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
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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-03-01
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| Series: | Metabolites |
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| Online Access: | https://www.mdpi.com/2218-1989/10/3/103 |
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| author | Ramji Rathod Bharat Gajera Kenneth Nazir Janne Wallenius Vidya Velagapudi |
| author_facet | Ramji Rathod Bharat Gajera Kenneth Nazir Janne Wallenius Vidya Velagapudi |
| author_sort | Ramji Rathod |
| collection | DOAJ |
| description | 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 × 100 mm, particle size 3-μm 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> > 0.980) for malic acid; 11.72 to 12,000 ng/mL (r<sup>2</sup> > 0.980) for cis-aconitic acid and L-aspartic acid; 29.30 to 30,000 ng/mL (r<sup>2</sup> > 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> > 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> > 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> > 0.980) for <span style="font-variant: small-caps;">d</span>-glucose; 23.44 to 24,000 ng/mL (r<sup>2</sup> > 0.980) for fumaric acid and succinic acid; and, 244.14 to 250,000 ng/mL (r<sup>2</sup> > 0.980) for pyruvic acid. Validation was carried out, as per European Medicines Agency (EMA) “guidelines on bioanalytical method validation”, for linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LLOQ), recovery, matrix effect, and stability. The recoveries from serum and tissue were 79−119% and 77−223%, 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 µmol/L), <span style="font-variant: small-caps;">l</span>-alanine (302.4 µmol/L), and serine (92.9 µmol/L). |
| first_indexed | 2024-04-12T09:25:56Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 2218-1989 |
| language | English |
| last_indexed | 2024-04-12T09:25:56Z |
| publishDate | 2020-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Metabolites |
| spelling | doaj.art-66cdc19de30f4db181229ce50cc337c22022-12-22T03:38:30ZengMDPI AGMetabolites2218-19892020-03-0110310310.3390/metabo10030103metabo10030103Simultaneous 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 TissueRamji Rathod0Bharat Gajera1Kenneth Nazir2Janne Wallenius3Vidya Velagapudi4Metabolomics Unit, Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Tukholmankatu 8, Biomedicum 2U, 00290 Helsinki, FinlandMetabolomics Unit, Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Tukholmankatu 8, Biomedicum 2U, 00290 Helsinki, FinlandMetabolomics Unit, Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Tukholmankatu 8, Biomedicum 2U, 00290 Helsinki, FinlandMetabolomics Unit, Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Tukholmankatu 8, Biomedicum 2U, 00290 Helsinki, FinlandMetabolomics Unit, Institute for Molecular Medicine Finland (FIMM), HiLIFE, University of Helsinki, Tukholmankatu 8, Biomedicum 2U, 00290 Helsinki, FinlandThe 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 × 100 mm, particle size 3-μm 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> > 0.980) for malic acid; 11.72 to 12,000 ng/mL (r<sup>2</sup> > 0.980) for cis-aconitic acid and L-aspartic acid; 29.30 to 30,000 ng/mL (r<sup>2</sup> > 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> > 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> > 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> > 0.980) for <span style="font-variant: small-caps;">d</span>-glucose; 23.44 to 24,000 ng/mL (r<sup>2</sup> > 0.980) for fumaric acid and succinic acid; and, 244.14 to 250,000 ng/mL (r<sup>2</sup> > 0.980) for pyruvic acid. Validation was carried out, as per European Medicines Agency (EMA) “guidelines on bioanalytical method validation”, for linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LLOQ), recovery, matrix effect, and stability. The recoveries from serum and tissue were 79−119% and 77−223%, 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 µmol/L), <span style="font-variant: small-caps;">l</span>-alanine (302.4 µmol/L), and serine (92.9 µmol/L).https://www.mdpi.com/2218-1989/10/3/103tricarboxylic acid (tca) cyclemass spectrometrychromatographymetabolitesbioanalytical method validation |
| spellingShingle | Ramji Rathod Bharat Gajera Kenneth Nazir Janne Wallenius Vidya Velagapudi 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 tricarboxylic acid (tca) cycle mass spectrometry chromatography metabolites bioanalytical method validation |
| 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | tricarboxylic acid (tca) cycle mass spectrometry chromatography metabolites bioanalytical method validation |
| url | https://www.mdpi.com/2218-1989/10/3/103 |
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