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Articles |
1
Biochemical Genetics Laboratory, Department of Laboratory Medicine & Pathology, Mayo Clinic, 200 First St. SW, Rochester, MN 55905.
a Author for correspondence. Fax 507-266-2888; e-mail rinaldo{at}mayo.edu
Background: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) with electrospray ionization is robust and allows accurate measurement of both low- and high-molecular weight components of complex mixtures. We developed a LC-MS/MS method for the analysis of methylmalonic acid (MMA), a biochemical marker for inherited disorders of propionate metabolism and acquired vitamin B12 deficiency.
Methods: We added 1 nmol of the internal standard MMA-d3 to 500 µL of plasma or 100 µL of urine before solid-phase extraction. After elution with 18 mol/L formic acid, the eluate was evaporated, and butyl ester derivatives were prepared with 3 mol/L HCl in n-butanol at 65 °C for 15 min. For separation, we used a Supelcosil LC-18, 33 x 4.6 mm column with 60:40 (by volume) acetonitrile:aqueous formic acid (1 g/L) as mobile phase. The transitions m/z 231 to m/z 119 and m/z 234 to m/z 122 were used in the selected reaction monitoring mode for MMA and MMA-d3, respectively. The retention time of MMA was 2.2 min in a 3.0-min analysis, without interference of a physiologically more abundant isomer, succinic acid.
Results: Daily calibrations between 0.25 and 8.33 nmol in 0.5 mL exhibited consistent linearity and reproducibility. At a plasma concentration of 0.12 µmol/L, the signal-to-noise ratio for MMA was 40:1. The regression equation for our previous gas chromatography-mass spectrometry (GC-MS) method (y) and the LC-MS/MS method (x) was: y = 1.030x - 0.032 (Sy|x = 1.03 µmol/L; n = 106; r = 0.994). Inter- and intraassay CVs were 3.8–8.5% and 1.3–3.4%, respectively, at mean concentrations of 0.13, 0.25, 0.60, and 2.02 µmol/L. Mean recoveries of MMA added to plasma were 96.9% (0.25 µmol/L), 96.0% (0.60 µmol/L), and 94.8% (2.02 µmol/L). One MS/MS system used only overnight (7.5 h) replaced two GC-MS systems (30 instrument-hours/day) to run 100–150 samples per day, with reductions of total cost (supplies plus equipment), personnel, and instrument time of 59%, 14%, and 75%, respectively.
Conclusions: This method is well suited for large-scale MMA
testing (
100 samples per day) where a shorter analytical time is
highly desirable. Reagents are less expensive than the
anion-exchange/cyclohexanol-HCl method, and sample preparation of
batches up to 100 specimens is completed in less than 8 h and is
automated.
The following articles in journals at HighWire Press have cited this article:
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  H.-A. Lakso, P. Appelblad, and J. Schneede Quantification of Methylmalonic Acid in Human Plasma with Hydrophilic Interaction Liquid Chromatography Separation and Mass Spectrometric Detection Clin. Chem., December 1, 2008; 54(12): 2028 - 2035. [Abstract] [Full Text] [PDF]
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G. la Marca, S. Malvagia, E. Pasquini, M. Innocenti, M. A. Donati, and E. Zammarchi Rapid 2nd-Tier Test for Measurement of 3-OH-Propionic and Methylmalonic Acids on Dried Blood Spots: Reducing the False-Positive Rate for Propionylcarnitine during Expanded Newborn Screening by Liquid Chromatography-Tandem Mass Spectrometry Clin. Chem., July 1, 2007; 53(7): 1364 - 1369. [Abstract] [Full Text] [PDF]
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A. Schmedes and I. Brandslund Analysis of methylmalonic Acid in plasma by liquid chromatography-tandem mass spectrometry. Clin. Chem., April 1, 2006; 52(4): 754 - 757. [Abstract] [Full Text] [PDF]
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 A. L. Bjorke Monsen and P. M. Ueland Homocysteine and methylmalonic acid in diagnosis and risk assessment from infancy to adolescence Am. J. Clinical Nutrition, July 1, 2003; 78(1): 7 - 21. [Abstract] [Full Text] [PDF]
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M. M. Kushnir, G. Komaromy-Hiller, B. Shushan, F. M. Urry, and W. L. Roberts Analysis of Dicarboxylic Acids by Tandem Mass Spectrometry. High-Throughput Quantitative Measurement of Methylmalonic Acid in Serum, Plasma, and Urine Clin. Chem., November 1, 2001; 47(11): 1993 - 2002. [Abstract] [Full Text] [PDF]
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R. E. Ford, M. J. Magera, K. M. Kloke, P. A. Chezick, A. Fauq, and J. P. McConnell Quantitative Measurement of Porphobilinogen in Urine by Stable-Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry Clin. Chem., September 1, 2001; 47(9): 1627 - 1632. [Abstract] [Full Text] [PDF]
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R. M. Kisabeth Laboratory Adaptations--Changing Expectations Clin. Chem., August 1, 2001; 47(8): 1509 - 1515. [Full Text] [PDF]
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 D. Matern and M. J. Magera Mass Spectrometry Methods for Metabolic and Health Assessment J. Nutr., May 1, 2001; 131(5): 1615S - 1620. [Abstract] [Full Text]
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 P. C. Kao, D. A. Machacek, M. J. Magera, J. M. Lacey, and P. Rinaldo Diagnosis of Adrenal Cortical Dysfunction by Liquid Chromatography-Tandem Mass Spectrometry Ann. Clin. Lab. Sci., April 1, 2001; 31(2): 199 - 204. [Abstract] [Full Text] [PDF]
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