Identification and Quantitation of Malonic Acid Biomarkers of In-Born Error Metabolism by Targeted Metabolomics.
Malonic acid (MA), methylmalonic acid (MMA), and ethylmalonic acid (EMA) metabolites are implicated in various non-cancer disorders that are associated with inborn-error metabolism. In this study, we have slightly modified the published 3-nitrophenylhydrazine (3NPH) derivatization method and applied it to derivatize MA, MMA, and EMA to their hydrazone derivatives, which were amenable for liquid chromatography- mass spectrometry (LC-MS) quantitation. 3NPH was used to derivatize MA, MMA, and EMA, and multiple reaction monitoring (MRM) transitions of the corresponding derivatives were determined by product-ion experiments. Data normalization and absolute quantitation were achieved by using 3NPH derivatized isotopic labeled compounds13C2-MA, MMA-D3, and EMA-D3. The detection limits were found to be at nanomolar concentrations and a good linearity was achieved from nanomolar to millimolar concentrations. As a proof of concept study, we have investigated the levels of malonic acids in mouse plasma with malonyl-CoA decarboxylase deficiency (MCD-D), and we have successfully applied 3NPH method to identify and quantitate all three malonic acids in wild type (WT) and MCD-D plasma with high accuracy. The results of this method were compared with that of underivatized malonic acid standards experiments that were performed using hydrophilic interaction liquid chromatography (HILIC)-MRM. Compared with HILIC method, 3NPH derivatization strategy was found to be very efficient to identify these molecules as it greatly improved the sensitivity, quantitation accuracy, as well as peak shape and resolution. Furthermore, there was no matrix effect in LC-MS analysis and the derivatized metabolites were found to be very stable for longer time. Graphical Abstract ᅟ.
Metabolomics Core Facility, Molecular and Cellular Biology, Alkek Center for Molecular Discovery, Baylor College of Medicine, Houston, TX, 77030, USA.
Chandra Shekar R
CS
Ambati
Department of Biochemistry, Baylor College of Medicine, Houston, TX, 77030, USA.
Furong
F
Yuan
Department of Biochemistry, Baylor College of Medicine, Houston, TX, 77030, USA.
Lutfi A
LA
Abu-Elheiga
Metabolomics Core Facility, Molecular and Cellular Biology, Alkek Center for Molecular Discovery, Baylor College of Medicine, Houston, TX, 77030, USA.
Yiqing
Y
Zhang
Metabolomics Core Facility, Molecular and Cellular Biology, Alkek Center for Molecular Discovery, Baylor College of Medicine, Houston, TX, 77030, USA. vivekananda.shetty@bcm.edu.
Vivekananda
0000-0001-9012-4944
V
Shetty
D000818:Animals; D015415:Biomarkers / Q000097:blood / Q000378:metabolism; D002262:Carboxy-Lyases / Q000097:blood / Q000172:deficiency* / Q000378:metabolism; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008314:Malonates / Q000097:blood* / Q000378:metabolism; D008316:Malonyl Coenzyme A / Q000097:blood / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods; D008661:Metabolism, Inborn Errors / Q000097:blood* / Q000378:metabolism; D055432:Metabolomics / Q000379:methods*; D008764:Methylmalonic Acid / Q000097:blood* / Q000378:metabolism; D008810:Mice, Inbred C57BL; D010659:Phenylhydrazines / Q000737:chemistry
C523491:3-nitrophenylhydrazine; D015415:Biomarkers; D008314:Malonates; D010659:Phenylhydrazines; C038080:ethylmalonic acid; D008316:Malonyl Coenzyme A; D008764:Methylmalonic Acid; C030290:malonic acid; D002262:Carboxy-Lyases
3NPH derivatization; Inborn-error metabolism; LC-MRM-MS; Malonic acids; Plasma; Quantitation