Simultaneous Analysis of Amino Acids and Acylcarnitines in DBS (Dried Blood Spot) with LCMS-8040

Importance of Amino Acid and Acylcarnitine Analysis

Analysis of amino acids and acylcarnitines in dried blood spots serves as a cornerstone for newborn screening and metabolic disorder monitoring. Simultaneous measurement of these biomarkers provides critical insight into amino acid metabolism and fatty acid oxidation, enabling early detection of inherited metabolic diseases and guiding timely therapeutic interventions.

Objectives and Study Overview

This application note describes a streamlined protocol for the simultaneous quantitation of multiple amino acids and acylcarnitines using a triple quadrupole LC-MS/MS system. Developed by the Clinical Chemistry and Pharmacology Unit of Meyer Children’s Hospital, the study aimed to validate sample preparation, chromatographic separation, and mass spectrometric detection conditions that deliver high sensitivity and throughput for clinical research applications.

Methodology and Instrumentation

Sample Preparation

• Use 3.2 mm discs punched from dried blood spot cards into a 96-well plate
• Extract with methanolic solution containing stable-isotope internal standards
• Derivatize with hydrazine solution at 37 °C for 25 minutes
• Evaporate under nitrogen and reconstitute in 0.05% formic acid in water/acetonitrile (30/70)

Chromatographic Conditions

• Mobile phases: 0.1% formic acid in water (A) and in acetonitrile (B)
• Gradient or isocratic ratio: 30:70 A:B
• Flow rate: 0.07 mL/min; injection volume: 40 µL; analysis time: 2.2 minutes

Mass Spectrometric Conditions

• Ionization mode: positive electrospray (ESI+), probe voltage 4.5 kV
• Nebulizing gas: 3.0 L/min; drying gas: 20.0 L/min
• DL temperature: 300 °C; block heater: 500 °C
• Acquisition modes: neutral loss scanning for amino acids, precursor ion scanning for acylcarnitines, and multiple reaction monitoring for selected analytes

Main Results and Discussion

Control samples analyzed with the neonate quality control solution produced concentrations within predefined notice limits, confirming method accuracy. Seven clinical samples (A–G), each representing a distinct metabolic disorder, exhibited characteristic deviations in key analytes and metabolite ratios. For example sample A (Argininosuccinic aciduria) showed elevated argininosuccinate, sample B (VLCAD deficiency) increased long-chain acylcarnitines, and sample F (tyrosinemia) a marked rise in tyrosine levels. Extracted-ion chromatograms demonstrated clear differences in signal profiles between control and pathological samples, underscoring method specificity.

Benefits and Practical Applications

• High sensitivity and selectivity for a broad panel of metabolites using minimal sample volume
• Rapid throughput with sub-3-minute analytical cycle, suitable for large-scale screening
• Simplified sample handling avoiding laborious cleanup steps
• Internal standardization ensures reproducible quantitation across runs
• Applicability to newborn screening, metabolic profiling, and research studies

Future Trends and Potential Applications

Advances may include further automation of sample preparation, expansion of analyte panels to include novel biomarkers, integration with high-resolution mass spectrometry for untargeted screening, and application of machine learning for pattern recognition. Developing tighter coupling with laboratory information systems will enhance data management and accelerate clinical decision-making.

Conclusion

The validated LC-MS/MS protocol enables reliable, high-throughput quantification of amino acids and acylcarnitines from dried blood spots. Its robustness and efficiency make it a valuable tool for research and screening of inherited metabolic diseases.

Reference

Shimadzu Application Note No C94 First Edition June 2014