A Simple and Sensitive Method for Quantitative Measurement of Methylmalonic Acid by Turbulent Flow Chromatography and Tandem Mass Spectrometry

Abstract

Background: Methylmalonic acid (MMA) is elevated in patients with inherited defects in methylmalonyl CoA mutase or due to inherited or acquired deficiencies of vitamin B12. MMA levels are measured in serum to evaluate individuals with deficiencies of vitamin B12 or to assess children with symptoms of methylmalonic acidemia. Although methods to measure MMA by mass spectrometery have been previously developed, we utilized turbulent flow liquid chromatography (TFLC) to simplify the sample preparation procedure. The sample was prepared by precipitating serum proteins using methanol and directly injecting the supernatant into the turbulent flow column after centrifugation. The objective was to develop an MMA assay performed by TFLC-MS/MS with the following characteristics: analytically sensitive with a clinically useful dynamic range; good specificity with no cross-reactivity with succinic acid (SA); and suitable analytical transferability for a high volume clinical laboratory. Methods: Serum samples were prepared by protein precipitation using methanol containing deuterated MMA as an internal standard. TFLC-MS/MS analyses were performed on a Thermo Scientific TLX-2 HPLC system (TurboFlow technology) interfaced to a TSQ Quantum Ultra mass spectrometer operated in the negative ion ESI mode. Chromatographic separation was achieved using a Cyclone MAX TurboFlowcolumn (50 X 0.5 mm) and an Allure Organic Acids analytical column (150 X 3 mm). The HPLC elution occurred with a mobile phase composition of 50% methanol that was held for 1.3 minutes. Calibrators (6) were prepared in blank human serum. Results: The analytical measurement range (AMR) for MMA was 30-1000 nmol/L with a CV less than 20% at the lower limit of quantitation (LLOQ); the calibration curves were linear over the AMR with correlation coefficients R2>= 0.995. Dilutions of 1:2, 1:10 and 1:20 were validated giving a clinically reportable range of 30-105 nmol/L. The accuracy of the MMA assay was evaluated by comparing results of 63 residual patient specimens to the results obtained from a national reference laboratory utilizing LC-MS/MS methodology. The accuracy was further evaluated through recovery experiments. The slopes of the linear regression curves comparing the assays was +/- 1% with excellent correlation coefficients. MMA recoveries at concentrations spanning the AMR were between 96 and 111%. Within-day and between-day (N=20) CVs at concentrations spanning the AMR were less than 12%. Conclusion: We have developed an accurate and sensitive assay to measure MMA levels in serum by TFLC-MS/MS. The method showed excellent correlation with previously established mass spectrometry-based methods and has been fully validated for imprecision, accuracy, linearity, recovery, carryover, specificity and matrix effects. The assay is more simple to perform than previously published methods and has proven very accurate and robust and cost effective.