Paper Information

Journal:   REPORTS OF BIOCHEMISTRY AND MOLECULAR BIOLOGY   APRIL 2016 , Volume 4 , Number 2; Page(s) 104 To 109.
 
Paper: 

DEVELOPMENT AND VALIDATION OF A GC-FID METHOD FOR DIAGNOSIS OF METHYLMALONIC ACIDEMIA

 
 
Author(s):  KEYFI FATEMEH, VARASTEH ABDOLREZA*
 
* PARDIS CLINICAL AND GENETIC LABORATORY, 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 60oC with a 30 min reaction time. Standard solutions and derivatized samples were stable for 7 days at 4-8oC. 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 R2=0.997.
Conclusions: 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.

 
Keyword(s): GAS CHROMATOGRAPHY/FLAME IONIZATION DETECTOR (GC-FID), METHOD DEVELOPMENT, METHYLMALONIC ACIDEMIA DISORDER, URINE ORGANIC ACID ANALYSIS
 
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