定量sildenafil citrate 的高壓液相層析法之開發與生物檢體之應用

Development of a high performance liquid chromatographic method for biological application with sildenafil citrate

夏安琪(Angel hsia)

指導教授 ： 何秀娥

臺北醫學大學/藥學院/藥學研究所/碩士(2002年)

摘要

本研究主要目的在於開發一簡單方便且對於sildenafil靈敏度高的HPLC分析方法來偵測血漿中微量sildenafil的含量。並將其應用於評估當併服葡萄柚汁後，是否可有效縮短sildenafil達到最高血漿濃度所需要的時間 (Tmax)，以改善sildenafil容易為普遍存在於肝臟及小腸中CYP 3A4酵素代謝的問題。經比較各種分析條件後選定以Inersil 5, ODS-2, 4.6* 150mm之層析管柱為固定相，30mM pH6.0 KH2PO4緩衝液/acetonitrile (45/55, v/v)的混合溶媒為移動相，流速0.5 ml /min，樣品注射量為100ul，使用UV檢測器於230nm下偵測結果。結果顯示此分析方法對於血漿當中sildenafil濃度的定量具有相當好的靈敏度與再現性，在檢量線濃度10-1000 ng/ml的範圍之內呈現良好的線性關係，其回收率可達80%。經密度評估異日間及同日內確效的偏差各在0.41%~11.15%及0.36%~8.05%之間；準確度評估異日間及同日內確效的偏差則各在-8.72%~6.81%及0.36~6.53之間，顯示此分析條件適合用於分析血漿中微量的sildenafil。 將上述分析條件實際應用於人體試驗以評估sildenafil在血漿中濃度的變化，四位受試者分兩次試驗併服25mg sildenafil citrate膠囊與白開水及葡萄柚汁後，進行為時8小時的試驗及血液檢品採樣，經HPLC分析結果發現當sildenafil citrate與葡萄柚汁併服後，其AUC值明顯的比併服白開水為高，但Tmax及T1/2則有延長的現象，顯示葡萄柚汁的確增加了sildenafil的生體可用率。此機轉可預期的是葡萄柚汁內的furano- coumarins抑制腸道內CYP 3A4所致，因而增加sildenafil的吸收速率，但結果顯示其Tmax並未如預期有縮短的現象。

關鍵字

威而剛 ； 高壓液相層析系統 ； 細胞色素P450 3A4 ； 葡萄柚汁

並列摘要

This study aimed at developing and validating an HPLC method for the assay of sildenafil in biological samples such as plasma. The application of this method to examine the potentials of correcting the drawback of a longer Tmax during the clinical use of sildenafil by the inhibition of cytochrome P450 enzymes (CYP3A4), a primary metabolizing enzyme located in intestine and liver, with grapefruit juice was conducted. Results demonstrated that chromatographic systems comprising a Inersil 5-ODS2 column, a mobile phase of potassium dihydrogen phosphate (30 mM, pH 6.0) — acetonitrile (45:55 v/v), a flow rate of 0.5 mL/min, and an injection volume of 100 uL and a UV detection at 230 nm was satisfactory for chromatographic separation of sildenafil. A linear relationship was found in the range from 10 ng/mL to 1000 ng/mL with absolute recovery about 80% for all concentrations examined. Precision for interday and intraday is from 0.41% to 11.15% and from 0.36% to 8.05%, respectively. Accuracy for interday and intraday is from —8.72% to 6.81% and from 0.36% to 6.53%, respectively. In the pharmacokinetic study, 4 healthy male volunteers received a single dose of sildenafil (25 mg/cap) with either 250 mL grapefruit juice or water in separate occasion. Plasma concentrations of sildenafil were determined up to 8 hours post dose with the use of the validated HPLC method described above. The result showed that co-administration of grapefruit juice was able to significantly increase both AUC0-8 and AUC0-inf, but to prolong Tmax and T1/2, of sildenafil in comparison to that of water. The increase of bioavailability of sildenafil was expectable as a result of inhibiting metabolizing enzymes of CYP3A4 located in the intestine by furanocoumarins containing in grapefruit juice. However, a shorter Tmax for sildenafil with the inhibition of CYP3A4 in the intestine did not accomplished.

並列關鍵字

sildenafil ； HPLC ； CYP 3A4 ； grapefruit juice

參考文獻

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