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% v/v) and acetonitrile. Linearity was clearly seen over a calibration range of: 6.25 to 6400 ng/mL (r² > 0.980) for malic acid; 11.72 to 12,000 ng/mL (r² > 0.980) for cis-aconitic acid and L-aspartic acid; 29.30 to 30,000 ng/mL (r² > 0.980) for isocitric acid, l-serine, and l-glutamic acid; 122.07 to 125,000 ng/mL (r² > 0.980) for citric acid, glycine, oxo-glutaric acid, l-alanine, and l-glutamine; 527.34 to 540,000 ng/mL (r² > 0.980) for l-lactic acid; 976.56 to 1,000,000 ng/mL (r² > 0.980) for d-glucose; 23.44 to 24,000 ng/mL (r² > 0.980) for fumaric acid and succinic acid; and, 244.14 to 250,000 ng/mL (r² > 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 (n = 6) of glycine (246.4 µmol/L), l-alanine (302.4 µmol/L), and serine (92.9 µmol/L).

中文翻译：

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液相色谱-串联质谱法同时测量不同生物基质中的三羧酸循环中间体 人血清，血浆，Kasumi-1细胞和小鼠肝组织中TCA循环中间体的定量和比较

三羧酸（TCA）循环是碳和能量代谢的核心部分，也与糖酵解，氨基酸和脂质代谢有关。由于对细胞和组织中中心碳代谢谱的兴趣，在一种方法中对TCA循环中间体的定量是有利可图的。另外，已经发现血清中的TCA循环中间体作为生物标志物对应于各种潜在的病理状况。在这项工作中，开发并验证了一种基于液相色谱-质谱/质谱的定量方法，该方法利用了快速，特异，灵敏且经济高效的沉淀萃取技术。使用Atlantis dC18 2.1 mm×100 mm，可实现色谱分离，v / v）和乙腈。在苹果酸的6.25至6400 ng / mL（r ² > 0.980）的校准范围内可以清楚地看到线性。顺式乌头酸和L-天门冬氨酸11.72至12,000 ng / mL（r ² > 0.980）; 异柠檬酸，1-丝氨酸和1-谷氨酸为29.30至30,000 ng / mL（r ² > 0.980）; 柠檬酸，甘氨酸，氧代戊二酸，1-丙氨酸和1-谷氨酰胺为122.07至125,000 ng / mL（r ² > 0.980）; 1-乳酸为527.34至540,000 ng / mL（r ² > 0.980）；976.56至1,000,000 ng / mL（r ²d-葡萄糖> 0.980）; 富马酸和琥珀酸为23.44至24,000 ng / mL（r ² > 0.980）; 丙酮酸为244.14至250,000 ng / mL（r ² > 0.980）。根据欧洲药品管理局（EMA）的“生物分析方法验证指南”进行了线性，精密度，准确性，检出限（LOD），定量限（LLOQ），回收率，基质效应和稳定性的验证。血清和组织的回收率分别为79–119％和77–223％。使用这种方法，我们测量了血清，血浆（NIST 1950 SRM）和小鼠肝脏样品中的TCA中间体。NIST SRM 1950（n = 6）中甘氨酸（246.4 µmol / L）的浓度，l-丙氨酸（302.4 µmol / L）和丝氨酸（92.9 µmol / L）。