Fatemeh Keyfi1, Abdolreza Varasteh2. 1. Immunobiochemistry Lab, Immunology Research Center, School of medicine, Mashhad University of Medical Sciences, Mashhad, Iran; Pardis Clinical and Genetic Laboratory, Mashhad, Iran. 2. Pardis Clinical and Genetic Laboratory, Mashhad, Iran; Immunobiochemistry Lab, Allergy Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Abstract
BACKGROUND: Urinary organic acids are water-soluble intermediates and end products of the metabolism of amino acids, carbohydrates, lipids, and a number of other metabolic processes. In the hereditary diseases known as organic acidurias, an enzyme or co-factor defect in a metabolic pathway leads to the accumulation and increased excretion of one or more of these acidic metabolites. Gas chromatography is the most commonly-used technology to separate and identify these metabolites. In this report the analytical conditions for the determination of methylmalonic acid using a gas chromatography/flame ionization detector (GC-FID) are studied with the aim to establish a method to analyze organic acids in human urine. METHODS: Studies included the GC-FID method development, the conditions of the derivatization (trimethylsilylation) reaction, and the stability of the methylmalonic acid standard solution and trimethylsilyl derivatives during storage. Also, a systematic comparison between GC-FID and gas chromatography/mass spectrometry (GC-MS) was performed. RESULTS: The highest resolution and sensitivity were obtained at 60 °C with a 30 min reaction time. Standard solutions and derivatized samples were stable for 7 days at 4-8 °C. Relative standard deviations of within-day and day-to-day assay results were less than 5%. Methylmalonic acid was detected in thirty human urine samples by the proposed GC-FID, and the results were compared with gold standard technique GC-MS. The correlation coefficient between GC-MS and GC-FID was obtained with R(2)= 0.997. CONCLUSION: The developed method was applied to the quantitative analysis of methylmalonic acid in urine from hospitalized children with methylmalonic acidemia. With this method we aim to support pediatric clinics in Iran and assist in clinical diagnostics.
BACKGROUND: Urinary organic acids are water-soluble intermediates and end products of the metabolism of amino acids, carbohydrates, lipids, and a number of other metabolic processes. In the hereditary diseases known as organic acidurias, an enzyme or co-factor defect in a metabolic pathway leads to the accumulation and increased excretion of one or more of these acidic metabolites. Gas chromatography is the most commonly-used technology to separate and identify these metabolites. In this report the analytical conditions for the determination of methylmalonic acid using a gas chromatography/flame ionization detector (GC-FID) are studied with the aim to establish a method to analyze organic acids in human urine. METHODS: Studies included the GC-FID method development, the conditions of the derivatization (trimethylsilylation) reaction, and the stability of the methylmalonic acid standard solution and trimethylsilyl derivatives during storage. Also, a systematic comparison between GC-FID and gas chromatography/mass spectrometry (GC-MS) was performed. RESULTS: The highest resolution and sensitivity were obtained at 60 °C with a 30 min reaction time. Standard solutions and derivatized samples were stable for 7 days at 4-8 °C. Relative standard deviations of within-day and day-to-day assay results were less than 5%. Methylmalonic acid was detected in thirty human urine samples by the proposed GC-FID, and the results were compared with gold standard technique GC-MS. The correlation coefficient between GC-MS and GC-FID was obtained with R(2)= 0.997. CONCLUSION: The developed method was applied to the quantitative analysis of methylmalonic acid in urine from hospitalized children with methylmalonic acidemia. With this method we aim to support pediatric clinics in Iran and assist in clinical diagnostics.