Original Article
Volume: 2 | Issue: 2 | Published: Jan 23, 2020 | Pages: 93 - 98 | DOI: 10.24911/JBCGenetics/183-1563341940
Analysis of tricarboxylic acid cycle intermediates in dried blood spots by ultraperformance liquid chromatography-tandem mass spectrometry
Authors:
Lamia Alhammadi
, Maitha Aal Abdulla
, Naila Ahli
, Nahid Al Dhahouri
, Anas Al Aidaros
, Fatma Al-Jasmi
, Osama Y. Al-Dirbashi
Article Info
Authors
Lamia Alhammadi
UAE University, Al-Ain, United Arab Emirates
Maitha Aal Abdulla
UAE University, Al-Ain, United Arab Emirates
Naila Ahli
UAE University, Al-Ain, United Arab Emirates
Nahid Al Dhahouri
UAE University, Al-Ain, United Arab Emirates
Anas Al Aidaros
UAE University, Al-Ain, United Arab Emirates
Fatma Al-Jasmi
UAE University, Al-Ain, United Arab Emirates, Tawam Hospital, Al-Ain, United Arab Emirates
Osama Y. Al-Dirbashi
UAE University, Al-Ain, United Arab Emirates, University of Ottawa, Ottawa, ON, Canada, Children's Hospital of Eastern Ontario, Ottawa, ON, Canada
Publication History
Received: July 17, 2019
Revised: November 22, 2019
Accepted: December 12, 2019
Published: January 23, 2020
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.
Keywords: Tricarboxylic acid cycle, citric acid, 2-ketoglutaric acid, succinic acid, fumaric acid, malic acid, DBS, LC-MS/MS, DAABD-AE
