Measurement of Methylmalonic Acid, 3-OH Propionic Acid and Succinic Acid in DBS (Dried Blood Spot) with LCMS-8040

Significance of the Method

Dried blood spot analysis of methylmalonic acid (MMA), 3-hydroxypropionic acid (3-OH PA) and succinic acid (SA) provides critical insight into disorders of propionate metabolism and enzyme deficiencies. The use of liquid chromatography–tandem mass spectrometry (LC–MS/MS) enables precise quantification of these biomarkers with minimal sample volumes and high throughput.

Study Objectives and Overview

This work outlines a validated protocol for simultaneous measurement of MMA, 3-OH PA and SA in dried blood spots using a Shimadzu LCMS-8040 triple quadrupole system. The method was developed in collaboration with the Mass Spectrometry Laboratory, Meyer Children’s Hospital (Florence, Italy), and is intended to support expanded newborn screening and diagnostic research.

Methodology and Instrumentation

Sample preparation involves punching a 3.2 mm disc from each DBS, extracting with 0.05 % formic acid in water/acetonitrile (30:70) and adding deuterated 13C-MMA as an internal standard. Chromatographic separation is achieved on a Gemini C6-Phenyl column (100 × 2.0 mm, 3 µm) with a water/acetonitrile gradient containing 0.1 % formic acid at 0.2 mL/min. Detection is performed by electrospray ionization in positive mode using multiple reaction monitoring for each analyte.

Used Instrumentation

• Shimadzu LCMS-8040 triple quadrupole mass spectrometer
• Gemini C6-Phenyl column (100 mm × 2.0 mm, 3 µm)
• ESI source: +4.5 kV spray voltage, 15 L/min drying gas, 2.5 L/min nebulizing gas
• MRM transitions: MMA 116.9 > 73.1, d3-MMA 119.9 > 76.1, SA 116.9 > 73.2, 3-OH PA 89 > 58.9

Key Results and Discussion

Extracted-ion chromatograms demonstrate clear baseline separation of MMA, 3-OH PA and SA within a 5-minute run. In samples lacking enzyme activity, corresponding analyte peaks are absent. The method successfully distinguishes 3-OH PA from lactic acid despite identical MRM transitions by exploiting retention time differences.

Practical Benefits and Applications

• Rapid turnaround: 5 min per sample
• Minimal sample volume: single 3.2 mm punch
• High sensitivity and specificity for newborn screening
• Reduced false-positive rates for propionylcarnitine anomalies

Future Trends and Opportunities

Continuous advancements in high-resolution mass spectrometry and automated DBS handling promise further improvements in throughput, multiplexing and diagnostic accuracy. Expansion of target panels and integration with informatics will enhance metabolic disorder screening.

Conclusion

The described LC–MS/MS protocol provides a reliable, efficient approach for quantifying key organic acid biomarkers in dried blood spots, supporting clinical research and expanded newborn screening initiatives.

References

• la Marca G, et al. Progress in expanded newborn screening for metabolic conditions by LC-MS/MS in Tuscany: JIMD Short Report #127 (2008)
• la Marca G, et al. Rapid 2nd-Tier Test for Measurement of 3-OH-Propionic and Methylmalonic Acids on Dried Blood Spots. Clin Chem. 53:1364–1369 (2007)