利用液相層析和毛細管電泳對錠劑中thiopurine藥物及全血中mercaptopurine及其代謝物之分析研究

Analysis of thiopurine drugs in tablets, and mercaptopurine and its metabolites in whole blood by liquid chromatography and capillary electrophoresis

李宗達(Chung-Ta Li)

指導教授 ： 吳秀梅

高雄醫學大學/藥學院/藥學研究所/碩士(2004年)

摘要

本論文分別以高效液相層析法及毛細管電泳法建立thiopurine類藥物之分析法，並應用於錠劑含量分析及全血中藥物濃度監測。
(1)高效液相層析法--
利用C18層析管(250 × 4 mm，5 μm)進行兩種抗癌藥mercaptopurine (MP)和thioguanine (TG)的分析，移動相含磷酸鹽緩衝溶液 (10 mM, pH 3.50)及乙腈 ( 93：7, v/v)，流速0.7 ml/min，紫外光偵測器波長為200 nm 。本法偵測極限，TG和MP皆為25 nM。MP和TG的檢量線範圍為 2-100 μM，其相關係數皆大於0.999；同日內及異日間相對誤差 (RE)及相對標準偏差 (RSD)皆小於5％；製劑回收率大於97％，可知本法具有良好的線性、準確度及精密度；經應用於製劑的分析，所得之結果皆符合美國藥典第二十五版所規定（TG為93-107 %，MP為93-110 %）。
由於待測物具氧化還原能力，為提高感度，另配合電化學偵測器(ECD)進行分析，並應用於全血藥物監測；取100 μL全血，經前處理後，利用HPLC-ECD分析MP、代謝物TG及thioxanthine (TX)。所得理想化條件：層析管為C18的管柱(150 × 3.9 mm，5 μm)，移動相含borate緩衝溶液 (50 mM, pH 8.00)，流速0.7 ml/min，使用電化學偵測器，電位設為800 mV，範圍為10 nA。檢量線範圍，MP為0.2 - 3 μM，TG或TX為0.4 - 3 μM，其相關係數皆大於0.99；同日內及異日間RE及RSD介於-18.40~11.78％；本法偵測極限，MP、TG或TX皆為50 nM (S/N＝3)；經應用於一位六歲患急性淋巴性白血病之男童，投藥MP 50 mg，1 hr後作其全血藥物監測，所得MP為0.24 ?搟gM，但未測得其代謝物。
(2)毛細管電泳法--
首先利用未處理之熔矽毛細管（全長40 cm，有效分析長度
30 cm；內徑50 μm）對三種抗癌藥TG、MP及azathioprine(AZA)進行同時分析，經檢討影響分離之因素後，所得最佳分析條件為glycine溶液（200 mM, pH 9.00），使用25 kV的分離電壓，偵測波長為214 nm。本方法偵測極限，TG和AZA為5 μM，MP為15 μM （S/N=3，壓力取樣5 s）。經確效所建立之分析方法，待測物TG和AZA之檢量線範圍為20.0 ~ 400.0 μM，MP為50.0 ~ 400.0 μM，相關係數（r）皆大於0.999；測試同日內及異日間的準確度及精密度，其RE及RSD均小於5%，回收率皆大於95%；本法已順利應用於測定市售製劑（Lanvis®、Azapress®和Purinethol®）之含量，所得含量百分率皆符合美國藥典XXV版之規定（TG或AZA為93-107%; MP為93-110%）。
為開發CE法偵測病人血中藥物濃度，又針對於全血中MP及其四個代謝物thioguanosine (rTG), thioguanine (TG), methyl – mercaptopurine (MMP)和methyl-mercaptopurine riboside (rMMP)建立毛細管電泳法，所得分析條件為使用未處理之熔矽毛細管（全長40.2 cm，有效分析長度30 cm；內徑50 μm），以borate緩衝溶液（50 mM, pH 8.5），使用20 kV的分離電壓，並以300 nm紫外光偵測，其偵測極限分別為MMP、 rMMP 、MP為20 μM；TG、 rTG為100 μM (S/N = 3；injection : 0.5 psi，5 s)。由於本法之感度不足以偵測全血中的待測物，目前另開發線上濃縮CE分析法。

關鍵字

硫嘌呤

並列摘要

This study describes the establishment of (1) high performance liquid chromatography, and (2) capillary electrophoresis methods for the determination of thiopurines in available commercial formulations, and the analysis of mercaptopurine and its metabolites in whole blood.
(1) High performance liquid chromatography---
The liquid chromatograph consisted of a module 501 pump
connected with UV detector (200 nm). Mercaptopurine (MP) and
thioguanine (TG) were eluted using a mobile phase consisting of monobasic phosphate buffer (10 mM, pH 3.5) and acetonitrile (93:7, v/v). A LiChroCART® RP-18 column (250 × 4.0 mm; 5 μm) was used. Thiouracil was used as an internal standard. Based on the optimized LC conditions, the method validation was evaluated over the range of 2-100 μM. The correlation coefficients of linear
regression lines (n=3) were all greater than 0.999. The method was validated and applied to the analysis of MP and TG in tablets. The RE and RSD were all less than 5％ in intra- and inter-day assay. All of the recoveries were greater than 97 %. The limits of detection were 25 nM for MP or TG (S/N=3). This method was feasible for the application of commercial tablets (Purinethol®, Lanvis®). All of the analytical values fell within labeled amount required by the USP XXV (TG, AZA: 93-107%; MP: 93-110%).
In order to increase the sensitivity, electrochemical detector (ECD) was used for detection of MP and its metabolites, TG and thioxanthine (TX), in whole blood (100 μL). Effects of parameters on the separation were investigated. The analytes were eluted using a mobile phase consisting of borate buffer (50 mM, pH 8.00) in LC RP-18 column (150 mm × 3.9 mm; 5 μm), and detected by ECD. The method validation was evaluated over the range of 0.2 – 3 μM for MP and 0.5 – 3 μM for TG or TX. The correlation coefficients of calibration curves(n=3) were all greater than 0.99. The RE and RSD were all less than 11％ in intra- and inter-day assay. The limits of detection were 50 nM for MP or TG (S/N=3). This method was applied for monitoring blood MP in a patient with acute lymphoblastic leukemia.
(2) Capillary electrophoresis---
A simple CZE method was established for simultaneous determination of MP、AZA and TG. The untreated fused-silica capillary was used (40 cm, effective length 30 cm, 50 μm I.D.) for the analysis. The analysis was optimized using glycine buffer (200 mM, pH 9.00) at 25 kV and detected under 214 nm. On the method validation, the calibration curves were linear (r≧0.999) over 20.0- 400.0 μM for TG or AZA, and 50.0-400.0 μM for MP. The RSD and RE were all less than 5% for the intra- and inter-day assay. All of the recoveries were greater than 95 %. The limits of detection were 5 μM for TG or AZA and 15 μM for MP (S/N=3, hydrodynamic injection 5 sec). This method was feasible for the application of commercial tablets (Lanvis®, Azapress® and Purinethol®). All of the analytical values fell within labeled amount required by the USP XXV.（TG, AZA: 93-107 %; MP: 93-110 %）.
For monitoring drug levels, capillary zone electrophoresis method was also developed for the determination of MP and its four metabolites, including thioguanosine (rTG), thioguanine (TG), methyl–mercaptopurine (MMP) and methyl-mercaptopurine riboside (rMMP) in whole blood. The untreated fused-silica capillary was used (40 cm, effective length 30 cm, 50 μm I.D.). The analysis was optimized using borate buffer (50 mM, pH 8.50) at 25 kV and detected under 214 nm. The limits of detection were 20 μM for MMP, rMMP and MP, and 100 μM for TG and rTG (S/N=3, hydrodynamic injection 5 sec). This sensitivity can not afford for drug monitoring in whole blood. On-line concentration - CE method is still under investigation.

並列關鍵字

mercaptopurine ； azathioprine ； thioguanine

參考文獻

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