UHPLC-MS/MS Analysis of Amino Acids in Dried Blood Spots using Waters Kairos Amino Acid Kit for Clinical Research

Importance of the Topic

Accurate quantification of amino acids in dried blood spots (DBS) is vital for clinical research, newborn screening, and nutritional studies. Traditional optical detection approaches often require complex buffer systems, ion-pair reagents, and lengthy run times. A streamlined liquid chromatography-tandem mass spectrometry (LC-MS/MS) method reduces sample preparation complexity and analysis time while maintaining high precision and accuracy.

Objectives and Study Overview

The study aimed to develop and validate a rapid UHPLC-MS/MS workflow for the measurement of 42 amino acids in DBS using the Waters Kairos™ Amino Acid Kit combined with AccQ•Tag™ Ultra “3X” derivatization chemistry. Key performance parameters, including linearity, sensitivity, carryover, matrix effects, precision, and accuracy, were evaluated using commercial calibrators, CDC reference materials, and in-house quality controls.

Methodology and Instrumentation Used

• Sample Preparation: Single 3.2 mm DBS punches (~3.1 µL blood) were extracted with the Kairos internal standard, followed by borate buffer addition and AccQ•Tag Ultra “3X” derivatization.
• Chromatography: ACQUITY UPLC I-Class PLUS system equipped with a CORTECS™ UPLC C18 column, 9-minute gradient run.
• Detection: Xevo™ TQ-S micro triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode.
• Data Processing: MassLynx™ v4.2 for acquisition, TargetLynx™ XS v4.2 and waters_connect™ for quantitation (user factor 6.45 applied to DBS data).

Main Results and Discussion

• Carryover: Negligible (<20% of LOQ) between high calibrator and blank injections.
• Matrix Effects: Internal standard response in six independent DBS sources showed <±15% deviation (CV <10%) except histidine.
• Linearity: R2 >0.99 across the calibration range for all 42 amino acids (1/x weighting).
• Sensitivity: LOQs below Calibrator 1 levels for most analytes; a few histidine and methyl-histidine isomers exhibited higher LOQs.
• Precision and Accuracy: Solvent QCs achieved ≤14% total CV and ±15% accuracy; in-house DBS QCs (n=25) met ≤15% CV and <±15% bias for 37 amino acids, with slightly elevated CVs (up to 18.6%) for histidine, anserine, and 3-methyl-histidine.
• Recovery: Calculated recoveries ranged from 65.5% to 135.2% across spiked DBS levels.
• CDC Reference Material: Twelve amino acids measured in external DBS showed ≤14.4% total CV and ±15% agreement with target values.
• Chromatographic Separation: Effective resolution of isobaric leucines (leucine, isoleucine, allo-isoleucine) and consistent TIC profiles across QC levels.

Benefits and Practical Applications

• Rapid 9-minute analysis per sample without the need for mobile-phase buffers or ion-pair reagents.
• High throughput compatible 350 µL plate format supports large-scale studies.
• Robust performance for clinical research, newborn screening, nutritional profiling, and metabolic disorder studies.
• Simplified workflow reduces reagent complexity and operational cost.

Future Trends and Applications

• Extension of the platform for targeted small-molecule panels beyond amino acids.
• Automation of sample preparation and integration with laboratory information management systems (LIMS) for high-throughput screening.
• Application to non-invasive matrices (e.g., saliva, urine) for broader clinical and forensic investigations.
• Development of multiplexed kits for combined metabolite and biomarker quantitation in DBS.

Conclusion

The Waters Kairos Amino Acid Kit paired with AccQ•Tag Ultra “3X” chemistry and UHPLC-MS/MS instrumentation provides a rapid, accurate, and buffer-free method for quantifying up to 42 amino acids in DBS. The workflow demonstrates excellent linearity, low carryover, minimal matrix effects, and reliable precision and accuracy, enabling streamlined clinical research and high-throughput metabolic profiling.

References

• Waters Corporation. Kairos Amino Acid Kit Care and Use Manual, p/n 720006448.
• Waters Corporation. Application Note: UHPLC-MS/MS Analysis of Amino Acids in Dried Blood Spots, June 2025.