Determination of Unbound Urinary Amino Acids Incorporated with Creatinine Normalization by LC-MS/MS Method with CLAM-2000 Online Sample Pre-treatment

Significance of the topic

Free amino acids in urine reflect metabolic status and are critical in diagnosing metabolic disorders and assessing protein assimilation. Normalizing to creatinine compensates for hydration variability and is standard in clinical and research settings.

Study objectives

This work aimed to develop a fully automated, derivatization-free LC-MS/MS method with online sample pretreatment for quantifying unbound urinary amino acids and creatinine, enabling robust normalization and high-throughput analysis.

Methodology and instrumentation used

• Online sample preparation with CLAM-2000: automatic addition of internal standards, solvent mixing, protein precipitation, filtration, and transfer to LC-MS/MS.
• LC-MS/MS analysis using the Shimadzu LCMS-8040 triple quadrupole system.
• Chromatographic separation on Intrada Amino Acid column (100 × 3 mm, 3 µm) with gradient elution using acetonitrile/THF/ammonium formate/formic acid and acetonitrile/ammonium formate mobile phases.
• MS detection by electrospray ionization in positive MRM mode (SIM for glycine).

Results and discussion

• Established quantitation for 22 analytes (20 proteinogenic amino acids, citrulline, ornithine, and creatinine) with calibration ranges of 0.1–100 µM (up to 50 µM for some), correlation coefficients (R²) ≥ 0.991, LOQs between 0.07–5 µM, accuracy 89–113 %, and RSD ≤ 16 %.
• Analyzed 28 urine samples from healthy volunteers (20–30 years). Total amino acid and creatinine concentrations ranged widely (AA: ~543–16 640 µM; CRE: ~438–2 261 µM).
• Defined Total Creatinine-Normalized Amino acids (TCNA) and classified samples into low, medium, and high TCNA groups. Statistical and PCA analyses revealed significant differences (p < 0.05) for most amino acids between defined groups, highlighting TCNA as a discriminating metric.

Benefits and practical applications

This automated workflow reduces manual handling and analysis time (3–5 min per sample), enhances reproducibility, and provides reliable profiling of urinary amino acids normalized to creatinine. It is suitable for clinical diagnostics, metabolic research, QA/QC, and high-throughput studies.

Future trends and potential applications

• Extending the panel to include additional metabolites and biomarkers.
• Integration with bioinformatics tools for large-scale cohort studies.
• Application in personalized medicine for metabolic disorder screening and dietary monitoring.
• Adaptation to other biological matrices (e.g., plasma or saliva) using the automated CLAM-2000 platform.

Conclusion

A robust, automated LC-MS/MS method combining CLAM-2000 and Shimadzu LCMS-8040 enables accurate, high-throughput quantification of free urinary amino acids with creatinine normalization. The approach streamlines sample preparation, offers strong analytical performance, and facilitates metabolic profiling for research and clinical use.

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