A Rapid, Simple, Trace, Cost-Effective, and High-Throughput Stable Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry Method for Serum Methylmalonic Acid Quantification and Its Clinical Applications

HIGHLIGHTS: What are the main findings? An established method for trace, simple, rapid, cheap, sensitive, accurate, robust, and high-throughput for methylmalonic acid quantification. Good chromatographic separation of MMA and its intrinsic isomer and good signals of MMA were achieved using a simple isocratic elution strategy. Materials and reagents that are complex or not always accessible and procedures in previous methods such as derivatization, multistep SPE, incubations, evaporations, drying, or reconstitutions are not required in this MMA quantification method. What is the implication of the main finding? This method is suitable for large-scale MMA testing. ABSTRACT: Background: Methylmalonic acid (MMA) is an essential indicator of vitamin B12 (VB12) deficiency and inherited metabolic disorders (IMDs). The increasing number of requests for MMA testing call for higher requirements for convenient MMA testing methods. This study aims to develop a convenient quantification method for serum MMA. Methods: The method was established based on the stable isotope-dilution liquid chromatography–tandem mass spectroscopy (ID-LC-MS/MS) technique. The LC-MS/MS parameters and sample preparation were optimized. Specificity, sensitivity, robustness, accuracy, and clinical applicability were validated according to CLSI C62-A guidelines. MMA levels in VB12-sufficient subjects and VB12-deficient subjects were measured. Results: MMA and its intrinsic isomer, i.e., succinic acid (SA), were completely separated. The average slope, intercept, and correlation relationship (R) with 95% confidence intervals, during the two months, were 0.992 (0.926–1.059), −0.004 (−0.012–0.004), and 0.997 (0.995–0.999), respectively. The limit of detection and quantification were <0.058 μmol/L and 0.085 μmol/L, respectively. Intra-run, inter-run, and total imprecisions were 1.42–2.69%, 3.09–5.27%, and 3.22–5.47%, respectively. The mean spiked recoveries at the three levels were 101.51%, 92.40%, and 105.95%, respectively. The IS-corrected matrix effects were small. The VB12-deficient subjects showed higher MMA levels than VB12-sufficient subjects. Conclusions: A convenient LC-MS/MS method for serum MMA measurement was developed and validated, which could be suitable for large-scale MMA testing and evaluating MMA levels in VB12-deficient patients.