bims-metlip Biomed News
on Methods and protocols in metabolomics and lipidomics
Issue of 2020‒11‒29
seventeen papers selected by
Sofia Costa
Cold Spring Harbor Laboratory


  1. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Nov 17. pii: S1570-0232(20)31320-9. [Epub ahead of print]1161 122444
    van de Velde B, Guillarme D, Kohler I.
      Metabolomics, which consists of the comprehensive analysis of metabolites within a biological system, has been playing a growing role in the implementation of personalized medicine in modern healthcare. A wide range of analytical approaches are used in metabolomics, notably mass spectrometry (MS) combined to liquid chromatography (LC), gas chromatography (GC), or capillary electrophoresis (CE). However, none of these methods enable a comprehensive analysis of the metabolome, due to its extreme complexity and the large differences in physico-chemical properties between metabolite classes. In this context, supercritical fluid chromatography (SFC) represents a promising alternative approach to improve the metabolome coverage, while further increasing the analysis throughput. SFC, which uses supercritical CO2 as mobile phase, leads to numerous advantages such as improved kinetic performance and lower environmental impact. This chromatographic technique has gained a significant interest since the introduction of advanced instrumentation, together with the introduction of dedicated interfaces for hyphenating SFC to MS. Moreover, new developments in SFC column chemistry (including sub-2 µm particles), as well as the use of large amounts of organic modifiers and additives in the CO2-based mobile phase, significantly extended the application range of SFC, enabling the simultaneous analysis of a large diversity of metabolites. Over the last years, several applications have been reported in metabolomics using SFC-MS - from lipophilic compounds, such as steroids and other lipids, to highly polar compounds, such as carbohydrates, amino acids, or nucleosides. With all these advantages, SFC-MS is promised to a bright future in the field of metabolomics.
    Keywords:  Bioanalysis; Lipidomics; Mass spectrometry; Metabolomics; Supercritical fluid chromatography; Unified chromatography
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122444
  2. J Proteome Res. 2020 Nov 25.
    Southam AD, Pursell H, Frigerio G, Jankevics A, Weber RJM, Dunn WB.
      Metabolic phenotyping of tissues uses metabolomics and lipidomics to measure the relative polar and nonpolar (lipid) metabolite levels in biological samples. This approach aims to understand disease biochemistry and identify biochemical markers of disease. Sample preparation methods must be reproducible, sensitive (high metabolite and lipid yield), and ideally rapid. We evaluated three biphasic methods for polar and nonpolar compound extraction (chloroform/methanol/water, dichloromethane/methanol/water, and methyl tert-butyl ether [MTBE]/methanol/water), a monophasic method for polar compound extraction (acetonitrile/methanol/water), and a monophasic method for nonpolar compound extraction (isopropanol/water). All methods were applied to mammalian heart, kidney, and liver tissues. Polar extracts were analyzed by hydrophilic interaction chromatography (HILIC) ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) and nonpolar extracts by C18 reversed-phase UHPLC-MS. Method reproducibility and yield were assessed using multiple annotated endogenous compounds (putatively and MS/MS annotated). Monophasic methods had the highest yield and high reproducibility for both polar (positive ion: median relative standard deviation (RSD) < 18%; negative ion: median RSD < 28%) and nonpolar (positive and negative ion: median RSD < 15%) extractions for heart, kidneys, and liver. The polar monophasic method extracted higher levels of lipid than biphasic polar extractions, and these lipids caused minimal detection suppression for other compounds during HILIC UHPLC-MS. The nonpolar monophasic method had similar or greater detection responses of all detected lipid classes compared to biphasic methods (including increased phosphatidylinositol, phosphatidylserine, and cardiolipin responses). Monophasic methods are quicker and simpler than biphasic methods and are therefore most suited for future automation.
    Keywords:  metabolic profiling; metabolite profiling; tissue extraction; tissue preparation
    DOI:  https://doi.org/10.1021/acs.jproteome.0c00660
  3. J Chromatogr A. 2020 Nov 13. pii: S0021-9673(20)30982-1. [Epub ahead of print] 461708
    Palyzová A, Řezanka T.
      A combination of two chromatographic and two enzymatic methods was used for the analysis of molecular species of lipids from Gram-positive bacteria of the genus Kocuria. Gram-positive bacteria contain a majority of branched fatty acids (FAs), especially iso- and/or anteiso-FAs. Two strains K. rhizophila were cultivated at three different temperatures (20, 28, and 37°C) and the majority phospholipid, i.e., the mixture of molecular species of phosphatidylglycerols (PGs) was separated by means of hydrophilic interaction liquid chromatography (HILIC). After enzymatic hydrolysis of PGs by phospholipase C and derivatization of the free OH group, the sn-1,2-diacyl-3-acetyl triacylglycerols (AcTAGs) were separated by reversed phase HPLC. Molecular species such as i-15:0/i-15:0/2:0, ai-15:0/ai-15:0/2:0, and 15:0/15:0/2:0 (straight chains) were identified by liquid chromatography-positive electrospray ionization mass spectrometry. The tandem mass spectra of both standards and natural compounds containing iso, anteiso and straight chain FAs with the same carbons were identical. Therefore, for identification of the ratio of two regioisomers, i.e. i-15:0/ai-15:0/2:0 vs. ai-15:0/i-15:0/2:0, they were cleavage by pancreatic lipase. The mixture of free fatty acids (FFAs) and 2-monoacylglycerols (2-MAGs) was obtained. After their separation by TLC and esterification and/or transesterification, the fatty acid methyl esters were quantified by GC-MS and thus the ratio of regioisomers was determined. It has been shown that the ratio of PG (containing as majority i-15: 0 / i-15: 0, i-15: 0 / ai-15: 0 and / or ai-15: 0 / i-15: 0 and ai-15: 0 / ai-15: 0 molecular species) significantly affected the membrane flow of bacterial cells cultured at different temperatures.
    Keywords:  Gram-positive bacteria; RP-HPLC/MS-ESI(+); branched fatty acids; diacylglycerols; enzymatic hydrolysis; phosphatidylglycerols
    DOI:  https://doi.org/10.1016/j.chroma.2020.461708
  4. J Chromatogr A. 2020 Nov 13. pii: S0021-9673(20)30980-8. [Epub ahead of print] 461706
    Sorensen MJ, Kennedy RT.
      LC-MS is an important tool for metabolomics due its high sensitivity and broad metabolite coverage. The goal of improving resolution and decreasing analysis time in HPLC has led to the use of 5 - 15 cm long columns packed with 1.7 - 1.9 µm particles requiring pressures of 8 - 12 kpsi. We report on the potential for capillary LC-MS based metabolomics utilizing porous C18 particles down to 1.1 µm diameter and columns up to 50 cm long with an operating pressure of 35 kpsi. Our experiments show that it is possible to pack columns with 1.1 µm porous particles to provide predicted improvements in separation time and efficiency. Using kinetic plots to guide the choice of column length and particle size, we packed 50 cm long columns with 1.7 µm particles and 20 cm long columns with 1.1 µm particles, which should produce equivalent performance in shorter times. Columns were tested by performing isocratic and gradient LC-MS analyses of small molecule metabolites and extracts from plasma. These columns provided approximately 100,000 theoretical plates for metabolite standards and peak capacities over 500 in 100 min for a complex plasma extract with robust interfacing to MS. To generate a given peak capacity, the 1.1 µm particles in 20 cm columns required roughly 75% of the time as 1.7 µm particles in 50 cm columns with both operated at 35 kpsi. The 1.1 µm particle packed columns generated a given peak capacity nearly 3 times faster than 1.7 µm particles in 15 cm columns operated at ~10 kpsi. This latter condition represents commercial state of the art for capillary LC. To consider practical benefits for metabolomics, the effect of different LC-MS variables on mass spectral feature detection was evaluated. Lower flow rates (down to 700 nL/min) and larger injection volumes (up to 1 µL) increased the features detected with modest loss in separation performance. The results demonstrate the potential for fast and high resolution separations for metabolomics using 1.1 µm particles operated at 35 kpsi for capillary LC-MS.
    Keywords:  mass spectrometry; metabolomics; peak capacity; sub-2 micron particles; ultrahigh-pressure liquid chromatography
    DOI:  https://doi.org/10.1016/j.chroma.2020.461706
  5. J Anal Toxicol. 2020 Nov 24. pii: bkaa177. [Epub ahead of print]
    Oh J, Park MS, Chun MR, Hwang JH, Lee JY, Jee JH, Lee SY.
      Measuring nicotine metabolites is the most objective method for identifying smoke exposure. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can measure multiple metabolites and is sensitive enough to detect low concentrations of metabolites. Therefore, we developed a simple and high-throughput method for measuring nicotine, cotinine, trans-3'-hydroxycotinine (3-OH cotinine), nornicotine, and anabasine for population-based studies using LC-MS/MS. Each 30 µl of urine sample was diluted with 90 µL of acetonitrile containing five deuterated internal standards. Chromatographic separation used a C18 column and LC-MS/MS analysis was performed with a multiple reaction monitoring mode. The chromatographic run time for each sample was 6.5 min. The method was validated by evaluating selectivity, interference, limit of detection, lower limit of quantification, precision, accuracy, linearity, extraction recovery, matrix effect, and carry-over according to guidelines. Our methods required a short preparation time (about 20 minutes) while simultaneously measuring five markers for smoking status. No endogenous or exogenous interference was found. Our method showed excellent precision and accuracy: within-run CV 2.9-9.4%, between-run CV 4.8-8.7%, and bias -10.1 to 5.3%. Linear dynamic ranges were 1-10,000 ng/mL for nicotine, nornicotine, and anabasine; 2-5,000 ng/mL for cotinine; and 5-15,000 ng/mL for 3-OH cotinine. Extraction recovery was consistent (87-109%) across concentrations. No significant matrix effect or carry-over was observed. The validated method was applied to 849 urine samples. In samples from the 125 current smokers, nicotine, cotinine, 3-OH cotinine, nornicotine, and anabasine were detected in 97.6%, 99.2%, 98.4%, 96.8%, and 87.2%, respectively. No markers were detected in 93.9% of 609 non-smokers. The overlapping detection of multiple markers made it possible to identify smoking status even in current smokers with low concentration of cotinine. Our LC-MS/MS method using a simple sample preparation technique is sensitive and effective for screening of smoking status in the general population.
    Keywords:  High-throughput; Mass spectrometry; Nicotine metabolites; Smoking; Urine
    DOI:  https://doi.org/10.1093/jat/bkaa177
  6. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Oct 12. pii: S1570-0232(20)31267-8. [Epub ahead of print] 122391
    Lefeuvre S, Bois-Maublanc J, Mongeois E, Policarpo V, Formaux L, Francia T, Billaud EM, Got L.
      Urinary catecholamines and their methylated metabolites are biochemical indicators of pheochromocytoma, paraganglioma and neuroblastoma. A rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to high-resolution mass spectrometry (LC-HRMS) was developed and validated to measure urinary catecholamines (epinephrine (E), norepinephrine (NorE), dopamine (D)) and total methylated metabolites (normetanephrine (NorMN), metanephrine(MN) and 3-methoxytyramine (3-MT)) in a clinical setting. Results of 51 urine specimens measured using this LC-HRMS method were compared with a liquid chromatography assay with electrochemical detection (LC-EC). Urine samples (200 μL) were spiked with an internal standard solution followed by SPE purification. In the case of total methylated metabolites, urine was hydrolyzed before SPE purification. Separation was achieved on an Acclaim Mixed Mode WCX column, with an 8.5 min runtime. All compounds were detected in electrospray positive ionization mode with a parallel reaction monitoring acquisition and quantified with a linear regression (r2 > 0.998) between 2 and 200 µg/L (10.9-1090; 11.8-1182 nmol/L) for E and NorE respectively and between 10 and 1000 µg/L for others (65.2-6520; 50.7-5070; 54.5-5450 ; 59.8-5980 nmol/L for D, M, NorMN and 3-MT, respectively). Overall imprecision and bias did not exceed 15%. No significant matrix effect was observed. Correlation between the two assays was good except for epinephrine. Epinephrine concentrations measured by LC-EC method were slightly higher than values obtained with LC-HRMS method but without impact on clinical decision. This LC-HRMS assay provides a new tool for simultaneous quantitative catecholamine determination and was successfully applied in routine for the screening or follow up of pheochromocytoma, paraganglioma and neuroblastoma. LC-HRMS method offers significant advantages compared to LC-EC with good sensitivity, an unambiguous analyte determination and high sample throughput.
    Keywords:  LC-HRMS; Neuroblastoma; Paraganglioma; Pheochromocytoma; Urinary cathecolamines; Urinary metanephrines
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122391
  7. Nat Biotechnol. 2020 Nov 23.
    Dührkop K, Nothias LF, Fleischauer M, Reher R, Ludwig M, Hoffmann MA, Petras D, Gerwick WH, Rousu J, Dorrestein PC, Böcker S.
      Metabolomics using nontargeted tandem mass spectrometry can detect thousands of molecules in a biological sample. However, structural molecule annotation is limited to structures present in libraries or databases, restricting analysis and interpretation of experimental data. Here we describe CANOPUS (class assignment and ontology prediction using mass spectrometry), a computational tool for systematic compound class annotation. CANOPUS uses a deep neural network to predict 2,497 compound classes from fragmentation spectra, including all biologically relevant classes. CANOPUS explicitly targets compounds for which neither spectral nor structural reference data are available and predicts classes lacking tandem mass spectrometry training data. In evaluation using reference data, CANOPUS reached very high prediction performance (average accuracy of 99.7% in cross-validation) and outperformed four baseline methods. We demonstrate the broad utility of CANOPUS by investigating the effect of microbial colonization in the mouse digestive system, through analysis of the chemodiversity of different Euphorbia plants and regarding the discovery of a marine natural product, revealing biological insights at the compound class level.
    DOI:  https://doi.org/10.1038/s41587-020-0740-8
  8. Xenobiotica. 2020 Nov 21. 1-42
    Jiang S, Wang S, Dong P, Shi L, Li Q, Wei X, Gao P, Zhang J.
      Liquiritin (LQ), a main component of liquorice, exerts various biological activities. However, insufficient attentions have been paid to the metabolism study on this natural compound until now. Our present study was conducted to investigate the LQ metabolites in rats urine, faeces and plasma using UHPLC-LTQ-Orbitrap mass spectrometer in both positive and negative ion modes. Meanwhile, post-acquisition data-mining methods including high-resolution extracted ion chromatogram (HREIC), multiple mass defect filters (MMDFs), neutral loss fragments (NLFs) and diagnostic product ions (DPIs) were utilized to screen and identify LQ metabolites from HR-ESI-MS to ESI-MS n stage. As a result, a total of 49 metabolites were detected and characterized unambiguously or tentatively. These metabolites were presumed to generate through glucuronidation, sulfation, deglucosylation, dehydrogenation, methylation, hydrogenation, hydroxylation, ring cleavage and their composite reactions. Our results not only provided novel and useful data to better understand the biological activities of LQ, but also indicated that the proposed strategy was reliable for a rapid discovery and identification drug-related constituents in vivo.
    Keywords:  Liquiritin; UHPLC-LTQ-Orbitrap mass spectrometer; data-mining methods; metabolites
    DOI:  https://doi.org/10.1080/00498254.2020.1854366
  9. Biomed Chromatogr. 2020 Nov 23. e5036
    Gravitte A, Archibald T, Kennard B, Cobble A, Brown S.
      Liquid chromatography, coupled with tandem mass spectrometry, presents a powerful tool for the quantification of the sex steroid hormones 17-β estradiol, progesterone, and testosterone from biological matrices. The importance of accurate quantification with these hormones, even at endogenous levels, has evolved with our understanding of the role these regulators play in human development, fertility, and disease risk and manifestation. Routine monitoring of these analytes can be accomplished by immunoassay techniques, which face limitations on specificity and sensitivity, or using gas chromatography-mass spectrometry. LC-MS/MS is growing in capability and acceptance for clinically relevant quantification of sex steroid hormones in biological matrices and is able to overcome many of the limitations of immunoassays. Analyte specificity has improved through the use of novel derivatizing agents, and sensitivity has been refined through use of high resolution chromatography and mass spectrometric technology. This review highlights these innovations, among others, in LC-MS/MS steroid hormone analysis captured in the literature over the last decade.
    Keywords:  LC-MS/MS; Steroid hormone bioanalysis; estradiol; progesterone; testosterone
    DOI:  https://doi.org/10.1002/bmc.5036
  10. Mass Spectrom Rev. 2020 Nov 25.
    Han W, Li L.
      Determining metabolomic differences among samples of different phenotypes is a critical component of metabolomics research. With the rapid advances in analytical tools such as ultrahigh-resolution chromatography and mass spectrometry, an increasing number of metabolites can now be profiled with high quantification accuracy. The increased detectability and accuracy raise the level of stringiness required to reduce or control any experimental artifacts that can interfere with the measurement of phenotype-related metabolome changes. One of the artifacts is the batch effect that can be caused by multiple sources. In this review, we discuss the origins of batch effects, approaches to detect interbatch variations, and methods to correct unwanted data variability due to batch effects. We recognize that minimizing batch effects is currently an active research area, yet a very challenging task from both experimental and data processing perspectives. Thus, we try to be critical in describing the performance of a reported method with the hope of stimulating further studies for improving existing methods or developing new methods.
    Keywords:  NMR; batch effect; mass spectrometry; metabolome analysis; metabolomics
    DOI:  https://doi.org/10.1002/mas.21672
  11. J Chromatogr A. 2020 Nov 14. pii: S0021-9673(20)30995-X. [Epub ahead of print] 461721
    Weggler BA, Dubois LM, Gawlitta N, Gröger T, Moncur J, Mondello L, Reichenbach S, Tranchida P, Zhao Z, Zimmermann R, Zoccali M, Focant JF.
      Comprehensive two-dimensional gas chromatography (GC × GC) is amongst the most powerful separation technologies currently existing. Since its advent in early 1990, it has become an established method which is readily available. However, one of its most challenging aspects, especially in hyphenation with mass spectrometry is the high amount of chemical information it provides for each measurement. The GC × GC community agrees that there, the highest demand for action is found. In response, the number of software packages allowing for in-depth data processing of GC × GC data has risen over the last couple of years. These packages provide sophisticated tools and algorithms allowing for more streamlined data evaluation. However, these tools/algorithms and their respective specific functionalities differ drastically within the available software packages and might result in various levels of findings if not appropriately implemented by the end users. This study focuses on two main objectives. First, to propose a data analysis framework and second to propose an open-source dataset for benchmarking software options and their specificities. Thus, allowing for an unanimous and comprehensive evaluation of GC × GC software. Thereby, the benchmark data includes a set of standard compound measurements and a set of chocolate aroma profiles. On this foundation, eight readily available GC × GC software packages were anonymously investigated for fundamental and advanced functionalities such as retention and detection device derived parameters, revealing differences in the determination of e.g. retention times and mass spectra.
    Keywords:  Chocolate, Fragrances and Allergens; Data Processing; Open Source Data
    DOI:  https://doi.org/10.1016/j.chroma.2020.461721
  12. Drug Test Anal. 2020 Nov 26.
    Iannella L, Colamonici C, Curcio D, Botre F, de la Torre X.
      The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography tandem mass spectrometry (GC-MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/mL and not originated from pregnant female athletes or female treated with 19-norethisterone. 19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TC), while androsterone (A), pregnanediol (PD) and pregnanetriol (PT) were selected as endogenous reference compounds (ERC). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone based formulations. Before the instrumental analysis, urine samples (< 25 mL) were hydrolyzed with β-glucuronidase from E. coli and extracted with n-pentane. Compounds of interest were purified through a single (for PT) or double (for 19-NE, 19-NA, A, and PD) liquid chromatographic step, to reduce the background noise and eliminate interferences that could have affect the accuracy of δ13 C values. The limit of quantification (LOQ) of 2 ng/mL was ensured for both 19-NA and 19-NE. The 19-NE determination could be helpful in case of "unstable" urine samples, in late excretion phases or when co-administration with 5α-reductase inhibitors occur.
    Keywords:  19-norandrosterone; 19-noretiocholanolone; Isotope Ratio Mass Spectrometry; doping control; nandrolone
    DOI:  https://doi.org/10.1002/dta.2985
  13. J Am Soc Mass Spectrom. 2020 Nov 24.
    Ousji O, Sleno L.
      Bisphenol A (BPA) structural analogs are increasingly used as alternatives in many industrial applications, due to growing evidence of BPA-related toxicity. Despite their widespread use, little is known about the biotransformation of these BPA analogs in the body. In this study, the in vitro metabolism of five BPA analogs (bisphenol AF, bisphenol F, bisphenol S, cumylphenol, and tetramethylbisphenol F) were investigated, using human and rat liver fractions, to evaluate the formation of phase I and phase II metabolites. Liquid chromatography high-resolution tandem mass spectrometry was employed to separate and characterize over 50 metabolites, many of which were not previously reported. The structures of all detected oxidative metabolites, dimers, GSH adducts, glucuronide, and sulfate conjugates were elucidated. A biphenyl solid-core chromatographic column was utilized for the separation of all metabolites, with a subsequent method, on a F5 column, specifically optimized for the separation of dimers formed via oxidative metabolism. There are several examples in this work where the combination of high chromatographic resolution and tandem mass spectrometry were necessary to distinguish between isomeric metabolites and conjugates.
    DOI:  https://doi.org/10.1021/jasms.0c00301
  14. J Chromatogr B Analyt Technol Biomed Life Sci. 2020 Oct 12. pii: S1570-0232(20)31279-4. [Epub ahead of print] 122403
    Heydari P, Martins MLF, Rosing H, Hillebrand MJX, Gebretensae A, Schinkel AH, Beijnen JH.
      The aim of this study was to develop and to validate a UPLC-MS/MS method for the quantification of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in mouse plasma and tissue homogenates to support preclinical pharmacokinetic studies. The sample preparation consisted of protein precipitation with cold (2-8 °C) methanol:acetonitrile (1:1, v/v), evaporation of the supernatant to dryness, and reconstitution of the dry-extracts in 4 mM ammonium formate pH 3.5. Separation was achieved on a Waters UPLC HSS T3 column (150 × 2.1 mm, 1.8 µm) maintained at 50 °C and using gradient elution with a total runtime of 6.7 min. Mobile phase A consisted of 4 mM ammonium formate pH 3.5 and mobile phase B of 0.1% formic acid in methanol:acetonitrile (1:1, v/v). Detection was carried out by tandem mass spectrometry with electrospray ionization in the positive ion mode. The method was validated within a linear range of 1-2,000 ng/mL, 10-20,000 ng/mL, and 0.5-200 ng/mL for morphine, morphine-3-glucuronide, and morphine-6-glucuronide, respectively. In human plasma, the intra- and inter-run precision of all analytes, including the lower limit of quantification levels, were ≤ 15.8%, and the accuracies were between 88.1 and 111.9%. It has been shown that calibration standards prepared in control human plasma can be used for the quantification of the analytes in mouse plasma and tissue homogenates. The applicability of the method was successfully demonstrated in a preclinical pharmacokinetic study in mice.
    Keywords:  Bioanalysis; Morphine; Morphine-3-glucuronide; Morphine-6-glucuronide; Tandem mass spectrometry; Ultra performance liquid chromatography
    DOI:  https://doi.org/10.1016/j.jchromb.2020.122403
  15. J AOAC Int. 2020 Jul 01. 103(4): 1140-1147
    Lee I, Vo J, Gao Q, Purohit P, Zarraga V, Babajanian S, Chang P, Swanson G.
      BACKGROUND: A quantitative NMR (qNMR) method can provide rapid analysis compared to chromatographic methods. Sample preparation steps are relatively simpler and run time is shorter. Rapid analysis methods for release tests in quality control laboratories are very important for laboratory efficiency. Here, we describe a single-laboratory validation study for a rapid qNMR analysis of L-arginine, L-citrulline, and taurine in powdered and tablet dietary supplement products.OBJECTIVES: This validation work is to provide documented evidence for the qNMR method validity as well as method performance.
    METHODS: The method used Bruker 400 MHz high-resolution proton NMR spectroscopy for simultaneous determination of L-arginine, L-citrulline, and taurine contents in dietary supplement product 1 (powder) and dietary supplement product 2 (tablet). The absolute NMR quantitation is based on a principle of universal proton response intensity correlation with the number of protons in each target analyte (amino acids) vs. that of a reference standard (maleic acid).
    RESULTS: The test method performance was validated with dietary supplement-1 (powder) and dietary supplement-2 (tablet). The linearity of the method was studied from about 360 mg/g to about 675 mg/g of L-arginine; from about 15 mg/g to about 30 mg/g of L-citrulline; and from about 20 mg/g to about 40 mg/g of taurine in dietary supplement-1, and from about 15 mg/g to about 30 mg/g of taurine in dietary supplement-2. The coefficients of determination (R2) are 1.0000 for L-arginine, 0.9967 for L-citrulline, and 0.9995 for taurine in dietary supplement-1 and 0.9903 for taurine in dietary supplement-2. The accuracies measured from the sample matrices are 102%, 101%, and 100% average recoveries for 80%, 100%, and 120% concentration levels of L-arginine, 105%, 105%, and 103% average recoveries for 80%, 100%, and 120% concentration level of L-citrulline, and 101%, 102%, and 100% average recoveries of taurine for 80%, 100%, 120% concentration levels in dietary supplement-1; and 95, 98%, and 93% average recoveries of taurine for 80%, 100%, 120% concentration levels in dietary supplement-2, respectively. The precisions (RSD) are 1% for L-arginine, 5% for L-citrulline, and 2% for taurine in dietary supplement -1, respectively; and 4% for taurine in dietary supplement-2. The ruggedness of the test method is within 2%, 4%, and 2% for L-arginine, L-citrulline, and taurine for dietary supplement -1, respectively, and within 4% for dietary supplement-2. The method is specific for the quantitation of each nutrient with no background interference from the matrix for the proton peaks of L-arginine, L-citrulline, taurine, and maleic acid (standard).
    CONCLUSIONS: The test method is proven to be specific, precise, accurate, rugged, and suitable for intended quantitative analysis of L-arginine, L-citrulline, and taurine in powdered and tablet finished products.
    HIGHLIGHTS: The simultaneous determination of all three nutrients of L-arginine, L-citrulline, and taurine using proton NMR provides rapid analysis for quality control release tests that is more efficient versus that of two HPLC methods. Previously, our laboratory was using one HPLC method to analyze L-arginine and L-citrulline while using a second HPLC method to analyze taurine. That approach required two HPLC instruments and two analysts for parallel analysis that takes 2 days using volatile and flammable solvents for extraction and chemical derivatization. This rapid NMR method can analyze the sample "as is" with results obtained in less than 4 h, and is efficient, safe, and environmentally friendly. The initial higher NMR instrument investment versus two HPLC instruments is rewarded with high returns for continued quality control tests.
    DOI:  https://doi.org/10.1093/jaoacint/qsaa002
  16. Bioinformatics. 2020 Nov 27. pii: btaa998. [Epub ahead of print]
    Bach E, Rogers S, Williamson J, Rousu J.
      MOTIVATION: Identification of small molecules in a biological sample remains a major bottleneck in molecular biology, despite a decade of rapid development of computational approaches for predicting molecular structures using mass spectrometry (MS) data. Recently, there has been increasing interest in utilizing other information sources, such as liquid chromatography (LC) retention time (RT), to improve identifications solely based on MS information, such as precursor mass-per-charge and tandem mass spectra (MS2).RESULTS: We put forward a probabilistic modelling framework to integrate MS and RT data of multiple features in an LC-MS experiment. We model the MS measurements and all pairwise retention order information as a Markov random field and use efficient approximate inference for scoring and ranking potential molecular structures. Our experiments show improved identification accuracy by combining MS2 data and retention orders using our approach, thereby outperforming state-of-the-art methods. Furthermore, we demonstrate the benefit of our model when only a subset of LC-MS features have MS2 measurements available besides MS1.
    AVAILABILITY AND IMPLEMENTATION: Software and data is freely available at https://github.com/aalto-ics-kepaco/msms_rt_score_integration.
    DOI:  https://doi.org/10.1093/bioinformatics/btaa998
  17. Iran J Pharm Res. 2020 ;19(2): 274-282
    Gholami A, Bahrami F, Faraji M.
      A simple, rapid, and ultra sensitive dispersive solid phase extraction based on nano graphene oxide was developed for simultaneous measurement of trace amounts of metformin (MET) and linagliptin (LIN) in plasma samples by HPLC-UV-Vis. Affecting factors on the extraction of these drugs, including adsorbent weight, extraction time, organic solvent type, desorption situations, and composition of solvent were examined and optimized. In optimum conditions, the LOD (limit of detection) and LOQ (limit of quantification) of the suggested technique were 2.0 ngmL-1 and 6.1 (ngmL-1) for LIN and 3.0 ngmL-1 and 9.2 ngmL-1 for MET, respectively. Suitable linear behavior in the considered ranges of concentration (10-2000 ngmL-1) and good correlation coefficient of 0.9901 and 0.9903 (r2) for LIN and MET were obtained, respectively. The RSD (relative standard deviations) according to three replicate measurements at 2, 20, 200 ngmL-1 levels of these drugs was less than 8.0%. In the last step, applicability of the suggested technique was examined by analyzing the drugs in plasma samples and reasonable results were achieved.
    Keywords:  Dispersive solid phase extraction; HPLC; Linagliptin; Metformin; Nano graphene oxide; Plasma analysis
    DOI:  https://doi.org/10.22037/ijpr.2019.111659.13292