Method for simultaneously determining tryptophan and tyrosine in blood serum by high performance liquid chromatography and fluorescence method
A technology of high-performance liquid chromatography and fluorescence method, which is applied in the field of simultaneous determination of serum tryptophan and tyrosine by high-performance liquid chromatography-fluorescence method, can solve the problems of not meeting the detection requirements of clinical serum samples, narrow linear range, etc., and achieve Good linear relationship, wide linear range, and low reagent consumption
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Embodiment 1
[0024] Embodiment 1, the preparation of reagent and the collection processing of serum sample
[0025] 1. Preparation of standard solution
[0026] Accurately weigh Trp10.0mg, Tyr10.0mg, dissolve and dilute to 10.0mL with 2.5% (v / v) perchloric acid solution, mix well, and make 4900μmol / LTrp standard stock solution and 5500μmol / LTyr standard stock solution respectively, After aliquoting, store in a -30°C refrigerator for later use. Before use, take 2 kinds of standard stock solution and add 2.5% perchloric acid to make standard working solution (containing Trp 24.5 μmol / L, Tyr 55 μmol / L).
[0027] 2. Protein Precipitating Agent
[0028] Prepare 5% (v / v) perchloric acid solution with ultrapure water as serum protein precipitant.
[0029] 3. Preparation of mobile phase
[0030] A 10% (v / v) acetonitrile solution was prepared with ultrapure water, filtered through a 0.22 μm microporous membrane, and ultrasonically degassed for 20 minutes before use.
[0031] 4. Sample solution...
Embodiment 2
[0035] Embodiment 2, the optimization of chromatographic conditions
[0036] 1. Selection of detection wavelength
[0037] Dilute a certain concentration of Tyr and Trp standard solutions with the mobile phase, adjust to zero with the mobile phase blank, and scan the excitation and emission spectra of Tyr and Trp on the 2475-type fluorescence detector to obtain the maximum excitation light wavelength: λex Tyr =228nm, λex Trp =285nm; maximum emission wavelength: λem Tyr =306nm, λem Trp = 353nm.
[0038] 2. The effect of the proportion of organic matter in the mobile phase on the retention time
[0039] Mobile phases with different acetonitrile contents (5%, 8%, 10%, 12%) were prepared, and the standard solution was used as a sample for analysis. With the increase of acetonitrile content, the peak retention time of Tyr and Trp is shortened correspondingly, and the peak area response value of Tyr and Trp also increases correspondingly; when the content of acetonitrile is 5%,...
Embodiment 3
[0042] Embodiment 3, the detection of standard substance and serum sample
[0043] 1. Chromatographic separation of standard and serum samples
[0044]Mixed standard (containing Trp24.5μmol / L, Tyr 55μmol / L), mixed serum sample deproteinized supernatant 20μL was injected for determination. Detection conditions are: where chromatographic column: MegresC18 column (250mm×4.6mmi.d., 5μm); mobile phase: 10% (v / v) acetonitrile; flow rate: 1.2mL / min; fluorescence detection wavelength: 0-5min excitation light The wavelength of the emitted light is 228nm, and the wavelength of the emitted light is 306nm; after 5 minutes, the wavelength of the excited light is 285nm, and the wavelength of the emitted light is 353nm; the injection volume: 20μl; measured at room temperature. figure 1 , figure 2 The chromatograms of the mixed standard and normal human serum samples are shown respectively. The retention times of Tyr and Trp are about 3.4min and 7.6min, respectively. The peak shape is sta...
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