HPLC测定大鼠全血中虫草素药代动力学方法的建立
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摘要
建立一种灵敏、稳定的检测血液中虫草素及其代谢物的高效液相色谱方法,并报告采用该方法进行药代动力学的初步研究。大鼠全血0.5 mL,加入甲醇1 mL,涡旋振荡2 min,超声10 min,4℃条件下10 000 r·min~(-1)离心15 min,取上清室温条件下氮气吹干,用体积分数为0.06的乙腈溶液0.5 mL复溶,4℃条件下15 000 r·min~(-1)离心10 min,取上清过0.22μm微孔滤膜进行HPLC检测;色谱条件为Agilent TC-C18色谱柱(4.6 mm×250 mm,5μm),流动相为乙腈/水,6:94(V/V),流速0.8 mL·min~(-1),柱温30℃,紫外检测波长259 nm。虫草素在0.1～5.0μg·mL-1浓度范围内线性关系良好,线性回归方程为Y=10.97X-11.73,相关系数r=0.998 0血液样品中虫草素在高、中、低浓度的回收率分别为99.63%、95.37%和93.26%。全血中虫草素的药物代谢动力学初步研究的结果为给药后10～120 min内均未检测到虫草素原型,相同色谱条件下,保留时间为8.8 min左右检测到一虫草素的未知代谢物,且该物质在30 min时含量最高,随后逐渐降低。对该产物富集后通过质谱检测得未知代谢物分子量为252 M。该方法灵敏度高,重现性好,可用于大鼠全血中虫草素的代谢研究;研究结果同时表明,虫草素在大鼠体内代谢速度快,按40 mg·kg~(-1)剂量灌胃,即使灌胃给药后10 min也未检测到虫草素原型。
It was aimed to establish a sensitive and stable HPLC method for the determination of Cordycepin and its metabolites in blood and to report the preliminary study of pharmacokinetics.The rat whole blood 0.5 mL was added with 1 mL of methanol,vortexed for 2 min,sonicated for 10 min and centrifuged at 10 000 r·min~(-1) for 15 min at 4 ℃,the supernatant was dried under nitrogen at room temperature with volume fraction of 0.06 in acetonitrile of 0.5 m L reconstituted,centrifuged at 15 000 r·min~(-1) at 4℃for 10 min,the supernatant was filtered through a 0.22 μm microporous membrane for HPLC detection;the chromatographic conditions were Agilent TC-C18 column(4.6 mm×250 mm,5 μm).The mobile phase consisted of acetonitrile/water,6:94(V/V),the flow rate was 0.8 m L·min~(-1),the column temperature was 30 ℃ and the UV detection wavelength was 259 nm. Cordycepin had a good linearity in the range of 0.1～5.0 μg·mL-1 with a linear regression equation of Y=10.97 X-11.73 and a correlation coefficient of r=0.998 0.The recoveries of Cordycepin in the blood samples were 99.63%,95.37% and 93.26% at high,medium and low concentrations,respectively.Using this condition,the pharmacokinetics of Cordycepin in whole blood were studied preliminarily:no prototype of Cordycepin was detected within 10～120 min after administration,and the retention time was 8.8 min under the same chromatographic conditions Cordycepin unknown metabolites,and the substance at 30 min when the highest content,then gradually decreased. The mass of the unknown metabolite detected by mass spectrometry after enrichment of this product was 252 M.The method had high sensitivity and good reproducibility and could be used to study the metabolism of Cordycepin in rat whole blood.The results also showed that Cordycepin was metabolized rapidly in rats at a dose of 40 mg·kg~(-1),even if Cordycepin was not detected at 10 min after intragastric administration.
It was aimed to establish a sensitive and stable HPLC method for the determination of Cordycepin and its metabolites in blood and to report the preliminary study of pharmacokinetics.The rat whole blood 0.5 mL was added with 1 mL of methanol,vortexed for 2 min,sonicated for 10 min and centrifuged at 10 000 r·min~(-1) for 15 min at 4 ℃,the supernatant was dried under nitrogen at room temperature with volume fraction of 0.06 in acetonitrile of 0.5 m L reconstituted,centrifuged at 15 000 r·min~(-1) at 4℃for 10 min,the supernatant was filtered through a 0.22 μm microporous membrane for HPLC detection;the chromatographic conditions were Agilent TC-C18 column(4.6 mm×250 mm,5 μm).The mobile phase consisted of acetonitrile/water,6:94(V/V),the flow rate was 0.8 m L·min~(-1),the column temperature was 30 ℃ and the UV detection wavelength was 259 nm. Cordycepin had a good linearity in the range of 0.1～5.0 μg·mL-1 with a linear regression equation of Y=10.97 X-11.73 and a correlation coefficient of r=0.998 0.The recoveries of Cordycepin in the blood samples were 99.63%,95.37% and 93.26% at high,medium and low concentrations,respectively.Using this condition,the pharmacokinetics of Cordycepin in whole blood were studied preliminarily:no prototype of Cordycepin was detected within 10～120 min after administration,and the retention time was 8.8 min under the same chromatographic conditions Cordycepin unknown metabolites,and the substance at 30 min when the highest content,then gradually decreased. The mass of the unknown metabolite detected by mass spectrometry after enrichment of this product was 252 M.The method had high sensitivity and good reproducibility and could be used to study the metabolism of Cordycepin in rat whole blood.The results also showed that Cordycepin was metabolized rapidly in rats at a dose of 40 mg·kg~(-1),even if Cordycepin was not detected at 10 min after intragastric administration.
引文
[1]钟丽萍,李瑾,王凤忠,等.虫草素对ob/ob小鼠非酒精性脂肪肝的改善作用及机制研究[J].药学学报,2017,52(1):106-112.
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[9]潘传江,林莉,王竹青,等.微波-超声波协同作用蛹虫草虫草素提取条件的优化[J].贵州农业科学,2017,45(1):127-130.
[10]高玉玲,刘少斌,孙鹏.UHPLC-MS/MS法测定大米中丁草胺和吡虫啉残留[J].黑龙江八一农垦大学学报,2018,30(1):33-37.
[11]田野,王传喜,钱正明,等.在线固相萃取-HPLC测定4种虫草类药材中虫草素和2’-脱氧腺苷[J].中国中药杂志,2017,42(10):1932-1938.
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[13]谷珊山,李广领,郭梅燕,等.分散固相萃取—高效液相色谱检测花生中咪唑乙烟酸残留[J].河南科技学院学报:自然科学版,2014(2):31-34.
[14]张敬茹,于小桐,孙宁,等.桑黄素和乙酰化白藜芦醇对沙奎那韦在大鼠体内药代动力学的影响[J].中国药理学通报,2017,33(10):1414-1420.
[15]陈庆文,杨积慧,郝自新,等.高效液相色谱法同时测定独一味分散片中两种活性成分的含量[J].安徽科技学院学报,2017(6):82-85.
[16]杨云,赵克斌,杨泽华,等.利用操作过程规范图设计腺苷脱氨酶测定的室内质控方法[J].中国药物与临床,2012,12(2):263-264.
[17]吕丹.虫草素羧甲基壳聚糖纳米粒的制备、释药性能及体外抗肿瘤作用研究[D].扬州:扬州大学,2015.
[18] H P Wei,X L Ye,Z Chen,et al.Synthesis and pharmacokinetic evaluation of novel N-acyl-cordycepin derivatives with a normal alkyl chain[J].European Journal of Medicinal Chemistry,2009,44:665-669.
[19]齐真真,沈新南,吴岷.反相高效液相色谱法测定大鼠全血中虫草素含量[J].卫生研究,2015,44(4):678-680.
[20] Yung Jen Tsai,Lie Chwen Lin,Tung Hu Tsai. Pharmacokinetics of Adenosine and Cordycepin,a Bioactive Constituent of Cordyceps sinensis in Rat[J].J Agric. Food Chem,2010,58:4638-4643.
[21] E Szabados,R I Christopherson. Rapid radioassay for metabolites of adenosine and deoxyadenosine in erythrocytes[J].Journal of Chromatography B,1995,674:132-137.
[2]董宏鸿,李建军,周行,等.蛹虫草中虫草素的提取及药理活性研究进展[J].吉林医药学院学报,2016,37(6):465-467.
[3]散丹,贾海侠,嘎比日,等.HPLC法测定蒙药胡日查-6味丸中没食子酸的含量[J].内蒙古民族大学学报:自然科学版,2013(5):568-571.
[4]李婧.虫草素的体内代谢特点及药理作用[J].国外医学中医中药分册,2005,27(5):283-285.
[5]胡贤达,岳颖,武鹏,等.虫草素药理作用研究及展望[J].中国生化药物杂质,2015,12(35):180-183.
[6] Chang W.Cordycepin inhibits vascular smooth muscle cell proliferation[J].Eur J Pharmacol,2008,597(1-3):64-69.
[7]田香,玉华,白玉霞.HPLC法测定蒙药如达七味散中栀子苷的含量[J].内蒙古民族大学学报:自然科学版,2016(2):152-155.
[8]李娜,单媛媛,武德珍,等.虫草素抑制肝癌转移及分子机制研究[J].药学学报,2017(7):1117-1121.
[9]潘传江,林莉,王竹青,等.微波-超声波协同作用蛹虫草虫草素提取条件的优化[J].贵州农业科学,2017,45(1):127-130.
[10]高玉玲,刘少斌,孙鹏.UHPLC-MS/MS法测定大米中丁草胺和吡虫啉残留[J].黑龙江八一农垦大学学报,2018,30(1):33-37.
[11]田野,王传喜,钱正明,等.在线固相萃取-HPLC测定4种虫草类药材中虫草素和2’-脱氧腺苷[J].中国中药杂志,2017,42(10):1932-1938.
[12]李兵,张文成,王冰嵩,等.HPLC法同步测定蛹虫草中虫草素及腺苷的含量[J].安徽化工,2016,42(4):105-106.
[13]谷珊山,李广领,郭梅燕,等.分散固相萃取—高效液相色谱检测花生中咪唑乙烟酸残留[J].河南科技学院学报:自然科学版,2014(2):31-34.
[14]张敬茹,于小桐,孙宁,等.桑黄素和乙酰化白藜芦醇对沙奎那韦在大鼠体内药代动力学的影响[J].中国药理学通报,2017,33(10):1414-1420.
[15]陈庆文,杨积慧,郝自新,等.高效液相色谱法同时测定独一味分散片中两种活性成分的含量[J].安徽科技学院学报,2017(6):82-85.
[16]杨云,赵克斌,杨泽华,等.利用操作过程规范图设计腺苷脱氨酶测定的室内质控方法[J].中国药物与临床,2012,12(2):263-264.
[17]吕丹.虫草素羧甲基壳聚糖纳米粒的制备、释药性能及体外抗肿瘤作用研究[D].扬州:扬州大学,2015.
[18] H P Wei,X L Ye,Z Chen,et al.Synthesis and pharmacokinetic evaluation of novel N-acyl-cordycepin derivatives with a normal alkyl chain[J].European Journal of Medicinal Chemistry,2009,44:665-669.
[19]齐真真,沈新南,吴岷.反相高效液相色谱法测定大鼠全血中虫草素含量[J].卫生研究,2015,44(4):678-680.
[20] Yung Jen Tsai,Lie Chwen Lin,Tung Hu Tsai. Pharmacokinetics of Adenosine and Cordycepin,a Bioactive Constituent of Cordyceps sinensis in Rat[J].J Agric. Food Chem,2010,58:4638-4643.
[21] E Szabados,R I Christopherson. Rapid radioassay for metabolites of adenosine and deoxyadenosine in erythrocytes[J].Journal of Chromatography B,1995,674:132-137.