Development and Validation of a GC-FID Method for Diagnosis of Methylmalonic Acidemia
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...
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| Format: | Article |
| Language: | English |
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Varastegan Institute for Medical Sciences
2016-05-01
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| Series: | Reports of Biochemistry and Molecular Biology |
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| Online Access: | http://rbmb.net/article-1-78-en.pdf |
| _version_ | 1811261536216285184 |
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| author | Fatemeh Keyfi Abdolreza Varasteh |
| author_facet | Fatemeh Keyfi Abdolreza Varasteh |
| author_sort | Fatemeh Keyfi |
| collection | DOAJ |
| description | 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 oC with a 30 min reaction time. Standard solutions and derivatized samples were stable for 7 days at 4-8 oC. 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. |
| first_indexed | 2024-04-12T19:06:12Z |
| format | Article |
| id | doaj.art-00f0f11344f7439a858fe3def59c7220 |
| institution | Directory Open Access Journal |
| issn | 2322-3480 2322-3480 |
| language | English |
| last_indexed | 2024-04-12T19:06:12Z |
| publishDate | 2016-05-01 |
| publisher | Varastegan Institute for Medical Sciences |
| record_format | Article |
| series | Reports of Biochemistry and Molecular Biology |
| spelling | doaj.art-00f0f11344f7439a858fe3def59c72202022-12-22T03:20:00ZengVarastegan Institute for Medical SciencesReports of Biochemistry and Molecular Biology2322-34802322-34802016-05-0142104109Development and Validation of a GC-FID Method for Diagnosis of Methylmalonic AcidemiaFatemeh Keyfi0Abdolreza Varasteh1Immunobiochemistry Lab, Immunology Research Center, School of medicine, Mashhad University of Medical Sciences, Mashhad, Iran - Pardis Clinical and Genetic Laboratory, Mashhad, IranPardis Clinical and Genetic Laboratory, Mashhad, Iran - Immunobiochemistry Lab, Allergy Research Center, Mashhad University of Medical Sciences, Mashhad, IranBackground: 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 oC with a 30 min reaction time. Standard solutions and derivatized samples were stable for 7 days at 4-8 oC. 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.http://rbmb.net/article-1-78-en.pdfGas chromatography/Flame Ionization Detector (GC-FID)Method developmentMethylmalonic acidemia disorderUrine organic acid analysis |
| spellingShingle | Fatemeh Keyfi Abdolreza Varasteh Development and Validation of a GC-FID Method for Diagnosis of Methylmalonic Acidemia Reports of Biochemistry and Molecular Biology Gas chromatography/Flame Ionization Detector (GC-FID) Method development Methylmalonic acidemia disorder Urine organic acid analysis |
| title | Development and Validation of a GC-FID Method for Diagnosis of Methylmalonic Acidemia |
| title_full | Development and Validation of a GC-FID Method for Diagnosis of Methylmalonic Acidemia |
| title_fullStr | Development and Validation of a GC-FID Method for Diagnosis of Methylmalonic Acidemia |
| title_full_unstemmed | Development and Validation of a GC-FID Method for Diagnosis of Methylmalonic Acidemia |
| title_short | Development and Validation of a GC-FID Method for Diagnosis of Methylmalonic Acidemia |
| title_sort | development and validation of a gc fid method for diagnosis of methylmalonic acidemia |
| topic | Gas chromatography/Flame Ionization Detector (GC-FID) Method development Methylmalonic acidemia disorder Urine organic acid analysis |
| url | http://rbmb.net/article-1-78-en.pdf |
| work_keys_str_mv | AT fatemehkeyfi developmentandvalidationofagcfidmethodfordiagnosisofmethylmalonicacidemia AT abdolrezavarasteh developmentandvalidationofagcfidmethodfordiagnosisofmethylmalonicacidemia |