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Simple, Fast, and Simultaneous Detection of Plasma Total Homocysteine, Methylmalonic Acid, Methionine, and 2-Methylcitric Acid Using Liquid Chromatography and Mass Spectrometry (LC/MS/MS).

ABSTRACT: Cobalamin (Vitamin B12) plays an essential role both in the conversion of methylmalonyl-CoA to succinyl-CoA and in the synthesis of methionine (Met) from homocysteine (Hcy). Elevations of total homocysteine (tHcy), Met, methylmalonic acid (MMA), and 2-methylcitric acid (2MCA) are indicative of disorders in these related pathways, and can clinically present as methylmalonic acidemia, cobalamin defects or deficiency, propionic acidemia, homocystinuria, and hypermethioninemia. We have developed a fast, sensitive, and simple method for the simultaneous detection of plasma tHcy, MMA, Met, and 2MCA using liquid chromatography mass spectrometry (LC/MS/MS). All analytes were directly determined without the need of derivatization. Both positive and negative modes were used to achieve the best sensitivity and specificity. The two stereo isomers of 2MCA (2S, 3S) and (2R, 3S) were successfully separated and were designated as 2MCA1 and 2MCA2. The assays were linear up to a concentration of 800 ?Mol/l for tHcy, 2,000 ?Mol/l for Met, 80 ?Mol/l for MMA, 40 ?Mol/l for 2MCA1, and 40 ?Mol/l for 2MCA2 (80 ?Mol/l for total 2MCA), respectively. The recovery was between 84.42 % and 120.05 %. The intra-assay coefficient of variations (CVs) ranged from 2.1 % to 6.9 % (n = 20), and the inter-assay CVs ranged from 2.7 % to 11.6 % (n = 20). Reference intervals were established and verified (n = 125). A total of 15 patients with variable disorders in related pathway were successfully confirmed. The assay can be performed either in diagnostic laboratories or as second-tier, follow-up test in newborn screening laboratories.A fast, sensitive, and simple LC/MS/MS method was developed successfully for the simultaneous detection of plasma total homocysteine, methylmalonic acid, methionine, and 2-methylcitric acid for diagnosis of disorders in related pathways.

SUBMITTER: Fu X

PROVIDER: S-EPMC3755585 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Simple, Fast, and Simultaneous Detection of Plasma Total Homocysteine, Methylmalonic Acid, Methionine, and 2-Methylcitric Acid Using Liquid Chromatography and Mass Spectrometry (LC/MS/MS).

Fu Xiaowei X Xu Yan-Kang YK Chan Penny P Pattengale Paul K PK

JIMD reports 20130215

Cobalamin (Vitamin B12) plays an essential role both in the conversion of methylmalonyl-CoA to succinyl-CoA and in the synthesis of methionine (Met) from homocysteine (Hcy). Elevations of total homocysteine (tHcy), Met, methylmalonic acid (MMA), and 2-methylcitric acid (2MCA) are indicative of disorders in these related pathways, and can clinically present as methylmalonic acidemia, cobalamin defects or deficiency, propionic acidemia, homocystinuria, and hypermethioninemia. We have developed a f ...[more]

PMID: 23430805

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Project description:Background and aimsIncreased propionylcarnitine levels in newborn screening are indicative for a group of potentially severe disorders including propionic acidemia (PA), methylmalonic acidemias and combined remethylation disorders (MMACBL). This alteration is relatively non-specific, resulting in the necessity of confirmation and differential diagnosis in subsequent tests. Thus, we aimed to develop a multiplex approach for concurrent determination of 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid from the same dried blood spot (DBS) as in primary screening (second-tier test). We also set out to validate the method using newborn and follow-up samples of patients with confirmed PA or MMACBL.MethodsThe assay was developed using liquid chromatography-tandem mass spectrometry and clinically validated with retrospective analysis of DBS samples from PA or MMACBL patients.ResultsReliable determination of all three analytes in DBSs was achieved following simple and fast (<20 min) sample preparation without laborious derivatization or any additional pipetting steps. The method clearly distinguished the pathological and normal samples and differentiated between PA and MMACBL in all stored newborn specimens. Methylcitric acid was elevated in all PA samples; 3-hydroxypropionic acid was also high in most cases. Methylmalonic acid was increased in all MMACBL specimens; mostly together with methylcitric acid.ConclusionsA liquid chromatography-tandem mass spectrometry assay allowing simultaneous determination of the biomarkers 3-hydroxypropionic acid, methylmalonic acid and methylcitric acid in DBSs has been developed. The assay can use the same specimen as in primary screening (second-tier test) which may reduce the need for repeated blood sampling. The presented preliminary findings suggest that this method can reliably differentiate patients with PA and MMACBL in newborn screening. The validated assay is being evaluated prospectively in a pilot project for extension of the German newborn screening panel (‟Newborn screening 2020"; Newborn Screening Center, University Hospital Heidelberg).

Dataset Information

Simple, Fast, and Simultaneous Detection of Plasma Total Homocysteine, Methylmalonic Acid, Methionine, and 2-Methylcitric Acid Using Liquid Chromatography and Mass Spectrometry (LC/MS/MS).

Publications

Simple, Fast, and Simultaneous Detection of Plasma Total Homocysteine, Methylmalonic Acid, Methionine, and 2-Methylcitric Acid Using Liquid Chromatography and Mass Spectrometry (LC/MS/MS).

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