Measurement of Fumarylacetoacetate Activity in DBS (Dried Blood Spot) with LCMS-8040

Importance of the Topic

The enzymatic breakdown of fumarylacetoacetate, catalyzed by fumarylacetoacetate hydrolase, is a critical step in tyrosine catabolism. Deficiency of this enzyme leads to accumulation of toxic intermediates such as succinylacetone, a biomarker for Tyrosinemia type I. Early and reliable detection of succinylacetone in newborns is essential for prompt treatment and prevention of severe liver and renal complications. Utilizing dried blood spots (DBS) for sample collection streamlines logistics, reduces invasiveness, and supports large-scale screening programs.

Study Objectives and Overview

This application note presents a robust liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to quantify succinylacetone in DBS. The primary aim is to demonstrate the sensitivity, specificity, and rapid turnaround of the Shimadzu LCMS-8040 system for monitoring fumarylacetoacetate hydrolase activity indirectly via succinylacetone levels. Both negative (no enzyme activity) and positive controls (spiked succinylacetone) were analyzed to validate method performance.

Methodology

Sample Preparation:

• Punch a 3.2 mm disk from DBS filter paper.
• Add internal standard (13C4-succinylacetone) and 3 mM hydrazine solution.
• Extract metabolites using methanol and hydrazine at 37 °C for 25 minutes with shaking.
• Evaporate extracts under nitrogen at 45 °C.
• Reconstitute dried residues in 0.05% formic acid in water/acetonitrile (30/70, v/v).

Chromatographic and MS Conditions:
Analysis was completed in a 4-minute run using a Synergi Polar-RP column (150 × 2.0 mm, 4 µm). The mobile phase consisted of 0.1% formic acid in water (A) and methanol (B) at 80% B, 0.2 mL/min flow, and 30 °C column temperature. Multiple reaction monitoring targeted succinylacetone (m/z 154.8 > 136.9) and the 13C4 internal standard (m/z 158.9 > 141.0).

Instrumentation

• Liquid Chromatograph: Shimadzu high-performance LC system.
• Mass Spectrometer: Triple quadrupole LCMS-8040 with ESI in positive mode.
• Column: Synergi Polar-RP, 150 mm × 2 mm I.D., 4 µm.
• Interface settings: Probe voltage +4.5 kV, nebulizing gas 2.5 L/min, drying gas 15 L/min, DL temperature 250 °C, block heater 400 °C.

Results and Discussion

Extracted-ion chromatograms demonstrated clear separation and quantification of succinylacetone in DBS samples. In enzyme-deficient samples (‘Sample’), a succinylacetone peak was observed, whereas wild-type controls showed no peak. The positive control confirmed retention time and signal intensity for both analyte and internal standard. The method exhibited sufficient sensitivity to detect low nanomolar concentrations within a short analysis time, enabling high sample throughput.

Practical Benefits and Applications

• Newborn screening for Tyrosinemia type I using minimally invasive DBS sampling.
• Rapid assay time (4 min) supports large-scale clinical laboratories.
• High specificity through MRM reduces false positives.
• Stable isotope internal standard ensures accurate quantification.

Future Trends and Potential Applications

Advancements may include automation of DBS handling, integration with multiplex panels for expanded metabolic disorder screening, and application of high-resolution MS for broader metabolite coverage. Emerging microfluidic platforms could further reduce sample volume and increase assay speed. Data-driven analytics and machine learning may enhance interpretation of complex metabolic profiles.

Conclusion

The described LC-MS/MS method using DBS provides a reliable, sensitive, and rapid approach for monitoring fumarylacetoacetate hydrolase activity via succinylacetone measurement. Its adoption in newborn screening workflows can facilitate early diagnosis of Tyrosinemia type I and improve clinical outcomes.

References

1. la Marca G, et al. Progress in expanded newborn screening for metabolic conditions by LC-MS/MS in Tuscany: Update on methods to reduce false positives. JIMD Short Rep #127 (2008).
2. la Marca G, et al. Inclusion of succinylacetone as marker for tyrosinemia type I in expanded newborn screening programs. Rapid Commun Mass Spectrom. 22 (2008) 812–818.
3. la Marca G, et al. Successful use of succinylacetone as a marker of tyrosinemia type I in Tuscany newborn screening program. Rapid Commun Mass Spectrom. 23 (2009) 3891–3893.