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. 2022 Sep 20;12(10):2273.
doi: 10.3390/diagnostics12102273.

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

Lizi Jin  1   2 , Zhenni Liu  1   2 , Weiyan Zhou  1   2 , Jie Zeng  1 , Minhang Wu  3 , Yu Zhang  3 , Tianjiao Zhang  1   2 , Falin He  1   2 , Chuanbao Zhang  1   2
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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

Lizi Jin et al. Diagnostics (Basel). .

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.

Keywords: liquid chromatography–tandem mass spectrometry; method improvements; methylmalonic acid; vitamin B12 deficiency.

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Conflict of interest statement

The authors state no conflicts of interest.

Figures

Figure 1
Figure 1
Optimization results of reaction systems in sample preparation.
Figure 2
Figure 2
Representative chromatographs of: (A) calibrators; (B) healthy controls; (C) patients.
Figure 3
Figure 3
Methylmalonic acid levels in clinical subjects.
See this image and copyright information in PMC

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Grants and funding

The National Key Research and Development Program of China (2021YFC1005304 and 2018YFC1002204).

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