Introduction
The screening for elevated levels of methylmalonic acid (MMA) in serum is commonly used as a clinical diagnostic indicator of Cobalamin (Vitamin B12) deficiency in humans. MMA is commonly analyzed using LC-MS/MS with or without prior derivatization. This poster summarizes various sample preparation strategies for the extraction of MMA from serum without the necessity for derivatization, prior to LC-MS/MS analysis. A range of extraction techniques of varying complexity were evaluated: protein precipitation, phospholipid depletion, supported liquid extraction and solid phase extraction using both silica and polymer-based mixed-mode anion exchange chemistries.
Method performance was evaluated for evaporative effects, assay recovery, ion suppression, phospholipid removal and simplicity of transfer to an automated sample preparation platform.
Experimental
MMA, MMA 13C4 ISTD, formic acid, ammonium hydroxide, ammonium acetate, ethylene glycol and LC/MS grade solvents were obtained from Sigma-Aldrich Chemical Co. (Poole, UK). Water (18.2 MΩ.cm) was drawn fresh daily from a Direct-Q 5 water purifier (Merck Millipore, Watford, UK). Human serum and stripped serum was kindly donated by Golden West Biologicals Inc. (Ca, USA.) and serum calibrators purchased from Chromsystems (Munich, Germany).
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