Pathan Shadab A, Jain Gaurav K, Zaidi Syed M A, Akhter Sohail, Vohora Divya, Chander Prakash, Kole Prashant L, Ahmad Farhan J, Khar Roop K
Department of Pharmaceutics, Faculty of Pharmacy, Hamdard University, New Delhi, India.
Biomed Chromatogr. 2011 May;25(5):613-20. doi: 10.1002/bmc.1492. Epub 2010 Aug 23.
Thymoquinone (THQ) is known for its neuroprotective and anti-convulsant properties in preclinical studies. We herewith describe a simple, rapid, selective, sensitive and stability-indicating UPLC method for the estimation of THQ and its application to biopharmaceutical studies such as in vitro release from nanoparticulate system and in vivo pharmacokinetic study. The method employed gradient elution using a Waters Acquity HSS-T3 C(18) (100 × 2.1 mm, 1.8 µm) UPLC column. The mobile phase consisted of water and acetonitrile, pumped at a flow rate of 0.5 mL/min. The injection volume was 5 µL and THQ was monitored at 294 nm wavelength with a total run time of 6 min. In solution as well as in plasma, the method was found to be linear (r ≥ 0.998), precise (CV ≤ 2.45%) and accurate (recovery ≥ 84.8%) in the selected concentration range of 0.1-0.8 µg/mL. Forced degradation studies revealed that THQ undergoes degradation under acidic, basic, oxidation and UV light stress conditions. However, the developed UPLC method could effectively resolve degradation product peaks from THQ. Further, no interference was found at the retention time of THQ from any plasma components, indicating selectivity of the developed method. For solutions, the limits of detection and quantitation of the method were found to be 0.001 and 0.0033 µg/mL, respectively; while in plasma they were 0.006 and 0.02 µg/mL, respectively. The validated method was successfully applied to quantify THQ in dissolution medium as well as oral in vivo pharmacokinetic study of THQ suspension and THQ- solid lipid nanoparticle (THQ-SLN) formulation. A 2-fold increase in the relative bioavailability was observed with the THQ-SLN compared with THQ. The results indicate that the SLN significantly increased plasma concentrations and retention within the systemic circulation.
在临床前研究中，百里醌（THQ）以其神经保护和抗惊厥特性而闻名。我们在此描述一种简单、快速、选择性好、灵敏且能指示稳定性的超高效液相色谱（UPLC）方法，用于测定THQ，并将其应用于生物制药研究，如纳米颗粒系统的体外释放和体内药代动力学研究。该方法采用梯度洗脱，使用沃特世Acquity HSS-T3 C(18)（100×2.1 mm，1.8 µm）UPLC色谱柱。流动相由水和乙腈组成，流速为0.5 mL/min。进样体积为5 µL，在294 nm波长下监测THQ，总运行时间为6 min。在溶液和血浆中，该方法在所选择的0.1 - 0.8 µg/mL浓度范围内呈线性（r≥0.998）、精密度高（CV≤2.45%）且准确度高（回收率≥84.8%）。强制降解研究表明，THQ在酸性、碱性、氧化和紫外光胁迫条件下会发生降解。然而，所开发的UPLC方法能够有效分离THQ的降解产物峰。此外，在THQ的保留时间处未发现来自任何血浆成分的干扰，表明所开发方法的选择性良好。对于溶液，该方法的检测限和定量限分别为0.001和0.0033 µg/mL；而在血浆中分别为0.006和0.02 µg/mL。经过验证的方法成功应用于定量溶出介质中的THQ，以及THQ悬浮液和THQ - 固体脂质纳米粒（THQ - SLN）制剂的口服体内药代动力学研究。与THQ相比，THQ - SLN的相对生物利用度提高了2倍。结果表明，固体脂质纳米粒显著提高了血浆浓度并延长了在体循环中的保留时间。
