利用分散式液液微萃取法結合毛細管電泳線上濃縮技術進行尿液中finasteride及其代謝物之分析研究

雄性禿的成因為體內睪固酮受到5α-還原酶代謝形成二氫睪固酮進而阻斷頭皮毛囊合成蛋白質，使頭髮生長期縮短，在頭髮還尚未完全生長則進入休止期，最後導致毛囊萎縮並出現落髮症狀。Finasteride為第二型5α-還原酶的抑制劑，可以藉由抑制5α-還原酶作用進而降低二氫睪固酮生成，使頭皮毛囊恢復生髮機能。本研究利用毛細管電泳法建立測定尿液中finasteride及其活性代謝物finasteride carboxylic acid (M3)的分析方法，作為藥物動力學研究工具。本實驗以分散式液液微萃取法以萃取溶媒chloroform與分散溶媒acetonitrile (ACN)作為尿液樣品前處理方法增加濃縮倍率及去除基質干擾。結合毛細管電泳法field enhanced sample stacking (FESS)搭配sweeping線上濃縮技術以大量樣品進樣及負電微胞掃集線上樣品達到樣品堆積及分離效果。分離條件為高導電度緩衝溶液組成為200 mM phosphate (pH 3) 含25% (v/v) ACN，分離緩衝溶液組成為100 mM phosphate buffer (pH 3)含100 mM SDS以及25% (v/v) ACN，樣品溶液為100 mM phosphate (pH 3)，樣品進樣量以壓力5 psi進樣1.5分鐘。實驗結果中於8.5分鐘內可觀察到兩支完全分離且堆積的波峰。待測物定量之線性範圍為75 ng/mL - 1000 ng/mL，同日內及異日間相關係數r值皆大於0.999，具有良好的直線性。相對標準偏差及相對誤差分別小於5.28%及4.25%，表示分析方法具良好的精密度及準確度，最低偵測極限兩者均可達到20 ng/mL。在線上濃縮技術與傳統micellar electrokinetic chromatography (MEKC) 相比待測物finasteride及M3之靈敏度分別提升362及480倍。本研究已成功建立檢測finasteride及代謝物M3之分析方法並可應用於檢測服用治療雄性禿用藥finasteride患者之尿液樣品。

關鍵字

雄性禿； Finasteride ；毛細管電泳；線上濃縮技術；分散式液液微萃取法

並列摘要

Androgenetic alopecia leads to hair follicle miniaturization due to dihydrotestosterone (DHT) which is converted from testosterone by 5α-reductase. DHT affects the synthesis of protein in hair follicle and disrupts the growth of hair. In the treatment of androgenetic alopecia, finasteride binds irreversibly to type II 5α-reductase and inhibits the conversion of testosterone to DHT so as to reduce the symptom of hair loss. Capillary electrophoresis (CE) is applied to establish an analytical method aimed to investigate the pharmacokinetics by monitoring the concentrations of finasteride and its metabolite, finasteride carboxylic acid (M3). In this study, the urine sample pre-treatment method of dispersive liquid liquid microextraction (DLLME) is used to concentrate the analytes and eliminate the interference of urine sample with extracting solvent of chloroform and disperser solvent of acetonitrile (ACN). Field enhanced sample stacking (FESS) and sweeping are coupled to enhance stacking effect of analytes. In the part of separation condition, the condition of high conductivity buffer (HCB) is 200 mM phosphate (pH 3) with 25% (v/v) ACN and separation buffer is 100 mM phosphate (pH 3) with 100 mM SDS and 25% (v/v) ACN. The pretreatment urine sample is dissolved in 100 mM phosphate (pH 3). The sample injection is 5 psi for 1.5 min. In experiment, two stacking peaks are observed within 8.5 min and analytes with baseline separated is also obtained. The linear range of the two analytes is from 75 ng/mL to 1000 ng/mL. The correlation coefficients of two analytes in intra-day and inter-day analysis are both above 0.999 and the relative standard deviation and relative error are less than 5.28% and 4.25%, respectively. The limit of detection of analytes are 20 ng/mL. The sensitivity enrichment of finasteride and M3 are 362-fold and 480-fold compared with conventional MEKC method respectively. The analytical technique is established to be applied in the determination of urine sample from patient in the administration of finasteride for the treatment of androgenetic alopecia.

並列關鍵字

Androgenetic alopecia ； finasteride ； capillary electrophoresis ； on-line preconcentration ； dispersive liquid liquid microextraction

參考文獻

[1] D. Rathnayake, R. Sinclair, Male androgenetic alopecia, Expert Opin Pharmacother, 11 (2010) 1295-1304.

[4] A. Rebora, Pathogenesis of androgenetic alopecia, Journal of the American Academy of Dermatology, 50 (2004) 777-779.

[6] E. Arca, G. Acikgoz, H.B. Tastan, O. Kose, Z. Kurumlu, An open, randomized, comparative study of oral finasteride and 5% topical minoxidil in male androgenetic alopecia, Dermatology, 209 (2004) 117-125.

[7] A.B. Smith, C.C. Carson, Finasteride in the treatment of patients with benign prostatic hyperplasia a review, Therapeutics and Clinical Risk Management, (2009) 535-545.

[8] G. Gormley, E. Stoner, R. Rittmaster, H. Gregg, D. Thompson, K. Lasseter, P. Vlasses, E. Stein, Effects of finasteride (MK-906), a 5 alpha-reductase inhibitor, on circulating androgens in male volunteers, Journal of Clinical Endocrinology and Metabolism, 70 (1990) 1136-1141.

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