【作者】 王燕萍；

【导师】 钟大放；

【作者基本信息】 浙江工业大学 ， 药物分析， 2010， 硕士

【摘要】 1.血浆中辅酶Q10定量分析方法研究和应用背景辅酶Q10是人体能自身合成的脂类物质,是线粒体呼吸链的辅因子,具有抗氧化作用。它极性较弱,体内本底浓度较高。目的建立专属、易操作的液相色谱-串联质谱法,测定人血浆中的辅酶Q10,并用于辅酶Q10的生物等效性研究。方法LC-MS/MS法测定人血浆中的辅酶Q10。血浆样品经正己烷液-液萃取两次,以甲醇为流动相,Capcell Pak C18柱(35 mm×2.0 mm I.D.,5μm)进行分离,采用大气压化学电离源,以多反应监测(MRM)方式进行正离子检测。用于定量分析的离子反应分别为m/z 864→m/z 197(辅酶Q10)和m/z 796→m/z 197(内标辅酶Q9)。结果测定血浆中辅酶Q10的线性范围为10.0 ~ 1000 ng?mL-1,定量下限可达10.0 ng?mL-1,日内、日间精密度(RSD)均小于8.9%,准确度(RE)在-0.9% ~ 3.8%之间。单个样品分析时间为4.5 min。结论本文所建立的液相色谱-串联质谱法灵敏,专属,快速,适用于辅酶Q10的血药浓度测定及其生物等效性评价。2.血浆和尿中羟乙基淀粉定量分析方法研究和应用背景羟乙基淀粉是由不同链长度和不同羟乙基取代位点的葡萄糖多聚体组成。它作为代血浆,临床上主要用于补充血容量不足。在羟乙基淀粉浓度测定时,需要将其水解为单体,通过测定葡萄糖单体浓度,从而计算羟乙基淀粉浓度。因此,在其浓度测定时,需排除内源性本底浓度的干扰,建立灵敏、专属的分析方法。目的建立灵敏、专属的定量分析方法,测定人血浆和尿中的羟乙基淀粉,并用于羟乙基淀粉200/0.5的人体药代动力学研究。方法己糖激酶法间接测定人血浆和尿样中的羟乙基淀粉。血浆和尿样先用三氯乙酸沉淀去除血浆蛋白,再用丙酮沉淀上清液中的羟乙基淀粉,最后,用三氟乙酸(油浴1.5 h,100°C)水解,采用己糖激酶法进行测定,从而计算人血浆和尿样中的羟乙基淀粉。结果测定血浆和尿中羟乙基淀粉的线性范围均为0.250 ~ 10.0 mg?mL-1,定量下限均为0.25 mg?mL-1;测定血浆中羟乙基淀粉的日内、日间精密度(RSD)均小于13.2%,准确度(RE)在-2.0% ~ -0.7%之间;测定尿中羟乙基淀粉的日内精密度小于4.8%,准确度(RE)在-3.9% ~ -1.2%之间。平均8 min能测定50个经预处理后的样品。结论本文所建立的己糖激酶法灵敏,快速,已成功用于羟乙基淀粉200/0.5注射液的临床药代动力学研究。更多还原

【Abstract】 1. Quantitative analysis of coenzyme Q10 in human plasma and its applicationsBackground Coenzyme Q10 (CoQ10) is an endogenous enzyme cofactor that exsits in all living cells in humans. It is an electron carrier in the mitochondrial respiratory chain and act as a lipid-soluble antioxidant.Objective To develop and validate a specific and rapid liquid chromatographic?tandem mass spectrometric method for determination of coenzyme Q10 in human plasma and application to the bioequivalence evaluation.Method Determination of coenzyme Q10 in human plasma by liquid chromatography-tandem mass spectrometry. The plasma sample was extracted twice with n-hexane, then separated on a Capcell Pak C18(35 mm×2.0 mm I.D.,5μm)column using methanol as the mobile phase. A triple quadrupole mass spectrometry equipped with atmospheric pressure chemical ionization (APCI) source was used as the detector and operated in the positive ion mode. Quantitation was performed using multiple reaction monitoring (MRM) of the transitions of m/z 864→m/z 197 and m/z 796→m/z 197 for CoQ10 and the internal standard CoQ9, respectively.Results The linear concentration range of the calibration curve for coenzyme Q10 was 10.0 ? 1000 ng?mL-1, The lower limit of quantification was 10.0 ng?mL-1. The intra-day and inter-day relative standard deviation over the entire concentration range were less than 8.9%. The accuracy was in the range of -0.9% to 3.8% in terms of relative error. Each sample was chromatographed within 4.5 min.Conclusion The validated liquid chromatographic?tandem mass spectrometric method was sensitive, specific, rapid and suitable for the bioequivalence evaluation of coenzyme Q10 in humans.2. Quantitative analysis of hydroxyethyl starch in human plasma and urine and their applicationsBackground Hydroxyethyl starch is a mixture of glucose polymers of varying chain length and various substitution with hydroxyethyl groups. It is necessary to hydrolyze hydroxyethly starch (HES) into glucose or hydroxyethyl glucose monomers when analysis its concentration in samples. Glucose is an endogenous substance. It is fundamental to develop a sensitive and specific method to eliminate the interference of endogenous glucose and quantify the concenteation of glucose monomers after hydrolysis in human plasma and urine samples.Objective To develop and validate a specific, sensitive and rapid analytical method for indirect determination of hydroxyethly starch in human plasma and application to the clinical pharmacokinetic study. Method Indirect determination of hydroxyethly starch in human plasma and urine by hexokinase method. Plasma and urine samples were deproteinated by trichloroacetic acid followed by centrifugation. Supernatant centrifugate was precipitated by acetone, then acidly hydrolyzed with 4M trifluoroaceticacid (1.5 h, 100°C.) to produce monomers. Glucose determination performed using an enzymatic test kit based on hexokinase/glucose-6-phosphatese and hydroxyethly starch concentration was calculated according to the determined concentration.Results The linear concentration range of the calibration curve was 0.25-10.0 mg?mL-1 and the lower limit of quantification was 0.25 mg?mL-1. The intra-day and inter-day relative standard deviation of plasma samples’determination over the entire concentration range were less than 13.2%. The accuracy was in the range of -2.0% to -0.7% in terms of relative error. The intra-day relative standard deviation of urine samples’determination was less than 4.8%. The accuracy was in the range of -3.9% to -1.2%. The mean analysis time was just 8 min for 50 processed samples.Conclusion The validated hexokinase analytical method was sensitive, rapid and suitable for the clinical pharmacokinetic study of hydroxyethly starch in humans.更多还原