Methylmalonic acid quantification in low serum volumes by UPLC-MS/MS

Area of Science:

• Biochemistry
• Clinical Chemistry
• Analytical Chemistry

Background:

• Methylmalonic acid (MMA) is a key biomarker for assessing vitamin B12 status.
• Current MMA measurement methods require large serum volumes (100-1000 μL), limiting their use in sample-limited studies like those involving neonates and infants.

Purpose of the Study:

• To develop and validate a sensitive ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for measuring MMA in small serum volumes (25 μL).
• To enable accurate assessment of functional vitamin B12 status in sample-limited populations.

Main Methods:

• Modified LC-MS/MS protocols for MMA measurement using n-butyl esters and MMA-d(3) as an internal standard.
• Scaled down sample processing volumes and optimized UPLC-MS/MS analysis with an isocratic mobile phase for rapid separation (<4 min).
• Incorporated 1-cyclohexyl-urido-3-dodecanoic acid (CUDA) as an additional internal standard for MMA recovery assessment.

Main Results:

• Successfully measured MMA in 25 μL of human serum using water-based calibration standards, eliminating the need for plasma-based solutions.
• Achieved high signal-to-noise ratios (>100:1) for low normal MMA concentrations, indicating potential for even smaller sample volumes (as low as 5 μL).
• Demonstrated excellent assay performance with intra- and inter-assay variability under 10%, high MMA-d(3) surrogate recovery (93±14%), and MMA stability exceeding three years with minimal impact from freeze/thaw cycles.

Conclusions:

• A robust and sensitive UPLC-MS/MS method for MMA quantification in 25 μL of serum has been established.
• This method is suitable for assessing vitamin B12 status in sample-limited scenarios, including neonatal and infant nutritional studies.
• The assay's sensitivity allows for potential application in studies requiring extremely small sample volumes.