alkaloids, lipids, amino acids, and terpenoids for S. reginensis. S. kourouensis presents a similar profile with more sulfated compounds. In short, these results enrich the current knowledge about Starmerella yeast secondary metabolites and reveal their significant structural diversity of small molecules.

Keywords:  FTICR-MS; Starmerella yeast strains; UHPLC-MS/MS; metabolic profiling; molecular networking; natural products; untargeted metabolomics

DOI:  https://doi.org/10.1021/acs.jafc.1c03201
Biomed Chromatogr. 2021 Sep 13. e5242

Bifunctionalized isotopologues as calibrants for standard-free quantitation of drug metabolites in plasma by liquid chromatography coupled with radiometry and mass spectrometry.

Yong Gong, Jie Chen, Heng-Keang Lim, Naidong Weng, Rhys Salter.

  The following method describes a novel workflow that eliminates the need of authentic reference standards for the quantitation of drug metabolites in biological samples using a single multi-isotopically labeled compound bearing both radio and stable isotopes. The resulting radio and stable bifunctionalized isotopologue (RADSTIL) of parent drug is employed as a substrate for in vitro biotransformation to targeted RADSTILs of metabolites as calibrants. Inclusion of a radio label enables both radiometric and mass spectrometric detection. Addition of stable labels ensures subsequent isotopic interference-free quantitation of unlabeled metabolites in preclinical and clinical samples. This affords a more accurate quantitation workflow compared to current semi-quantitation method, which utilizes isotopic interfering radio isotopologues of metabolites alone as calibrants. The proof-of-concept is illustrated with (14 C,13 C2 )-acetaminophen where in vitro biotransformation produced (14 C,13 C2 )-sulfate and (14 C,13 C2 )-glucuronide calibrants. Absolute quantitation of the acetaminophen metabolites was then achieved by liquid chromatography coupled with radiometry and mass spectrometry (LC-RAD/MS). Quantitative data obtained by this method fell within 82-86% of the values from conventional LC-MS/MS method.

Keywords:  LC-MS/MS; biotransformation; calibrant; isotopologue; metabolite quantitation

DOI:  https://doi.org/10.1002/bmc.5242
J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Sep 03. pii: S1570-0232(21)00408-6. [Epub ahead of print]1181 122927

Development and validation of a LC-MS/MS method for in vivo quantification of meta-iodobenzylguanidine (mIBG).

Antonio J López Quiñones, Laura M Shireman, Joanne Wang.

  meta-iodobenzylguanidine (mIBG) is a radiopharmaceutical used for the diagnosis and treatment of neuroendocrine cancers. Previous quantification of mIBG in biodistribution and pharmacokinetic studies mainly relied on the use of radiolabeled mIBG, which involves the handling of highly radioactive materials. The goal of this study was to develop a nonradioactive analytical method for quantifying mIBG in mouse plasma and tissue homogenates using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples were prepared for analysis using a protein precipitation method. Mass spectrometry analysis was performed using 4-hydroxyphenformin as the internal standard, and the mass-to-charge transitions were 276.1 → 217.0 for mIBG and 222.1 → 121.0 for 4-hydroxyphenformin. The quantification limit of mIBG was 0.98 ng/mL, and the method was linear up to 500 ng/mL. The accuracy, inter-day and intra-day precision were 96-112%, 5.5-14.4%, and 3.7-14.1%, respectively, suggesting that the method was accurate and precise in quantifying mIBG at multiple concentrations in mouse plasma and liver homogenates. The extraction recovery was 96-106% and the matrix effect was 95-110%, indicating that the method was reproducible in quantifying mIBG with minimal impact from the biological matrices. In summary, we have developed and validated a fast, high-throughput quantification method of non-radiolabeled mIBG using LC-MS/MS. This method is reproducible, accurate, and precise, and can be used to quantify mIBG in plasma and tissue matrices to determine the pharmacokinetics and biodistribution of mIBG in preclinical animal models.

Keywords:  HPLC-MS/MS; Liver; Meta-Iodobenzylguanidine; Method Validation; Mouse; Plasma

DOI:  https://doi.org/10.1016/j.jchromb.2021.122927
J Chromatogr A. 2021 Sep 04. pii: S0021-9673(21)00655-5. [Epub ahead of print]1656 462531

Optimization and validation of capillary electrophoresis- and gas chromatography-tandem mass spectrometry methods for the analysis of intermediate metabolites in glycolysis and pentose phosphate pathways within biological samples.

Miso Nam, Min-Sun Kim, Geum-Sook Hwang.

  Highly selective methods for the analysis of intermediate metabolites involved in glycolysis and phosphate pentose pathways are essential for metabolism and metabolic flux studies. However, the successful separation of phosphorylated compounds is difficult due to their high polarity, as well as their structural isomers. In this study, phosphorylated compounds in spiked serum samples were analyzed using capillary electrophoresis tandem mass spectrometry (CE-MS/MS) and gas chromatography (GC)-MS/MS. Following liquid-liquid extraction, ultrafiltration and derivatization steps were needed to perform CE-MS/MS and GC-MS/MS, respectively. The CE-MS/MS method allowed for the identification and quantification of all 15 biologically important phosphorylated compounds, whereas only 13 compounds were identified and quantified by GC-MS/MS. Both methods demonstrated wide linear ranges, good interday (<9.6%: CE-MS/MS; <14.7%: GC-MS/MS) and intraday (<13.0%: CE-MS/MS; <14.9%: GC-MS/MS) variability, and limits of detection (LODs) in the ranges of 0.25-2 and 0.05-0.5 μmol/L for CE-MS/MS and GC-MS/MS, respectively. In the phosphorylated compound stability test, the instability of glyceraldehyde 3-phosphate (GA3P) and dihydroxyacetone phosphate (DHAP) was observed during freeze-thaw and long-term storage due to reversible isomerization. The results of CE-MS/MS and GC-MS/MS analysis showed that the concentrations of phosphorylated compounds determined using the two methods matched closely, while that of glycerol 3-phosphate (G3P) showed some variability in cell extracts. Therefore, while both CE-MS/MS and GC-MS/MS are suitable for analyzing metabolites resulting from the glycolysis and pentose phosphate pathways, additional validation is needed for some compounds, depending on the background matrix.

Keywords:  CE–MS/MS; Cell extract; GC–MS/MS; Human serum; Intermediates of glycolysis and pentose phosphate pathways; Method validation

DOI:  https://doi.org/10.1016/j.chroma.2021.462531
J Chromatogr A. 2021 Sep 03. pii: S0021-9673(21)00654-3. [Epub ahead of print]1655 462530

Acetonitrile adduct analysis of underivatised amino acids offers improved sensitivity for hydrophilic interaction liquid chromatography tandem mass-spectrometry.

Jake P Violi, David P Bishop, Matthew P Padula, Mika T Westerhausen, Kenneth J Rodgers.

  LC-MS/MS method development for native amino acid detection can be problematic due to low ionisation efficiencies, in source fragmentation, potential for cluster ion formation and incorrect application of chromatography techniques. This has led to the majority of the scientific community derivatising amino acids for more sensitive analysis. Derivatisation has several benefits including reduced signal-to-noise ratios, more efficient ionisation, and a change in polarity, allowing the use of reverse phase chromatography. However, derivatisation of amino acids can be expensive, requires additional sample preparation steps, is more time consuming and increases sample instability, due to the most derivatised amino acids only be stable for finite amount of time. While showing initial promise, development of reliable hydrophilic interaction liquid chromatography (HILIC) separation methods has presented difficulties for the analyst including irreproducible separation and poor sensitivity. This study aimed to find a means to improve the detection sensitivity of the 20 protein amino acids by HILIC-MS/MS. We describe the use of previously undescribed amino acid-acetonitrile (ACN) adducts to improve detection of 16 out of the 20 amino acids. While all amino acids examined did form an ACN adduct, 4 had low intensity adduct formation compared to their protonated state, 3 of which are classified as basic amino acids. For 15 of the 20 amino acids tested, we used the ACN adduct for both quantification and qualification ions and demonstrated a significant enhancement in signal-to-noise ratio, ranging from 23 to 1762% improvement. Lower LODs, LOQs and lower ranges of linearity were also achieved for these amino acids. The optimised method was applied to a human neuroblastoma cell line (SH-SY5Y) with the potential to be applied to other complex sample types. The improved sensitivity this method offers simplifies sample preparation and reduces the costs of amino acid analysis compared to those methods that rely on derivatisation for sensitivity.

Keywords:  Adduct; Amino acid; HILIC; HILIC-MS/MS; LC-MS/MS; Metabolites

DOI:  https://doi.org/10.1016/j.chroma.2021.462530
J Chromatogr A. 2021 Aug 27. pii: S0021-9673(21)00613-0. [Epub ahead of print]1655 462489

A generic sample preparation method for the multiplex analysis of seven therapeutic monoclonal antibodies in human plasma or serum with liquid chromatography-tandem mass spectrometry.

Mohsin El Amrani, Laszlo Gerencser, Alwin D R Huitema, C Erik Hack, Matthijs van Luin, Kim C M van der Elst.

  Due to the increasing number of therapeutic monoclonal antibodies (mAbs) used in the clinic, there is an increasing need for robust analytical methods to quantify total mAb concentrations in human plasma for clinical studies and therapeutic drug monitoring. We developed an easy, rapid, and robust sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The method was validated for infliximab (IFX), rituximab (RTX), cetuximab (CTX), dupilumab (DPL), dinutuximab (DNX), vedolizumab (VDZ), and emicizumab (EMZ). Saturated ammonium sulfate (AS) was used to precipitate immunoglobulins in human plasma. After centrifugation, supernatant containing albumin was decanted, and the precipitated immunoglobulin fraction was re-dissolved in buffer containing 6M guanidine. This fraction was then completely denatured, reduced, alkylated, and trypsin digested. Finally, signature peptides from the seven mAbs were simultaneously quantified on LC-MS/MS together with their internal standards stable isotopically labeled peptide counterparts. The linear dynamic ranges (1 - 512 mg/L) of IFX, CTX, RTX, and EMZ showed excellent (R2 > 0.999) linearity and those of DPL, DNX, and VDZ showed good (R2 > 0.995) linearity. The method was validated in accordance with the EMA guidelines. EDTA plasma, sodium citrate plasma, heparin plasma, and serum yielded similar results. Prepared samples were stable at room temperature (20°C) and at 5°C for 3 days, and showed no decline in concentration for all tested mAbs. This described method, which has the advantage of an easy, rapid, and robust pre-analytical sample preparation, can be used as a template to quantify other mAbs in human plasma or serum.

Keywords:  Ammonium sulfate; LC-MS/MS; Liquid chromatography-tandem mass spectrometry; Multiplex analysis; Quantification; Therapeutic monoclonal antibody

DOI:  https://doi.org/10.1016/j.chroma.2021.462489
Pract Lab Med. 2021 Nov;27 e00254

Optimizing oxytocin LC-MS/MS sensitivity by choosing the right column.

Mariëtte T Ackermans.

   Objective: Sensitivity is often an issue in bioanalytical LC-MS/MS applications. Commonly investigated parameters to improve it include additives to mobile phase, derivatization and sample-preparation. The nature of the column, however, is not frequently evaluated.
Design and Methods: The sensitivity is compared for 18 different reversed phase and 2 different HILIC columns using 2 different mobile phase compositions. Sensitivity was evaluated in terms of S/N for 1,5 pg oxytocin on column, using a scouting gradient.
Results: The measured signal to noise ranged from 55 to 1473, indicating a substantial difference in sensitivity. The most sensitive columns were the Synergi Hydro RP (reversed phase) and the Atlantis HILIC (HILIC).
Conclusions: This study shows that choosing the right column contributes to the sensitivity of the method.

Keywords:  HILIC; Liquid chromatography; Oxytocin; Reversed phase

DOI:  https://doi.org/10.1016/j.plabm.2021.e00254
J Pharm Anal. 2021 Aug;11(4): 523-528

A simplified LC-MS/MS method for the quantification of the cardiovascular disease biomarker trimethylamine-N-oxide and its precursors.

Katharina Rox, Silke Rath, Dietmar H Pieper, Marius Vital, Mark Brönstrup.

  Trimethylamine-N-oxide (TMAO) has emerged as a potential biomarker for atherosclerosis and the development of cardiovascular diseases (CVDs). Although several clinical studies have shown striking associations of TMAO levels with atherosclerosis and CVDs, TMAO determinations are not clinical routine yet. The current methodology relies on isotope-labeled internal standards, which adds to pre-analytical complexity and costs for the quantification of TMAO and its precursors carnitine, betaine or choline. Here, we report a liquid chromatography-tandem mass spectrometry based method that is fast (throughput up to 240 samples/day), consumes low sample volumes (e.g., from a finger prick), and does not require isotope-labeled standards. We circumvented the analytical problem posed by the presence of endogenous TMAO and its precursors in human plasma by using an artificial plasma matrix for calibration. We cross-validated the results obtained using an artificial matrix with those using mouse plasma matrix and demonstrated that TMAO, carnitine, betaine and choline were accurately quantified in 'real-life' human plasma samples from healthy volunteers, obtained either from a finger prick or from venous puncture. Additionally, we assessed the stability of samples stored at -20 °C and room temperature. Whereas all metabolites were stable at -20 °C, increasing concentrations of choline were determined when stored at room temperature. Our method will facilitate the establishment of TMAO as a routine clinical biomarker in hematology in order to assess the risk for CVDs development, or to monitor disease progression and intervention effects.

Keywords:  Atherosclerosis; Betaine; Biomarker; Carnitine; Choline; LC-MS/MS; TMAO

DOI:  https://doi.org/10.1016/j.jpha.2021.03.007
Talanta. 2021 Dec 01. pii: S0039-9140(21)00663-9. [Epub ahead of print]235 122742

Synthesis and characterization of 13C labeled carnosine derivatives for isotope dilution mass spectrometry measurements in biological matrices.

Marco Maspero, Ettore Gilardoni, Chiara Bonfanti, Graziella Messina, Luca Regazzoni, Marco De Amici, Marina Carini, Giancarlo Aldini, Clelia Dallanoce.

  Due to the physiological properties of l-carnosine (l-1), supplementation of this dipeptide has both a nutritional ergogenic application and a therapeutic potential for the treatment of numerous diseases in which ischemic or oxidative stress are involved. Quantitation of carnosine and its analogs in biological matrices results to be crucial for these applications and HPLC-MS procedures with isotope-labeled internal standards are the state-of-the-art approach for this analytical need. The use of these standards allows to account for variations during the sample preparation process, between-sample matrix effects, and variations in instrument performance over analysis time. Although literature reports a number of studies involving carnosine, isotope-labeled derivatives of the dipeptide are not commercially available. In this work we present a fast, flexible, and convenient strategy for the synthesis of the 13C-labeled carnosine analogs and their application as internal standards for the quantitation of carnosine and anserine in a biological matrix.

Keywords:  Anserine; Carnosine; Histidine dipeptides; Isotope dilution mass spectrometry; Quantitative analysis; Synthesis

DOI:  https://doi.org/10.1016/j.talanta.2021.122742
Talanta. 2021 Dec 01. pii: S0039-9140(21)00695-0. [Epub ahead of print]235 122774

Analysis of aristolochic acid I in mouse serum and tissues by using magnetic solid-phase extraction and UHPLC-MS/MS.

Wenjing Guo, Zhangsheng Shi, Jing Zhang, Ting Zeng, Yu He, Zongwei Cai.

  A method combining magnetic solid-phase extraction (MSPE) and ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed for the analysis of aristolochic acids I (AAI) in mouse serum and tissues. The magnetic covalent organic frameworks (MNP@COF)-based MSPE exhibited high adsorption capacity towards AAI (93.1 mg/g) in optimal conditions. After MSPE extraction, AAI was separated with C18 column using gradient elution and quantified (m/z 342.21 → 298.13) by UHPLC-MS/MS with monitor reaction monitoring (MRM) mode. This MSPE-based UHPLC-MS/MS method was validated with respected to lower limit of quantification (LLOQ), linearity, recovery, precision and accuracy of intra- and inter-day, and matrix effect. Good calibration linearities at the range of 1-500 ng/L for AAI in biological matrices (serum, kidney, and liver) with high correlation coefficient (R2) > 0.9970, and high enrichment factors (mean values from 1038 to 1045) were obtained. This method was highly sensitive to determine AAI with LLOQ within the range of 4.62-5.24 ng/L in extracted serum, kidney, and liver samples. Recoveries at 5, 50, 100 and 300 ng/L in biological samples ranged from 93.2 to 104.0%, and intra- and inter day accuracy and precision (defined as bias and coefficient of variation, respectively) were below ± 15%. The method was successfully applied in the analysis of biological samples collected from mice exposed with AAI with concentrations range of 0.007-0.041 μg/L for consecutive four days. The established method might be applied for the investigation of risk assessment and toxicity induced by long-time use of AAI-containing herbs or dietary supplements.

Keywords:  Aristolochic acid I; Biological samples; Magnetic solid-phase extraction; UHPLC-MS/MS

DOI:  https://doi.org/10.1016/j.talanta.2021.122774
Anal Bioanal Chem. 2021 Sep 17.

A highly sensitive, simple, and fast gas chromatography-mass spectrometry method for the quantification of serum short-chain fatty acids and their potential features in central obesity.

Miftakh Nur Rahman, Ajeng Diantini, Miswar Fattah, Melisa I Barliana, Andi Wijaya.

  Short-chain fatty acids (SCFAs) are the main gut microbe metabolites, which have no more than six carbons. SCFAs are an emerging biomarker in metabolic diseases, including central obesity. Commonly, SCFAs are measured in fecal samples, where they are highly abundant, but here they do not reflect direct interactions with related organs. Serum SCFAs are assumed to be more associated with metabolic disease than fecal SCFAs, albeit at very low concentrations. The aim of the present study is to develop a highly sensitive, simple, and fast method for measuring six SCFAs in the serum by gas chromatography-mass spectrometry (GCMS). The serum is mixed with meta-phosphoric acid and 2,2-dimethylbutyric acid, followed by homogenization and centrifugation. Supernatant is then injected into the fused silica capillary column. The method is linear from 0.12-500 μmol/L for all SCFAs with an accuracy of 90-117%. The total coefficient of variation for precision ranges from 3.8 to 14.1%. A preliminary study is performed with 32 centrally obese subjects and 17 lean subjects. The mean values of all SCFAs, including acetic, propionic, isobutyric, butyric, isovaleric, and valeric acid, in the centrally obese subjects are significantly higher compared with lean subjects. A significant correlation also exists between all SCFAs, with the waist circumference indicating that serum SCFAs have potential features with respect to metabolic diseases, especially central obesity. The validated GCMS method provides highly sensitive, fast, simple, and reliable SCFA quantitation in the serum and demonstrates the potential features of circulating SCFAs in central obesity.

Keywords:  Central obesity; Gas chromatography; Mass spectrometry; Method development; Protein precipitation; Short-chain fatty acids

DOI:  https://doi.org/10.1007/s00216-021-03639-3
Anal Methods. 2021 Sep 16.

A relative quantitation method for measuring DNA methylation and hydroxymethylation using guanine as an internal standard.

Krisztina Németh, Katalin Mészáros, Borbála Szabó, Henriett Butz, Tamás Arányi, Pál T Szabó.

Global DNA methylation and hydroxymethylation play an important role in gene expression. They can be connected with several diseases. The modification status could be a biomarker to determine the status of disease. A fast, easy and accurate liquid chromatography - tandem mass spectrometry method has been developed for the precise quantitation of 5-methylcytosine and 5-hydroxymethylcytosine. Formic acid was used for the hydrolysis of the DNA strand resulting in nucleobases. These polar hydrolysis products were separated on a normal phase column using reversed phase eluents in inverse gradient mode. Multiple reaction monitoring was applied to achieve high selectivity and sensitivity for the quantitation. A new relative quantitation model was developed by using guanine, as an internal standard, present in samples. The new method was successfully validated with excellent accuracy and precision values in the range of 0.005-0.5% for 5hmC and 1-15% for 5mC. The main advantages of this quantitation method are that, due to relative quantitation, calibration curves can be used without reacquiring the calibration points and no additional isotope labeled internal standards are required. The method was tested to identify the concentrations of 5mC and 5hmC in various sample types. The lowest level of DNA sample required in the case of 0.005% 5hmC is 0.5 μg.

DOI: https://doi.org/10.1039/d1ay00897h
Talanta. 2021 Dec 01. pii: S0039-9140(21)00641-X. [Epub ahead of print]235 122720

Using random forest to detect multiple inherited metabolic diseases simultaneously based on GC-MS urinary metabolomics.

Nan Chen, Hai-Bo Wang, Ben-Qing Wu, Jian-Hui Jiang, Jiang-Tao Yang, Li-Juan Tang, Hong-Qin He, Dan-Dan Linghu.

  Inborn errors of metabolism, also known as inherited metabolic diseases (IMDs), are related to genetic mutations and cause corresponding biochemical metabolic disorder of newborns and even sudden infant death. Timely detection and diagnosis of IMDs are of great significance for improving survival of newborns. Here we propose a strategy for simultaneously detecting six types of IMDs via combining GC-MS technique with the random forest algorithm (RF). Clinical urine samples from IMD and healthy patients are analyzed using GC-MS for acquiring metabolomics data. Then, the RF model is established as a multi-classification tool for the GC-MS data. Compared with the models built by artificial neural network and support vector machine, the results demonstrated the RF model has superior performance of high specificity, sensitivity, precision, accuracy, and matthews correlation coefficients on identifying all six types of IMDs and normal samples. The proposed strategy can afford a useful method for reliable and effective identification of multiple IMDs in clinical diagnosis.

Keywords:  GC-MS; Inherited metabolic diseases; Metabolomics analysis; Multi-classification; Random forest

DOI:  https://doi.org/10.1016/j.talanta.2021.122720
Nat Protoc. 2021 Sep 17.

Stable isotope tracing to assess tumor metabolism in vivo.

Brandon Faubert, Alpaslan Tasdogan, Sean J Morrison, Thomas P Mathews, Ralph J DeBerardinis.

Cancer cells undergo diverse metabolic adaptations to meet the energetic demands imposed by dysregulated growth and proliferation. Assessing metabolism in intact tumors allows the investigator to observe the combined metabolic effects of numerous cancer cell-intrinsic and -extrinsic factors that cannot be fully captured in culture models. We have developed methods to use stable isotope-labeled nutrients (e.g., [13C]glucose) to probe metabolic activity within intact tumors in vivo, in mice and humans. In these methods, the labeled nutrient is introduced to the circulation through an intravenous catheter prior to surgical resection of the tumor and adjacent nonmalignant tissue. Metabolism within these tissues during the infusion transfers the isotope label into metabolic intermediates from pathways supplied by the infused nutrient. Extracting metabolites from surgical specimens and analyzing their isotope labeling patterns provides information about metabolism in the tissue. We provide detailed information about this technique, from introduction of the labeled tracer through data analysis and interpretation, including streamlined approaches to quantify isotope labeling in informative metabolites extracted from tissue samples. We focus on infusions with [13C]glucose and the application of mass spectrometry to assess isotope labeling in intermediates from central metabolic pathways, including glycolysis, the tricarboxylic acid cycle and nonessential amino acid synthesis. We outline practical considerations to apply these methods to human subjects undergoing surgical resections of solid tumors. We also discuss the method's versatility and consider the relative advantages and limitations of alternative approaches to introduce the tracer, harvest the tissue and analyze the data.

DOI: https://doi.org/10.1038/s41596-021-00605-2
J Biosci Bioeng. 2021 Sep 08. pii: S1389-1723(21)00205-X. [Epub ahead of print]

Recent advances in metabolic engineering-integration of in silico design and experimental analysis of metabolic pathways.

Hiroshi Shimizu, Yoshihiro Toya.

  Microorganisms are widely used to produce valuable compounds. Because thousands of metabolic reactions occur simultaneously and many metabolic reactions are related to target production and cell growth, the development of a rational design method for metabolic pathway modification to optimize target production is needed. In this paper, recent advances in metabolic engineering are reviewed, specifically considering computational pathway modification design and experimental evaluation of metabolic fluxes by 13C-metabolic flux analysis. Computational tools for seeking effective gene deletion targets and recruiting heterologous genes are described in flux balance analysis approaches. A kinetic model and adaptive laboratory evolution were applied to identify and eliminate the rate-limiting step in metabolic pathways. Data science-based approaches for process monitoring and control are described to maximize the performance of engineered cells in bioreactors.

Keywords:  (13)C-metabolic flux analysis; Adaptive laboratory evolution; Flux balance analysis; Metabolic control analysis; Metabolic engineering

DOI:  https://doi.org/10.1016/j.jbiosc.2021.08.002