利用高效液相層析法串聯電化學偵測器對膠囊及病人血漿中Hydroxyurea之分析研究

Analysis of Hydroxyurea in Capsules and Patients’ Plasma by High-Performance Liquid Chromatography with Electrochemical Detector

徐秀娥(Hsiu-O Hsu)

指導教授 ： 吳秀梅 ； 鐘育志

高雄醫學大學/藥學院/藥學研究所/碩士(2003年)

摘要

本研究建立簡單、靈敏之高效液相層析法以分析hydroxyurea (HU)，並應用於製劑之含量測定及病人血中濃度監測。分析於移動相恆定系統及室溫下完成，所使用的分離管柱為逆相碳18的管柱(250 × 4 mm I.D.; 5 mm)，使用電化學偵測器，工作電極為玻璃石墨電極， Ag/AgCl的參考電極，range 設在10 nA，filter 設在0.1 Hz。
製劑分析時，移動相為醋酸鈉(10 mM)-乙腈(95：5, v/v；pH 6.5)，探討影響分離的參數，並加以條件理想化。方法驗證時，於同日內及異日間分析HU，檢量線範圍在0.05 ~ 2.00 μM，所得迴歸曲線之相關係數(r)為0.9999，顯示本法有良好的定量性。方法的精密度及準確度資料，同日內(n = 6)及異日間(n = 6)之相對標準偏差於1.32~ 4.00%間，相對誤差值在-3.06 ~ 2.10 %間，回收率大於96%，已應用於市售膠囊含量分析，以t-test檢驗本法與USP XXV方法之測定值，結果無顯著之差異性(p = 0.795)，顯示本法可為膠囊含量分析之替代方法。
血漿檢品分析時，取20 mL之血漿檢品，移動相為醋酸鈉(25 mM)-乙腈(97.5：2.5, v/v；pH 6.5)，針對影響分離之參數加以探討，所得理想條件加以驗證，檢量線範圍在1.0 ~ 300 μM間，其迴歸曲線之相關係數(r)皆大於0.9992，顯示有良好的線性關係。方法的精密度及準確度資料，同批內(n = 3)及異批間(n = 5)之相對標準偏差於1.34 ~ 4.39%間，相對誤差值在-1.89 ~ 4.87%間，回收率大於96%，偵測極限為0.3 μM (S/N=3, 注入量 10 μL)。將本法實際應用於真性紅血球增多症或骨髓纖維變性患者，其血中HU濃度監測，所得測定值接近文獻之數據；本分析方法將應用於檢測HU治療濃度與臨床療效之相關性研究。

關鍵字

電化學偵測器

並列摘要

This thesis includes two parts：
(1) Analysis of hydroxyurea in capsules by high performance liquid chromatography with electrochemical detection
A simple HPLC method with electrochemical detection was developed for the analysis of hydroxyurea (HU) in capsules. Isocratic separation was achieved at ambient temperature using LC RP-18 column (250 × 4 mm I.D.; 5 mm). The mobile phase was 10 mM sodium acetate as supporting electrolyte containing 5% acetonitrile (pH 6.5). The effluent was monitored with electrochemical detector consisted of a glassy carbon working electrode and an Ag/AgCl reference electrode. The influence of the chromatographic parameters on the separation was investigated. The regression equations were linear (r ≧ 0.9999) in intra- and inter-day assay. This method was validated and applied to the analysis of HU in capsules. Comparison by t-test between the assay results obtained from HPLC-UV (USP XXV) and this method, it shows no significant difference (p = 0.795). This method may be an alternative method for analysis of HU in capsules.
(2) Analysis of hydroxyurea in patients’ plasma by high performance liquid chromatography with electrochemical detection
An improved and sensitive reversed-phase HPLC method with electrochemical detection was developed for analysis of HU in patients’ plasma after simple extraction. The separation was achieved using LC RP-18 column (250 × 4 mm I.D.; 5 mm). The mobile phase was sodium acetate (25 mM) - acetonitrile (97.5 : 2.5, v/v; pH 6.5). Several parameters were investigated and optimized. On the method validation, the calibration curves were linear over the range of 1.0~300 μM (r ≧ 0.9992). The detection limit was 0.3 μM (S/N = 3, 10mL injection volume). It is successfully applied to therapeutic drug monitoring of patients with polycythemia vera or myelofibrosis. This method may be feasible for clinical drug monitoring and study of pharmacokinetics and pharmacodynamics of hydroxyurea in the future.

並列關鍵字

electrochemical detector ； hydrooxyurea

參考文獻

References

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2. Yabro JW. Mechanism of action of hydroxyurea. Semin Oncol 19(1992) 1-10.

3. Donovan PB, Kaplan ME, Goldberg JD, Tatarsky I, Najean Y, Silberstein, William H. Knospe, John Laszlo, Karen Mack, Poul D. Berk EB, Wasserman LR. Treatment of polycythemia vera with hydroxyurea. Am J Hematol 17(1984) 329-334.

4. Berk PD, Goldberg JD, Donovan PB, Fruchtman SM, Berlin NI, Wasserman LR. Therapeutic recommendations in polycythemia vera based on Polycythemia Vera Study Group Protocol. Semin Hematol 23(1986) 132-143.