利用毛細管電泳法分析Tiludronate、Zoledronate、Risedronate製劑含量與人體血漿中的濃度

Development of capillary electrophoresis for determination of tiludronate, zoledronate and risedronate in pharmaceuticals and human plasma

林聖傑(Sheng-Chieh Lin)

指導教授 ： 吳秀梅

高雄醫學大學/藥學院/藥學系碩士班/碩士(2019年)

摘要

本研究方法建立兩種毛細管電泳 (Capillary electrophoresis，CE) 分別應用於骨質疏鬆藥物中的雙磷酸鹽類製劑與血漿樣品分析。
第一部分以微胞電動毛細管電泳法搭配電場放大樣品堆積及微胞掃集之線上濃縮技術 (Field-amplified sample injection-sweeping-micellar electrokinetic chromatography, FASI-sweeping-MEKC)，可以同時分析3種藥物，包含 tiludronate (T)、risedronate (R)、zoledronate (Z)，經探討影響分離因素後，所得最佳分析條件為含有100.00 mM SDS的125.00 mM NaH2PO4 (pH 2.0) 緩衝液。待測物於本研究之線性範圍如下，tiludronate為10~1000 ng/mL；risedronate及zoledronate為20~2000 ng/mL，其相關係數 (r) 皆大於0.995；而待測物偵測極限如下，tiludronate為2.5 ng/mL，risedronate及zoledronate為5 ng/mL。在準確度與精密度的探討中，相對標準偏差 (Relative standard deviation, RSD) 與相對誤差(Relative error, RE)皆小於6.3%，表示具有良好再現性與理想準確度；相對回收率皆大於97%，本方法已經成功應用於市售risedronate及zoledronate製劑之含量分析。
第二部分以電場放大樣品推積 (FASI) 分析血漿中risedronate (R)及zoledronate (Z)，其原理透過注入water plug影響導電度進而濃縮待測物，經探討分離因素後(包括緩衝溶液之濃度、pH、water plug time、樣品注入時間) ，所得最佳分析條件為100.00 mM NaH2PO4 (pH 3.0) 緩衝液，water plug注射時間為1 psi，10 s，樣品注入時間為-10 kV，150 s。血漿樣品使用80%的甲醇含量去蛋白，經由高速離心後取出上清液後再使用0.22 μm過濾膜，進入毛細管電泳儀分析。Chlorbenzoic acid為內部標準品，對其他兩種待測物進行定量分析，檢量線範圍risedronate與zoledronate為10~100 μg/mL，其相關係數(r)皆大於0.995，顯示出濃度與面積比之間有良好關係，測試同日間及異日間的準確度及精密度，相對標準偏差小於6.8%；相對誤差小於6.4%，表示具有良好再現性與準確度。相對回收率皆大於97%，絕對回收率再17%~30%之間。其偵測極限risedronate及zoledronate為3 μg/mL，盼望此方法未來可應用於實際檢體中。

關鍵字

雙磷酸鹽類 ； 毛細管電泳 ； 電場放大樣品推積及微胞掃集線上濃縮技術

並列摘要

Capillary electrophoresis method had been developed for determination of bisphosphonates in available commercials formulations and plasma.
A new method has been developed for the determination of three bisphosphonates, including tiludronate, risedronate, zoledronate, using capillary electrophoresis (CE) coupled with field-amplified sample injection-sweeping-micellar electrokinetic chromatography (FASI-sweeping-MEKC). After optimization of the parameters, the background buffer was phosphate buffer monobasic (concentration: 125.00 mM , pH 2.0) containing 100.00 mM SDS. During the method validation, the calibration curves were linear over a range of tiludronate was 10~1000 ng/mL, risedronate and zoledronate were 20~2000 ng/mL and coefficient of correlation were all above 0.995. The limit of detection of tiludronate was 2.5 ng/mL, risedronate and zoledronate were 5 ng/mL. After validation, the relative standard deviation (RSD) and relative error (RE) were less than 6.3% which proved that this analysis could be reliable. All recoveries were greater than 97%. Moreover, this analysis had been successfully applied in commercial products (risedronate and zoledronate).
A novel method has been developed for the analysis of two bisphosphonates in plasma, including risedronate and zoledronate by field-amplified sample injection. After optimization of the parameters, the background buffer was phosphate buffer monobasic (concentration: 100.00 mM , pH 3.0), water plug time (1 psi, 10 s) and sample injection time (-10 kV, 150 s). The plasma was pretreated by using 80% methanol to deprotein and the supernatant was filtered using 0.22 μm filter. During validation, linear range of risedronate and zoledronate were 10~100 μg/mL and coefficient of correlation were all above 0.995. The limit of detection of risedronate and zoledronate were 3 μg/mL. After validation, the relative standard deviation (RSD) and relative error (RE) were less than 6.8% which proved that this analysis could be reliable. Relative recoveries were greater than 97% and absolute recoveries were between 17% to 30%. In future, the analysis will be applied in clinical samples.

並列關鍵字

bisphosphonates ； capillary electrophoresis ； FASI-sweeping MEKC

參考文獻

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