High-sensitivity, high-throughput quantitation of catecholamine metabolites in urine by LC/MS/MS for clinical research

Significance of the Topic

Urinary catecholamine metabolites such as vanillylmandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxyindolacetic acid (5-HIAA) serve as non-invasive biomarkers for neurotransmitter turnover and are widely employed in clinical research. Their elevated excretion levels compared with parent catecholamines, combined with simple sample collection, make them attractive targets for high-throughput analysis.

Study Objectives and Overview

This study aimed to develop and validate a rapid and sensitive LC/MS/MS method using a triple quadrupole system (Shimadzu LCMS-8060) to quantify urinary VMA, HVA and 5-HIAA. Key goals included minimizing sample preparation time, achieving robust linearity across clinically relevant concentration ranges, and demonstrating concordance with conventional HPLC analysis.

Methodology and Sample Processing

A dilute-and-shoot approach was adopted. Urine (20 μL) was mixed with 980 μL of methanol containing deuterated internal standards, vortexed, and directly injected (1 μL) into the LC/MS/MS system. The workflow is amenable to robotic automation, reducing manual handling and increasing throughput.

Analytical Conditions

• Chromatography: Shim-pack GISS C18 column (100×2.1 mm, 3 μm) at 40 °C.
• Mobile phase: A = 0.05% acetic acid in water; B = methanol; gradient from 2% to 100% B in 2.5 min; flow rate 0.6 mL/min.
• Mass spectrometry: Negative-ion MRM transitions for each analyte and its deuterated analog; collision energies optimized for sensitivity.

Used Instrumentation

• Shimadzu LCMS-8060 triple quadrupole mass spectrometer
• Shim-pack GISS C18 HPLC column (100 mm × 2.1 mm, 3 μm)
• Automated liquid handling compatible deep-well plates and autosampler

Main Results and Discussion

• Calibration curves for VMA, HVA and 5-HIAA were linear over 0.1–77.5 mg/L with R² ≥ 0.9994.
• Limits of quantitation in urine were in the low ng/mL range, with precision (%RSD) and accuracy (bias) within ±15% (<20% at LLOQ).
• Endogenous levels in human urine (6.2, 7.7 and 5.1 mg/L for VMA, HVA, 5-HIAA) were quantified without interference, demonstrating method selectivity.
• Pre-validation with matrix calibration showed accuracy of 87–103% and inter-assay precision <3.3% across spiked levels.
• Comparison with conventional HPLC on 180 urine samples yielded strong agreement (slopes ≈1.05–1.09, R² ≥ 0.96), confirming quantitative equivalence.

Benefits and Practical Applications

The developed workflow enables:

• Three-minute total analysis time per sample, facilitating high throughput.
• Minimal sample preparation without solid-phase extraction, reducing cost and turnaround.
• High sensitivity and a wide dynamic range suitable for both research and potential clinical screening.

Future Trends and Applications

Emerging opportunities include:

• Integration with automated sample preparation platforms for large-scale epidemiological studies.
• Expansion to additional neurochemical metabolites for multiplexed biomarker panels.
• Adapting the method for dried urine spots or microfluidic sampling to support remote or point-of-care testing.

Conclusion

A robust, high-throughput LC/MS/MS method has been established for precise quantitation of urinary VMA, HVA and 5-HIAA. The protocol combines rapid dilute-and-shoot preparation with sensitive MRM detection, delivering performance comparable to HPLC while significantly reducing analysis time and manual labor.