| Objective:1. To establish a simple and rapid method for simultaneous determination of tyrosine (Tyr) and tryptophan (Trp) in serum by high performance liquid chromatography-fluorescence detection (HPLC-FD).2. To explore the clinical significance of serum Tyr and Trp in lung cancer patients.Methods:1. A 20μL supernatant of a serum sample deproteinized by 5% perchloric acid solution was assayed and separated on a Megres C18 column (250mm×4.6mm internal diameter,5μm).Best results were achieved with an elution of 10%(v/v) acetonitrile, a flow rate of 1.2mL/min. The fluorescence was recorded at the optimal wavelength for Tyr (λex=228nm,λem=306nm) from 0 to 5 min, and the optimal wavelength for Trp (λex=285nm,λem=353nm) after 5 min. The total assay time of serum was within 9 minutes.2. The concentrations of Tyr and Trp in healthy controls (n=120) were determinate by HPLC-FD. The concentrations of Tyr and Trp in lung cancer patients were determinate, to explore the clinical significance of serum Tyr and Trp in patients with lung cancer.Results:1. Under optimal conditions excellent linearity was obtained in the range of 0.275-275μmol/L and 0.49-196μmol/L for Tyr and Trp, and the recovery was 90.5%-108.8% and 88.8%-97.2% for Tyr and Trp, respectively. The precision results showed that the relative standard deviation of intra-day and inter-day were<5%, respectively. The results indicated Phe,5-HT, KYNA, KYN, and Cre had no interfering effect to the determination of Tyr and Trp.2. The concentrations of Tyr and Trp in healthy controls were (mean±SD, 66.40±8.55)μmol/L and (mean±SD,49.93±5.43)μmol/L, respectively. There was a significant difference between males and females groups in the concentrations of Trp (p<0.01). Trp concentrations were significantly lower in patients with lung cancer than in healthy controls (39.26±5.44μmol/L vs.49.93±5.43μmol/L, respectively; p<0.01), while Tyr concentrations were no difference with healthy controls (65.38±7.94μmol/L vs.66.40±8.55μmol/L, respectively; p>0.05). In addition, patients in adenocarcinoma (ADC) group had significantly lower Trp and Tyr concentrations than those in squamous cell carcinoma (SCC) group. There was no difference between the early stages and advanced stages of lung cancer.Conclusions:1. The present method by HPLC with programmed fluorescence wavelength detection is simple, rapid and sensitive as compared to other conventional methods of separate or simultaneous determination of Tyr and Trp in serum and will be suited for high throughput screening. It needs neither sample pre-purification nor derivatization, which makes sample preparation simpler and improves the reproducibility. An external standard curve method can be used in quantification with satisfactory precision and accuracy. The method meets the requirements for routine analysis of Tyr and Trp in serum.2. It is of great value to determination of Tyr and Trp for defining clinical staging and identifying pathologic category of lung cancer. |
