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Original Article



Analysis of tricarboxylic acid cycle intermediates in dried blood spots by ultraperformance liquid chromatography-tandem mass spectrometry

Lamia Alhammadi, Maitha Aal Abdulla, Naila Ahli, Nahid Al Dhahouri, Anas Al Aidaros, Fatma Al-Jasmi, Osama Y. Al-Dirbashi.


Abstract

Background: We developed a novel method for measuring the concentrations of tricarboxylic acid (TCA) cycle intermediates in dried blood spots (DBS) using liquid chromatography-tandem mass spectrometry (LC-MS/ MS). Analytes were derivatized before analysis using 4-[2-(N,N-dimethylamino) ethylaminosulfonyl]-7-(2-aminoethylamino)-2,1,3-benzoxa-diazole (DAABD-AE), a reagent that imparts powerful chromatographic and mass spectrometric properties onto carboxyl group-containing analytes. Methodology: Extraction and derivatization of TCA cycle intermediates were achieved in a single step by incubating a 3.2 mm circle of the DBS samples with DAABD-AE for 1 hour at 65°C. From the resultant mixture, 1.0 µl was injected into the LC-MS/MS. Results: Total analytical run time to separate target analytes from other interfering components in the sample was 8 minutes. The peaks corresponding to malic, fumaric, citric, succinic, and 2-ketoglutaric acid appeared at 3.55, 3.62, 3.64, 3.67, and 3.68 minutes, respectively. The method was adequately reproducible with a coefficient of variation for intraday (n = 15) and inter-day (n = 13) studies of 5.2%–18.4%. Reference intervals in DBS from controls (n = 125) were as follow (µmol/l): citric (36.6–126.4), 2-ketoglutaric (9.1–42.1), succinic (1.2–2.4), fumaric (2.4–9.0), and malic acid (15.9–39.3). Compared to controls, the levels of citric, succinic, and malic acids were statistically different in patients (n = 7) with a p-value of< 0.05. No statistically significant difference was detected in concentrations of 2-ketoglutaric and fumaric acids. Conclusion: We describe a simple, quick, and sensitive method to measure TCA cycle intermediates in DBS samples. That TCA cycle plays a central role in cellular metabolism; this method should be useful in studying these metabolites in health and disease.

Key words: Tricarboxylic acid cycle, citric acid, 2-ketoglutaric acid, succinic acid, fumaric acid, malic acid, DBS, LC-MS/MS, DAABD-AE.






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