【作者】 曹玉；

【导师】 王春波； 李德爱；

【作者基本信息】 青岛大学 ， 药理学， 2008， 硕士

【摘要】 目的:建立专属、灵敏、简便的检测人血浆和尿液中左卡尼汀（L-carnitine,LC）及其酰化物乙酰左卡尼汀（Acetyl-L-cainitine,ALC）、丙酰左卡尼汀（Propionyl-L-cainitine,PLC）含量的高效液相色谱方法,并应用于中国健康人左卡尼汀口服液单次给药后左卡尼汀及其酰化物的人体药代动力学研究。方法:HPLC-UV法:血浆样本经蛋白沉淀处理后,用对-溴苯甲酰甲基溴化物（PBPB）将样本紫外衍生化,高效液相色谱法测定,采用Hypersil SiO2色谱柱（4.6×250mm;5μm）,流动相为乙腈:柠檬酸缓冲液（10:90）,流速1.2ml·min^-1,检测波长260nm。HPLC-FL法:血浆样本经蛋白沉淀处理后,用1-氨基蒽（1-AA）将样本荧光衍生化,高效液相色谱方法测定,色谱柱采用Hypersil C₁₈柱（4.6mm×200mm,5μm）,流动相为乙腈-0.1mol·L^-1乙酸铵（34:66）,流速为1.0ml·min^-1,检测波长:Ex248nm,Em418nm。绘制标准曲线,测定精密度、回收率和稳定性,检测正常人血浆及尿液的左卡尼汀及其酰化物的含量。12名健康受试者左卡尼汀口服液单次给药2g后,分别于服药前（0h）和服药后0.5h,1.0h,1.5h,2h,2.5h,3.0h,3.5h,4.0h,5.0h,6.0h,8.0h,12.0h,24.0h,取前臂静脉血4ml,置肝素试管中,3000r·min^-1离心,分离血浆,同时收集给药后0h,0～2h,2～4h,4～8h,8～12h,12～24 h的尿液,-20℃冷冻待测。采用荧光衍生化高效液相色谱法测定血浆和尿中LC,ALC,PLC的含量。采用DAS软件进行处理,计算药代动力学参数。结果:紫外高效液相色谱法的血浆LC线性范围为2.5～500μmol·L^-1,尿液LC的线性范围为2～1000μmol·L^-1,回收率、精密度及稳定性试验均符合血、尿检测方法学的要求。荧光高效液相色谱法的血浆和尿液LC、ALC和PLC样品的线性范围分别是2.5～500,0.5～50,0.1～20μmol·L^-1和2～1000,1～500,0.2～50μmol·L^-1。回收率、精密度及稳定性试验均符合血、尿检测方法学的要求。采用荧光高效液相色谱法对12名中国健康受试者左卡尼汀口服液单次给药2g后左卡尼汀及酰化物进行药代动力学研究,主要药代动力学参数LC:Cmax:（84.73±25.23）μmol·L^-1,t_1/2α:（1.66±1.20）h,t_1/2β:（60.33±14.97）h,AUC_（0-t）:（2676.41±708.33）μmol·L^-1·h,AUC_（0-∞）:（2676.41±708.33）μmol·L^-1·h,Tmax:（3.4±0.46）h。ALC:t_1/2α:（6.33±6.39）h,t_1/2β（5.90±28.86）h,AUC_（0-t）:（119.54±55.84）μmol·L^-1·h,AUC_（0-∞）:（166.20±77.41）μmol·L^-1·h,Tmax:（4±0.66）h,Cmax:（12.89±5.52）μmol·L^-1和PLC:t_1/2α:（3.10±3.93）h,t_1/2β:（25.74±30.33）h,AUC_（0-t）:（57.98±48.52）μmol·L^-1·h,AUC_（0-∞）:（155.57±264.22）μmol·L^-1·h,Tmax:（3.8±0.79）h,Cmax.（5.08±3.08）μmol·L^-1。LC、ALC、PLC 24小时尿药累计排泄量分别为:（613.47±161.72）μmol,（368.25±134.77）μmol,（61.29±37.75）μmol,LC24小时累计排泄率为6.05%。结论:建立了同时检测血浆和尿液中左卡尼汀浓度的紫外衍生化高效液相色谱法和同时检测血浆和尿液中左卡尼汀及酰化物的荧光衍生化高效液相色谱法。采用荧光衍生化高效液相色谱法,首次测定了12名中国健康受试者左卡尼汀口服液单次给药2g后左卡尼汀及酰化物的药代动力学参数。更多还原

【Abstract】 Subject:To develop specific,sensitive and simple HPLC methods to determine L-carnitine（LC） and its acyl esters Acetyl-L-cainitine（ALC） and Propionyl-L-cainitine（PLC） levels in human plasma and urine.And HPLC method was used in pharmacokinetic study of L-carnitine and its acyl esters after single oral administration of L-carnitine oral solution in Chinese healthy volunteers.METHODS HPLC-UV:The analytes were extracted by protein precipitation and then precolumn derivatization with 2,4’-Dibromoacetophenone（PBPB） were performed. The fluorescent derivatives were separated on a Hypersil SiO₂ column,and the mobile phase consisted of acetonitrile-citrate buffer solution（10:90）,the flow rate was 1.2 ml·min^-1,the derivatives were monitored with a UV detector set at 260nm.HPLC-FL:The analytes were extracted by protein precipitation and then precolumn derivatization with 1-aminoanthracene（1-AA） were performed.The fluorescent derivatives were separated on a Hypersil C₁₈ column,and the mobile phase consisted of acetonitrile-0.1mol·L^-1 ammonium acetate（34:66）,the flow rate was 1.0ml·min^-1.The derivatives were monitored with a fluorimetric detector set at 248 nm（excitation wavelength） and 418 nm（emission wavelength）.After single oral administration of 2g L-carnitine oral solution in 12 Chinese healthy volunteers,the plasma was collected at Oh,0.5h,1.0h,1.5h,2h,2.5h,3.0h,3.5h,4.0h,5.0h, 6.0h,8.0h,12.0h,24.0h,and the urine was collected at Oh,0～2h,2～4h,4～8h,8～12h, 12～24h.The concentration of LC,ALC and PLC in plasma and urine were measured by HPLC-FL and the pharmacokinetics parameters were calculated by DAS software.RESULT:The good linearity of the assay were observed over the concentration ranges investigated,2.5～500μmol·L^-1 in plasma and 2～1000μmol·L^-1 in urine for LC by HPLC-UV and 2～500μmol·L^-1 for LC,0.5～50μmol·L^-1 for ALC,0.1～20μmol·L^-1 for PLC in plasma and 2～1000μmol·L^-1 for LC,1～500μmol·L^-1 for ALC,0.2～50μmol·L^-1 for PLC in urine by HPLC-FL.The recoveries,precision and stability test of LC,ALC and PLC correspond the requirement in plasma and urine.The methods were proved to be stable,accurate and convenient to study the concentrations of LC,ALC and PLC in clinical plasma and urine.After single oral administration of 2g L-carnitine oral solution, the main pharmacokinetic parameter of LC Cmax is（84.73+25.23）μmol·L^-1,t_1/2β is （60.33+14.97）h,AUC_（0-t） is（2676.41+708.33）μmol·L^-1·h,Tmax is（3.4+0.46）h.ALC: t_1/2α:（6.33+6.39）h,t_1/2β:（35.90±28.86）h,AUC_（0-t）:119.54±55.84μmol·L^-1·h,AUC_（0-∞）: 166.20±77.41μmol·L^-1·h,Tmax:（2.4±0.66）h,Cmax:（12.89±5.52）μmol·L^-1,and PLC:t_1/2α:（3.10±3.93）h,t_1/2β::（25.74±30.33）h,AUC_（0-t）:（57.98±48.52）μmol·L^-1·h, AUC_（0-∞）:（155.57±264.22）μmol·L^-1·h,Tmax:（3.8±0.79）h,Cmax:（5.08±3.08）μmol·L^-1. The accumulated excretion of LC,ALC and PLC were（613.47±161.72）μmol, （368.25±134.77）μmol,（61.29±37.75）μmol in 24h,respectively and the accumulated excretion rate of LC was 6.05%in 24h after administration.CONCLUSION:The specific,sensitive and simple HPLC-UV and HPLC-FL methods were developed to determine L-carnitine（LC） and its acyl esters Acetyl-L-cainitine（ALC） and Propionyl-L-cainitine（PLC） levels in human plasma and urine.The HPLC-FL method was successfully used in pharmacokinetic study of L-carnitine and its acyl esters after single oral administration of L-carnitine oral solution in Chinese healthy volunteers.更多还原