Equipped to Succeed

Methylmalonic acid (MMA) is a biomarker for vitamin B12 deficiency. This application note describes a fast, simple, and sensitive method to detect MMA in plasma that uses a zwitterionic hydrophilic interaction chromatography (ZIC®-HILIC) column with LC-MS or LC-MS/MS. Introduction Methylmalonic acid (MMA) levels in serum, plasma and urine are used to monitor cobalamin (vitamin B12) deficiency1 and methylmalonic acidemia. Different methods have been developed to quantify MMA in biological samples, including GC-MS, LC-MS/MS, HPLC, and capillary electrophoresis (CE). The main challenges that must be overcome for accurate measurement are the low physiological concentrations of MMA in human serum (100-500 nM), and the fact that MMA is a hydrophilic non-volatile compound. Retention and separation of MMA on reversed phase liquid chromatographic columns is difficult since MMA is poorly retained, and the structural isomer, succinic acid (SA), causes ion suppression because the concentration SA in serum is usually considerably higher than MMA. Many laboratories have adopted protocols that require extraction of MMA plus steps to yield MMA-derivatives that are compatible with GC-MS or LC-MS/MS techniques using reversed phase columns2. This way, derivatives of MMA and SA may be differentiated due to their different fragmentation patterns. As a consequence however, the cost per MMA-test is usually considerably higher than standard immunological assays for B12. HILIC columns efficiently separate polar hydrophilic compounds, which are not retained on reversed phase columns. The base material of HILIC columns can be either silica or polymer, and may be modified with different types of polar functionalities such as zwitterionic sulfoalkylbetaine (ZIC®-HILIC column). Because of its highly polar nature, MMA is retained on a ZIC®-HILIC column without the need to generate MMAderivatives, making the workflow simpler, easier and faster3. This report describes a sensitive LC-MS/MS method to measure MMA using a ZIC®-HILIC column. Experimental Conditions Chromatography Conditions