Simultaneous Measurement of Tricarboxylic Acid Cycle Intermediates in Different Biological Matrices Using Liquid Chromatography–Tandem Mass Spectrometry; Quantitation and Comparison of TCA Cycle Intermediates in Human Serum, Plasma, Kasumi-1 Cell and Murine Liver Tissue

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dc.contributor.author Rathod, Ramji
dc.contributor.author Gajera, Bharat
dc.contributor.author Nazir, Kenneth
dc.contributor.author Wallenius, Janne
dc.contributor.author Velagapudi, Vidya
dc.date.accessioned 2020-03-13T13:09:39Z
dc.date.available 2020-03-13T13:09:39Z
dc.date.issued 2020-03-12
dc.identifier.citation Rathod, R.; Gajera, B.; Nazir, K.; Wallenius, J.; Velagapudi, V. Simultaneous Measurement of Tricarboxylic Acid Cycle Intermediates in Different Biological Matrices Using Liquid Chromatography–Tandem Mass Spectrometry; Quantitation and Comparison of TCA Cycle Intermediates in Human Serum, Plasma, Kasumi-1 Cell and Murine Liver Tissue. Metabolites 2020, 10, 103.
dc.identifier.uri http://hdl.handle.net/10138/313303
dc.description.abstract The tricarboxylic acid (TCA) cycle is a central part of carbon and energy metabolism, also connecting to glycolysis, amino acid, and lipid metabolism. The quantitation of the TCA cycle intermediate within one method is lucrative due to the interest in central carbon metabolism profiling in cells and tissues. In addition, TCA cycle intermediates in serum have been discovered to correspond as biomarkers to various underlying pathological conditions. In this work, an Liquid Chromatography-Mass Spectrometry/Mass Spectrometry-based quantification method is developed and validated, which takes advantage of fast, specific, sensitive, and cost-efficient precipitation extraction. Chromatographic separation is achieved while using Atlantis dC18 2.1 mm &times; 100 mm, particle size 3-&mu;m of Waters column with a gradient elution mobile phase while using formic acid in water (0.1% <i>v</i>/<i>v</i>) and acetonitrile. Linearity was clearly seen over a calibration range of: 6.25 to 6400 ng/mL (r<sup>2</sup> &gt; 0.980) for malic acid; 11.72 to 12,000 ng/mL (r<sup>2</sup> &gt; 0.980) for cis-aconitic acid and L-aspartic acid; 29.30 to 30,000 ng/mL (r<sup>2</sup> &gt; 0.980) for isocitric acid, <span style="font-variant: small-caps;">l</span>-serine, and <span style="font-variant: small-caps;">l</span>-glutamic acid; 122.07 to 125,000 ng/mL (r<sup>2</sup> &gt; 0.980) for citric acid, glycine, <i>oxo</i>-glutaric acid, <span style="font-variant: small-caps;">l</span>-alanine, and <span style="font-variant: small-caps;">l</span>-glutamine; 527.34 to 540,000 ng/mL (r<sup>2</sup> &gt; 0.980) for <span style="font-variant: small-caps;">l</span>-lactic acid; 976.56 to 1,000,000 ng/mL (r<sup>2</sup> &gt; 0.980) for <span style="font-variant: small-caps;">d</span>-glucose; 23.44 to 24,000 ng/mL (r<sup>2</sup> &gt; 0.980) for fumaric acid and succinic acid; and, 244.14 to 250,000 ng/mL (r<sup>2</sup> &gt; 0.980) for pyruvic acid. Validation was carried out, as per European Medicines Agency (EMA) &ldquo;guidelines on bioanalytical method validation&rdquo;, for linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LLOQ), recovery, matrix effect, and stability. The recoveries from serum and tissue were 79&ndash;119% and 77&ndash;223%, respectively. Using this method, we measured TCA intermediates in serum, plasma (NIST 1950 SRM), and in mouse liver samples. The concentration found in NIST SRM 1950 (<i>n</i> = 6) of glycine (246.4 &micro;mol/L), <span style="font-variant: small-caps;">l</span>-alanine (302.4 &micro;mol/L), and serine (92.9 &micro;mol/L).
dc.publisher Multidisciplinary Digital Publishing Institute
dc.title Simultaneous Measurement of Tricarboxylic Acid Cycle Intermediates in Different Biological Matrices Using Liquid Chromatography–Tandem Mass Spectrometry; Quantitation and Comparison of TCA Cycle Intermediates in Human Serum, Plasma, Kasumi-1 Cell and Murine Liver Tissue
dc.date.updated 2020-03-13T13:09:39Z
dc.type.uri http://purl.org/eprint/entityType/JournalArticle
dc.type.uri http://purl.org/eprint/entityType/Expression
dc.type.uri http://purl.org/eprint/entityType/Expression

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