Department of Pharmacy, University of Peshawar, Peshawar-25120, Pakistan.
J Chromatogr B Analyt Technol Biomed Life Sci. 2010 Sep 1;878(25):2339-47. doi: 10.1016/j.jchromb.2010.07.009. Epub 2010 Aug 7.
A novel, simple and fast reversed-phase HPLC/UV method was developed, optimized for various chromatographic conditions, and validated according to international guidelines for simultaneous determination of all-trans-retinol and alpha-tocopherol in human serum using retinyl acetate as internal standard in the concentration of 0.5 microg/ml. A liquid-phase extraction was applied to the 250 microl of serum with n-hexane-dichloromethane mixture (70:30, v/v), in two steps, using ethanol-methanol mixture (95:5, v/v) for protein precipitation and BHT (butylated hydroxy toluene) as stabilizer for sample preparation. Both analytes were analyzed on Kromasil 100 C(18) column (150 mm x 4.6 mm, 5 microm), Brownlee analytical (Perkin Elmer) C(18) column (150 mm x 4.6 mm, 5 microm), and Supelco (Supelcosil) LC-18 column (150 mm x 3 mm, 3 microm), protected by a Perkin Elmer C(18) (30 mm x 4.6 mm, 10 microm; Norwalk, USA) pre-column guard cartridge, at 292 nm wavelength, using methanol-water (99:1, v/v), in isocratic mode as mobile phase applied at flow rate of 1.5 ml/min and 1 ml/min for both 5 microm and 3 microm columns, respectively. Complete separation of all the analytes was achieved in 3 and 6 min on 3 microm and 5 microm columns, respectively by injecting 20 microl of sample into the HPLC system by autosampler, keeping column oven temperature at 25 degrees C. Different particulate reversed-phase chromatographic columns were evaluated in order to select the best column in terms of sensitivity, selectivity, resolution and short run time of both the analytes and it was concluded that 3 microm columns are better to be used in clinical set up as well as in laboratories for the separation of these analytes in a shorter time as compared with 5 microm columns. The method was validated and applied for the analysis of all-trans-retinol and alpha-tocopherol in the serum of human volunteers.
建立并优化了一种新颖、简单、快速的反相高效液相色谱/紫外检测法，同时测定人血清中全反式视黄醇和α-生育酚，以醋酸视黄酯为内标，浓度为 0.5μg/ml。采用液-液萃取法，用正己烷-二氯甲烷（70:30，v/v）混合溶剂分两步提取 250μl 血清，先用乙醇-甲醇（95:5，v/v）沉淀蛋白，然后用 BHT（叔丁基对羟基甲苯）作为稳定剂进行样品制备。在 Kromasil 100 C(18)柱（150mm×4.6mm，5μm）、Brownlee analytical（Perkin Elmer）C(18)柱（150mm×4.6mm，5μm）和 Supelco（Supelcosil）LC-18 柱（150mm×3mm，3μm）上分析两种分析物，均在 292nm 波长下，以甲醇-水（99:1，v/v）为流动相，在等度模式下以 1.5ml/min 和 1ml/min 的流速进行洗脱，5μm 和 3μm 柱的流速分别为 1.5ml/min 和 1ml/min。通过自动进样器将 20μl 样品注入 HPLC 系统，在 25℃下保持柱温，两种分析物在 3μm 和 5μm 柱上的分离度分别达到 3min 和 6min。评估了不同的颗粒反相色谱柱，以选择在灵敏度、选择性、分辨率和两种分析物短运行时间方面具有最佳性能的柱，结果表明 3μm 柱在临床和实验室中更适合用于分离这些分析物，因为其运行时间比 5μm 柱更短。对该方法进行了验证，并应用于人志愿者血清中全反式视黄醇和α-生育酚的分析。
