氨基葡萄糖制剂的生物利用度及其质量控制方法的研究
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摘要
本文旨在研究氨基葡萄糖的专属、灵敏的质量控制方法，首先建立了毛细管电泳-激光诱导荧光法(CE-LIF)测定两种制剂中氨基葡萄糖的含量；又建立液相色谱-串联质谱法(LC-MS／MS)测定了人血浆中的氨基葡萄糖的含量，并应用于盐酸氨基葡萄糖片的生物利用度试验中。
一、CE-LIF法测定氨基葡萄糖制剂的含量
建立了灵敏度高、专属性强的氨基葡萄糖制剂的CE-LIF法，测定盐酸氨基葡萄糖片和盐酸氨基葡萄糖胶囊两种制剂中氨基葡萄糖的含量。详细地优化了荧光衍生化的条件及毛细管电泳的分离条件。采用荧光素异硫氰酸酯(FITC)为衍生化试剂，与氨基葡萄糖样品在室温下暗处放置14h，进行衍生化反应。以荧光素钠为内标，15mmol／L硼酸盐缓冲液(pH9.2)为背景电解质溶液，输出电压为20kV，重力进样30s，进行毛细管电泳分离。激发波长为473nm，发射波长为520nm。该法线性范围为8.8～87.6μg／mL，样品的平均回收率为96.5％，精密度RSD为3.2％。
二、LC-MS／MS法测定氨基葡萄糖制剂的生物利用度
氨基葡萄糖的血浆样品很难测定。这是由于氨基葡萄糖作为一种内源性物质可以迅速地被用于其它成分的生物合成。因此，要求测定方法具有较高的灵敏度。本文建立了简便、灵敏、专属的LC-MS／MS法，测定人血浆中氨基葡萄糖的浓度。以法莫替丁为内标，血浆样品经乙腈沉淀蛋白后，以乙腈-水(70∶30，v／v)为流动相，流量0.5mL／min及Luna NH_2(150×4.6mm，I.D. 5μm)色谱柱分离，通过电喷雾离子化串联四极杆质谱，以选择离子反应监测(SRM)和正离子方式进行检测。以m／z 180→m／z 162和m／z 338→m／z 259的离子反应，分别对氨基葡萄糖和内标法莫替丁进行定量分析。氨基葡萄糖的线性范围为20.0～1000 ng／mL，定量下限为20.0 ng／mL。样品的提取回收率大于90％，日内、日间精密度(RSD)小于9％，准确度(RE)在±4.5％范围内。
本文用该法，对盐酸氨基葡萄糖片的生物利用度进行了研究。采用单剂量口服给药。18名受试者口服480 mg盐酸氨基葡萄糖后主要药动学参数T_(max)为3.1±0.7 h，C_(max)为636.5±310.1 ng／mL，t_(1／2)为0.9±0.2 h。用梯形法计算，AUC_(0-t)为1896±809.7，AUC_(0-∞)为1940±807.2 ng·h／mL，以AUC_(0-t)计算，盐酸氨基葡萄糖片的生物利用度为96.8±9.7％。该方法灵敏度高，线性范围宽，操作简便、快速，适用于药物动力学研究。
The objective is to study the sensitive and specific quantitative control methods of glucosamine in this thesis. Firstly, a capillary electrophoresis with laser-induced fluorescence detection method (CE-LIF) was developed to determine glucosamine in two preparations; Secondly, a liquid chromatography tandem mass spectrometry method (LC-MS/MS) for quantitative analysis of glucosamine levels in human plasma was established and successfully used in the bioavailability investigation of glucosamine hydrochloride tablets.1. A CE-LIF method for quantitative determination of glucosamine preparationA sensitive and specific CE-LIF method was developed for determination glucosamine in two preparations (glucosamine hydrochloride tablets and capsules). The optimization of the fluorescence derivatization and separation conditions was carried out. Fluorescein isothiocyanate (FITC) was used as the labeling reagent.The FITC solution was mixed with the glucosamine samples and laid to react for 14 hours in darkness at room temperature. Fluorescein sodium was chosen as the internal standard. The analytes were separated in background eletrolyte of 15 mmol/L borate buffer(pH9.2) by capillary electrophoresis.The voltage applied was 20 kV and sample injection was accomplished by gravity for 30 s. The excitation wavelength and the emission wavelength were 473 nm and 520 nm, respectively. The linear calibration curve was obtained in the concentration range of 8.8-87.6μg/mL. The average recovery of glucosamine in sample preparation was 96.5%. The relative standard deviation of precision was 3.2%.
2. A LC-MS/MS method for determination of the bioavailability of glueosamine preparation
The glucosamine level in human plasma was difficult to investigate. Because it is an endogenous substance which is rapidly utilized by the body for the biosynthesis of other normal constituents. The assay method available for glucosamine must have a sufficient sensitivity. A simple, sensitive and specific LC-MS/MS method was developed for determination of glucosamine in human plasma in this thesis. Glucosamine and the internal standard famotidine were extracted from plasma by protein precipitation with acetontrile. Then the analytes were chromatographed using Luna NH_2 column and a mobile phase consisted of acetontrile-water (70:30, v/v) at a flow-rate of 0.5 mL/min. Electrospray ionization (ESI) source was applied and operated in the positive ion detection mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 180→m/z 162 and m/z 338→m/z 259 was used to determine glucosamine and the internal standard famotidine. The linear calibration curve was obtained in the concentration range of 20.0～1000 ng/mL. The quantification limit of glucosamine was 20.0 ng/mL. The recovery of glucosamine in sample preparation was more than 90%.The inter-and intra-day precisions (RSD) were less than 9%, and the accuracy (relative error) was within±4.5%.
The method was applied on the study of the bioavailability of glucosamine hydrochloride tablets. Glucosamine hydrochloride(480 mg) was orally administered in a single-dose study conducted on 18 healthy volunteers. The main parameters were as follows: C_(max) of 636.5±310.1 ng/mL and T_(max) of 3.1±0.7 h were observed.The t_(1/2) value was 0.9±0.2 h. AUC_(0-t) and AUC_(0-∞) were calculated to be 1896±809.7 ng.h/mL and 1940±807.2 ng.h/mL by trapezia method, respectively. According to AUC_(0-∞), the bioavailability of glucosamine hydrochloride tablets was 96.8±9.7%. The method was sensitive, large ranged, simple, rapid and suitable to investigate glucosamine pharmacokinetics.
一、CE-LIF法测定氨基葡萄糖制剂的含量
建立了灵敏度高、专属性强的氨基葡萄糖制剂的CE-LIF法，测定盐酸氨基葡萄糖片和盐酸氨基葡萄糖胶囊两种制剂中氨基葡萄糖的含量。详细地优化了荧光衍生化的条件及毛细管电泳的分离条件。采用荧光素异硫氰酸酯(FITC)为衍生化试剂，与氨基葡萄糖样品在室温下暗处放置14h，进行衍生化反应。以荧光素钠为内标，15mmol／L硼酸盐缓冲液(pH9.2)为背景电解质溶液，输出电压为20kV，重力进样30s，进行毛细管电泳分离。激发波长为473nm，发射波长为520nm。该法线性范围为8.8～87.6μg／mL，样品的平均回收率为96.5％，精密度RSD为3.2％。
二、LC-MS／MS法测定氨基葡萄糖制剂的生物利用度
氨基葡萄糖的血浆样品很难测定。这是由于氨基葡萄糖作为一种内源性物质可以迅速地被用于其它成分的生物合成。因此，要求测定方法具有较高的灵敏度。本文建立了简便、灵敏、专属的LC-MS／MS法，测定人血浆中氨基葡萄糖的浓度。以法莫替丁为内标，血浆样品经乙腈沉淀蛋白后，以乙腈-水(70∶30，v／v)为流动相，流量0.5mL／min及Luna NH_2(150×4.6mm，I.D. 5μm)色谱柱分离，通过电喷雾离子化串联四极杆质谱，以选择离子反应监测(SRM)和正离子方式进行检测。以m／z 180→m／z 162和m／z 338→m／z 259的离子反应，分别对氨基葡萄糖和内标法莫替丁进行定量分析。氨基葡萄糖的线性范围为20.0～1000 ng／mL，定量下限为20.0 ng／mL。样品的提取回收率大于90％，日内、日间精密度(RSD)小于9％，准确度(RE)在±4.5％范围内。
本文用该法，对盐酸氨基葡萄糖片的生物利用度进行了研究。采用单剂量口服给药。18名受试者口服480 mg盐酸氨基葡萄糖后主要药动学参数T_(max)为3.1±0.7 h，C_(max)为636.5±310.1 ng／mL，t_(1／2)为0.9±0.2 h。用梯形法计算，AUC_(0-t)为1896±809.7，AUC_(0-∞)为1940±807.2 ng·h／mL，以AUC_(0-t)计算，盐酸氨基葡萄糖片的生物利用度为96.8±9.7％。该方法灵敏度高，线性范围宽，操作简便、快速，适用于药物动力学研究。
The objective is to study the sensitive and specific quantitative control methods of glucosamine in this thesis. Firstly, a capillary electrophoresis with laser-induced fluorescence detection method (CE-LIF) was developed to determine glucosamine in two preparations; Secondly, a liquid chromatography tandem mass spectrometry method (LC-MS/MS) for quantitative analysis of glucosamine levels in human plasma was established and successfully used in the bioavailability investigation of glucosamine hydrochloride tablets.1. A CE-LIF method for quantitative determination of glucosamine preparationA sensitive and specific CE-LIF method was developed for determination glucosamine in two preparations (glucosamine hydrochloride tablets and capsules). The optimization of the fluorescence derivatization and separation conditions was carried out. Fluorescein isothiocyanate (FITC) was used as the labeling reagent.The FITC solution was mixed with the glucosamine samples and laid to react for 14 hours in darkness at room temperature. Fluorescein sodium was chosen as the internal standard. The analytes were separated in background eletrolyte of 15 mmol/L borate buffer(pH9.2) by capillary electrophoresis.The voltage applied was 20 kV and sample injection was accomplished by gravity for 30 s. The excitation wavelength and the emission wavelength were 473 nm and 520 nm, respectively. The linear calibration curve was obtained in the concentration range of 8.8-87.6μg/mL. The average recovery of glucosamine in sample preparation was 96.5%. The relative standard deviation of precision was 3.2%.
2. A LC-MS/MS method for determination of the bioavailability of glueosamine preparation
The glucosamine level in human plasma was difficult to investigate. Because it is an endogenous substance which is rapidly utilized by the body for the biosynthesis of other normal constituents. The assay method available for glucosamine must have a sufficient sensitivity. A simple, sensitive and specific LC-MS/MS method was developed for determination of glucosamine in human plasma in this thesis. Glucosamine and the internal standard famotidine were extracted from plasma by protein precipitation with acetontrile. Then the analytes were chromatographed using Luna NH_2 column and a mobile phase consisted of acetontrile-water (70:30, v/v) at a flow-rate of 0.5 mL/min. Electrospray ionization (ESI) source was applied and operated in the positive ion detection mode. Selected reaction monitoring (SRM) mode using the transitions of m/z 180→m/z 162 and m/z 338→m/z 259 was used to determine glucosamine and the internal standard famotidine. The linear calibration curve was obtained in the concentration range of 20.0～1000 ng/mL. The quantification limit of glucosamine was 20.0 ng/mL. The recovery of glucosamine in sample preparation was more than 90%.The inter-and intra-day precisions (RSD) were less than 9%, and the accuracy (relative error) was within±4.5%.
The method was applied on the study of the bioavailability of glucosamine hydrochloride tablets. Glucosamine hydrochloride(480 mg) was orally administered in a single-dose study conducted on 18 healthy volunteers. The main parameters were as follows: C_(max) of 636.5±310.1 ng/mL and T_(max) of 3.1±0.7 h were observed.The t_(1/2) value was 0.9±0.2 h. AUC_(0-t) and AUC_(0-∞) were calculated to be 1896±809.7 ng.h/mL and 1940±807.2 ng.h/mL by trapezia method, respectively. According to AUC_(0-∞), the bioavailability of glucosamine hydrochloride tablets was 96.8±9.7%. The method was sensitive, large ranged, simple, rapid and suitable to investigate glucosamine pharmacokinetics.
引文
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[2] Muller-Fassbender H, Bach GL, Haase W, et al. Glucosamine sulfate compared to ibuprofen in osteoarthritis of the knee. Osteoarthritis Cartilage. 1994, 2(1), 61-69.
[3] Noack W, Fischer M, Forster KK, et al. Glucosamine sulfate in osteoarthritis of the knee. Osteoarthritis Cartilage. 1994, 2(1), 51-59.
[4] Delafuente JC. Glucosamine in the treatment of osteoarthritis. Rheum Dis Clin North Am. 2000, 26(1), 1-11, ⅶ.
[5] Zupanets IA, Bezdetko NV, Dedukh NV, et al. Experimental study of the effect of glucosamine hydrochloride on metabolic and repair processes in connective tissue structures. Eksp Kiln Farmakol. 2002, 65(6), 67-69.
[6] Houpt JB, McMillan R, Wein C, et al. Effect of glucosamine hydrochloride in the treatment of pain of osteoarthritis of the knee. J Rheumatol. 1999, 26(11), 2423-2430.
[7] Lu F, Guo H. Study on the role of glucosamine hydrochloride in the pathogenesis of osteoarthritis. Wei Sheng Yan Jiu. 2003, 32(6), 594-597.
[8] Pavelka K, Gatterova J, Olejarova M, et al. Glucosamine sulfate use and delay of progression of knee osteoarthritis: a 3-year, randomized, placebo-controlled, double-blind study. Arch Intern Med. 2002, 162(18), 2113-2123.
[9] Dodge GR, Jimenez SA. Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes. Osteoarthritis Cartilage. 2003, 11(6), 424-432.
[10] 盐酸氨基葡萄糖国家药品标准(WS1-XG-028-2001) 国家药品监督管理局发布
[11] 冉兰，张榕，文霞等．比色法测定盐酸氨基葡萄糖片的含量．华西药学杂志．2001，16(3)，217-218．
[12] United states pharmacopeia(27), 2011
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