Application of Capillary Electrophoresis with Laser-Induced Fluorescence Detection for Determination of Methylmalonic Acid in Human Serum

Abstract

Methylmalonic acid (MMA) in serum is an established marker of cobalamin deficiency. MMA and other short-chain dicarboxylic acids react with 1-pyrenyldiazomethane to form stable, highly fluorescent 1-pyrenylmethyl monoesters. We have analyzed these esters in human blood by capillary electrophoresis (CE) combined with laser-induced fluorescence detection, and here we describe our approach to achieve long-term reproducibility, which is a prerequisite for routine clinical application. To stabilize CE performance and to minimize solute adsorption to the capillary wall, we coated capillaries with linear polyacrylamide, used hydroxypropyl methylcellulose and dimethylformamide as buffer additives, and extensively diluted derivatized samples prior to injection. A discontinuous buffer system was used for sample stacking. Separation was performed in Tris-citrate buffer, pH 6.4, under reversed polarity conditions (negative potential at the inlet vial). The assay was linear for serum MMA concentrations in the range 0.1-200 mumol/L, the total run time was 26 min, the sample output was about 50 samples/24 h, and the coefficients of variation ranged between 3 and 12%, depending on the MMA concentration. Comparison of our assay with two established GC/MS methods demonstrated good correlation and measuring agreement.

Quantification of Methylmalonic Acid in Human Plasma with Hydrophilic Interaction Liquid Chromatography Separation and Mass Spectrometric Detection

“…First, MMA has 2 pK a values in an aqueous matrix (pK a1 ϭ 2.83; pK a2 ϭ 5.69), and the difference between these pK a values is considerably greater than for SA (pK a1 ϭ 4.19; pK a2 ϭ 5.57). From these pK a values for the 2 carboxylic groups of MMA and SA, we expected a mobile-phase pH in the interval between pH 3 and pH 5 to be optimal for separation (10,26 ). pK a values may change in mixtures of aqueous and organic solvents, however, and are difficult to predict (27 ).…”

“…The different methods that have been developed for measuring MMA in serum, plasma, urine, and cerebrospinal fluid include GC-MS, liquid chromatographytandem mass spectrometry (LC-MS/MS), HPLC, and capillary electrophoresis (7)(8)(9)(10). The main obstacles to overcome are related to the low physiological concentrations of MMA in human serum (11 ) and the fact that MMA is a hydrophilic, nonvolatile compound.…”

“…Current methods require extraction and derivatization steps to yield MMA derivatives that are compatible with GC-MS techniques (7 ) or for increased selectivity and sensitivity in HPLC/capillary electrophoresis methods (9,10 ). More recently, investigators have introduced LC-MS/MS methods (8,11,13,14 ), in which MMA and SA derivatives may be distinguished by differences in their fragmentation patterns (8 ).…”

Relationship between methylmalonic acid, homocysteine, vitamin B12 intake and status and socio-economic indices, in a subset of participants in the British National Diet and Nutrition Survey of people aged 65 y and over

“…MMA was measured by capillary electrophoresis (Schneede & Ueland, 1995). The local reference range of the assay for non-elderly adults is 0.05 -0.26 mmol=l and the CV is below 10% for MMA concentration within the normal range.…”

Homocysteine and methylmalonic acid levels in pregnant Nepali women. Should cobalamin supplementation be considered?

“…The between-day coefficient of variation for the method is less than 5%. MMA in serum was determined by capillary electrophoresis with laser-induced fluorescence detection (Schneede & Ueland, 1995). The between-day coefficient of variation of the method is 5 -10% within the physiological range of MMA concentrations.…”