Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, The University of Houston, 1441 Moursund Street, Houston, TX 77030, USA.
J Pharm Biomed Anal. 2013 Nov;85:245-52. doi: 10.1016/j.jpba.2013.07.030. Epub 2013 Aug 5.
The purpose of this study is to develop an UPLC-MS/MS method to quantify 3-hydroxyflavone (3-HF) and its metabolite, 3-hydroxyflavone-glucuronide (3-HFG) from biological samples. A Waters BEH C8 column was used with acetonitrile/0.1% formic acid in water as mobile phases. The mass analysis was performed in an API 5500 Qtrap mass spectrometer via multiple reaction monitoring (MRM) with positive scan mood. The one-step protein precipitation by acetonitrile was used to extract the analytes from blood. The results showed that the linear response range was 0.61-2500.00 nM for 3-HF and 0.31-2500.00 nM for 3-HFG. The intra-day variance is less than 16.5% and accuracy is in 77.7-90.6% for 3-HF and variance less than 15.9%, accuracy in 85.1-114.7% for 3-HFG. The inter-day variance is less than 20.2%, accuracy is in 110.6-114.2% for 3-HF and variance less than 15.6%, accuracy in 83.0-89.4% for 3-HFG. The analysis was done within 4.0 min. Only 10 μl of blood is needed due to the high sensitivity of this method. The validated method was successfully used to pharmacokinetic study in A/J mouse, transport study in the Caco-2 cell culture model, and glucuronidation study using mice liver and intestine microsomes. The applications revealed that this method can be used for 3-HF and 3-HFG analysis in blood as well as in bioequivalent buffers such HBSS and KPI.
本研究旨在开发一种 UPLC-MS/MS 方法，用于从生物样品中定量测定 3-羟基黄酮（3-HF）及其代谢物 3-羟基黄酮-葡萄糖醛酸苷（3-HFG）。采用 Waters BEH C8 柱，以乙腈/0.1%甲酸水溶液为流动相。质谱分析采用 API 5500 Qtrap 质谱仪，通过正离子扫描模式下的多重反应监测（MRM）进行。采用乙腈一步蛋白沉淀法从血液中提取分析物。结果表明，3-HF 的线性响应范围为 0.61-2500.00 nM，3-HFG 的线性响应范围为 0.31-2500.00 nM。日内变异小于 16.5%，准确度在 77.7-90.6%之间，3-HF 的日内变异小于 15.9%，准确度在 85.1-114.7%之间。日间变异小于 20.2%，准确度在 110.6-114.2%之间，3-HF 的日间变异小于 15.6%，准确度在 83.0-89.4%之间。分析时间在 4.0 分钟内。由于该方法灵敏度高，仅需 10 μl 血液。该方法已成功应用于 A/J 小鼠的药代动力学研究、Caco-2 细胞培养模型的转运研究以及小鼠肝肠微粒体的葡萄糖醛酸化研究。这些应用表明，该方法可用于血液中 3-HF 和 3-HFG 的分析，以及 HBSS 和 KPI 等生物等效缓冲液中 3-HF 和 3-HFG 的分析。
