Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut, 71526, Egypt.
Urology Department, Faculty of Medicine, Al-Azhar University, Assiut, Egypt.
Sci Rep. 2024 Nov 17;14(1):28371. doi: 10.1038/s41598-024-79611-9.
The kidney is considered the main site for the net release of Tyrosine (Tyr) to the circulation through hydroxylation of Phenylalanine (Phe) using phenylalanine hydroxylase enzyme. In chronic kidney disease (CKD) patients the enzyme activity is impaired, therefore the serum concentration ratio of Tyr/Phe is reduced compared to healthy individuals. This motivates us to develop a cost effective, green, simple, sensitive, and selective HPTLC method to measure the levels of Tyr and Phe in serum samples. The analysis was carried out using acetonitrile: ethanol: 25% ammonia solution: ethyl acetate (6.5: 1.5: 1: 0.5) as a mobile phase. Rf values were 0.55 ± 0.05 for Phe and 0.39 ± 0.05 for Tyr. Densitometry scanning was performed using UV detector and dual wavelengths of 210 and 225 nm were obtained. A linear correlation was observed between the levels of Phe and Tyr, ranging from 50 to 700 ng band and 50 to 600 ng band, respectively, under the optimum conditions. The method selectivity, linearity, precision, accuracy, and robustness were all confirmed in accordance with ICH recommendations. Calculations of the separation and resolution factors, number of theoretical plates, and height equivalent to theoretical plates prove to the chromatographic system accuracy and high separation efficiency. The developed method exhibits an acceptable eco-scale when measuring the method greenness using AGREE and GAPI softwares. It was applied for the determination of Phe and Tyr concentrations in human serum samples.
肾脏被认为是通过苯丙氨酸羟化酶将苯丙氨酸羟化产生酪氨酸（Tyr）并释放到循环系统中的主要部位。在慢性肾脏病（CKD）患者中，该酶的活性受损，因此与健康个体相比，血清中 Tyr/Phe 的浓度比值降低。这促使我们开发了一种经济高效、绿色、简单、灵敏和选择性的 HPTLC 方法来测量血清样品中 Tyr 和 Phe 的水平。分析使用乙腈：乙醇：25%氨溶液：乙酸乙酯（6.5：1.5：1：0.5）作为流动相进行。Phe 的 Rf 值为 0.55±0.05，Tyr 的 Rf 值为 0.39±0.05。使用紫外检测器进行密度扫描，得到 210 和 225nm 的双波长。在最佳条件下，观察到 Phe 和 Tyr 的水平之间存在线性相关性，范围分别为 50 至 700ng 带和 50 至 600ng 带。该方法的选择性、线性、精密度、准确度和稳健性均按照 ICH 建议得到了确认。分离和分辨率因子、理论板数和理论板高度等效数的计算证明了色谱系统的准确性和高分离效率。使用 AGREE 和 GAPI 软件测量方法的绿色度时，该方法表现出可接受的生态规模。该方法已应用于人血清样品中 Phe 和 Tyr 浓度的测定。
