Application of liquid chromatography-tandem mass spectrometry (LC–MS/MS) for the analysis of stable isotope enrichments of phenylalanine and tyrosine

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2009, Vol.877 (1), p.43-49
• Main Authors: Meesters, Roland J.W., Wolfe, Robert R., Deutz, Nicolaas E.P.
• Format: Article
• Language: English
• Subjects: Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Chromatography, Liquid - methods; Derivatization; Fundamental and applied biological sciences. Psychology; General pharmacology; Humans; LC–MS/MS; Medical sciences; Pharmacology. Drug treatments; Phenylalanine; Phenylalanine - chemistry; Protein turnover; Reproducibility of Results; Stable isotope labeled amino acids; Tandem Mass Spectrometry - methods; Tracer–tracee ratio; Tyrosine; Tyrosine - chemistry
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2008.11.018

Whole-body protein synthesis and breakdown are measured by a combined tracer infusion protocol with the stable isotope amino acids l-[ ring- 2H 5]-phenylalanine, l-[ ring- 2H 2]-tyrosine and l-[ ring- 2H 4]-tyrosine that enable the measurement of the phenylalanine to tyrosine conversion rate. We describe a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the measurement of very low tracer–tracee ratios (TTR) of the amino acids l-phenylalanine and l-tyrosine in human plasma. TTR calibration curves of the tracers l-[ ring- 2H 5]-phenylalanine, l-[ ring- 2H 2]-tyrosine and l-[ ring- 2H 4]-tyrosine were linear ( r 2 > 0.99) in the range between 0.01% and 5.0% TTR and lowest measurable TTR for the tracers was 0.01% at a physiological concentration of 60 μM. The method was applied successfully to plasma samples from a clinical study reaching a steady state enrichment plateau (mean ± SD) of 3.33 ± 0.19% for l-[ ring- 2H 5]-phenylalanine, 2.40 ± 0.43% for l-[ ring- 2H 2]-tyrosine and 0.29 ± 0.07% for l-[ ring- 2H 4]-tyrosine, respectively. The LC–MS/MS method can be applied for measurement of very low plasma enrichments of phenylalanine and tyrosine for the determination of whole-body protein synthesis and breakdown rates in humans.