A Validated Bioanalytical Method for the Quantification of Biogenic Amines Using UPLC-MS/MS

Importance of the Topic

Amino acids and biogenic amines play critical roles in metabolic pathways and their blood levels can indicate pathological conditions such as metabolic disorders, diabetes, obesity, cancer, and toxicity. Accurate quantification in plasma or serum is essential for metabolic phenotyping, clinical diagnostics, and therapeutic monitoring. However, their polar and amphoteric nature challenges direct reverse-phase chromatography, necessitating optimized derivatization strategies for reliable high-throughput analysis.

Objectives and Study Overview

This study aimed to develop and validate a rapid, sensitive, and specific UPLC-ESI-MS/MS assay for over 50 amino acids and biogenic amines in human plasma and serum. Derivatization with AccQ•Tag Ultra reagent was employed to improve chromatographic retention and MS detectability. Validation followed FDA bioanalytical guidance to ensure accuracy, precision, specificity, and stability.

Methodology and Used Instrumentation

Sample preparation:

• Mix 10 µL plasma/serum with 10 µL water, 5 µL internal standard mix, and 40 µL cold isopropanol–0.1% formic acid to precipitate proteins.
• After incubation at –20 °C and centrifugation, derivatize 10 µL supernatant with 70 µL borate buffer (pH 8.6) and 20 µL AccQ•Tag Ultra reagent; heat at 55 °C for 10 min, then dilute 1:100.

Chromatography and detection:

• System: Waters ACQUITY UPLC I-Class with HSS T3 column (2.1×150 mm, 1.8 µm).
• Mobile phase A: 0.1% formic acid in water; B: 0.1% formic acid in acetonitrile. Gradient from 4% to 95% B over 6 min; total run time 7.5 min; flow 0.6 mL/min; column 45 °C; sample 4 °C; injection 2 µL.
• Detector: Waters Xevo TQ-S, ESI+ MRM (capillary voltage 1 kV, cone 50 V, analyte-specific collision energies).
• Data processing with MassLynx and TargetLynx Application Manager.

Main Results and Discussion

Derivatization produced stable 6-aminoquinoline (AMQ) derivatives yielding a common fragment m/z 171, facilitating selective MRM detection. Calibration in plasma was linear from 2–800 µM (R≥0.993). LLOQs were 2 µM for 17 analytes, 6 µM for eight, and 20 µM for seven; ULOQ was 800 µM. Intra-day CVs ranged 0.9–16.9%, inter-day CVs 2.1–15.9%; accuracy was within ±15.6% (intra) and ±13.7% (inter). Carryover <11% for analytes, <1% for internal standards. Underivatized stocks stable 6 h at room temperature, 48 h at –20 °C; derivatized samples stable up to one week at ambient or 4 °C. Chromatographic resolution achieved baseline separation of key isomers such as leucine/isoleucine, methylhistidines, GABA, BAIBA, AABA, and sarcosine.

Benefits and Practical Applications

• High throughput: 7.5 min/sample supports large-scale studies.
• Enhanced sensitivity and specificity by derivatization and MRM.
• Comprehensive coverage of >50 amino acids and related amines.
• Robust validation in plasma/serum per FDA guidelines.
• Applicable to metabolomics, clinical diagnostics, pharmaceutical research, and QC.

Future Trends and Opportunities

• Extension to additional metabolites and conjugated derivatives.
• Integration with high-resolution MS for structural elucidation.
• Automation of derivatization and sample preparation for higher reproducibility.
• Multiplexed assays combining targeted and untargeted workflows.
• Microflow LC and miniaturization to reduce sample and solvent use.
• Development of real-time monitoring and point-of-care platforms.

Conclusion

The validated UPLC-ESI-MS/MS assay using AccQ•Tag Ultra derivatization provides a rapid, sensitive, and specific platform for quantifying biogenic amines and amino acids in human plasma and serum. Its robust performance and broad analyte range make it well suited for diverse applications in bioanalytical, clinical, and research laboratories.

References

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3. Kaspar H, Dettmer K, Gronwald W, Oefner PJ. Automated GC-MS analysis of free amino acids in biological fluids. J Chromatogr B. 2008;870(2):222–232.