高效液相色谱—荧光法测定他莫西芬及其代谢物血药浓度及应用
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摘要
目的
建立高效液相荧光法检测他莫西芬及其主要代谢物血药浓度,并应用于人体生物利用度研究,为临床合理用药及新药报批提供参考依据。
方法
上部主要内容为他莫西芬及其主要代谢物的血药浓度检测方法的建立。实验方法为血浆样品经样品前处理后,以正己烷-正丁醇(98:2,v/v)提取。应用离线紫外照射(254 nm,10 min)激发其荧光后,再以HPLC进行分离,荧光检测器进行检测。色谱条件为:流动相:甲醇-1%三乙胺水溶液(82:18,v/v);流速:1 ml·min~(-1);色谱柱:Agilent Extend C_(18)(4.6 mm×150 mm,5μm);柱温:50℃。荧光检测波长:激发波长(λ_(ex))260 nm,发射波长(λ_(em))375 nm。
下部为枸橼酸他莫西芬分散片的人体药代动力学及相对生物利用度研究。以市售枸橼酸他莫西芬片为标准对照,进行生物等效性试验。采用上部所建立的高效液相色谱荧光法来测定20名健康受试者随机双交叉口服枸橼酸他莫西芬分散片与标准对照后,他莫西芬的血药浓度变化。应用非房室模型法估算药代动力学参数,统计学软件进行分析,比较两种药物的人体生物等效性。
结果
上部结果:在上述色谱条件下,慢心律(内标)、4-羟基他莫西芬、N-去甲他莫西芬、他莫西芬的出峰时间分别在2.4 min、3.9 min、9.9min、11 min左右。各目标化合物峰形良好,母体化合物和各代谢物能有效分离,无杂峰干扰测定,基线平稳。本方法具有较高的特异性,能准确测定血浆中他莫西芬及代谢物的浓度,灵敏度较高。血浆中杂质不干扰样品的测定。标准曲线线性范围分别为:TAM:0.5ng·mL~(-1)～200ng·mL~(-1),DMT:0.5ng·mL~(-1)～200ng·mL~(-1),OHT:0.1ng·mL~(-1)～10ng·mL~(-1),标准曲线线性关系(r_(TAM)=0.9994,r_(OHT)=0.9994,r_(DMT)=0.9988)良好。定量下限分别为0.5 ng·mL~(-1),0.5 ng·mL~(-1),0.1ng·mL~(-1)。各化合物高、中、低三种浓度的批间、批内变异均小于15%,绝对回收率在92.1%～95.5%范围内,相对回收率在101.1%～113.0%范围内,样品稳定性良好,符合GCP及药物(化学药品)制剂人体生物利用度和生物等效性试验指导原则生物样品分析要求。
下部结果:20名健康受试者口服20mg受试枸橼酸他莫西芬分散片后,估算的他莫西芬的消除半衰期为(143.26±24.52)h,达峰时间和达峰浓度分别为(6.3±2.2)h和(71.82±14.35)ng·mL~(-1),AUC_(0-τ)为(4608.43±2054.50)ng·h·mL~(-1);口服对照枸橼酸他莫西芬片后,估算的他莫西芬的消除半衰期为(153.00±32.91)h,达峰时间和达峰浓度分别为(6.7±2.4)h和(68.54±15.77)ng·mL~(-1),AUC_(0-τ)为(4586.05±2026.23)ng·h·mL~(-1),两种枸橼酸他莫西芬制剂的药代动力学参数相近,相对生物利用度为(101.3±12.9)%,生物利用度符合要求。对参数C_(max)、AUC_(0-∞)进行双单侧t检验,t_(max)进行非参数法检验,其主要药动学参数无显著性差异(P＞0.05),结果表明两种枸橼酸他莫西芬制剂生物等效。
结论
本研究建立的检测他莫西芬及其主要代谢物血药浓度的高效液相色谱-荧光分析法,具有快速、灵敏、专属性强、操作简便等特点,可为该类药物临床血药浓度监测提供科学可行的技术基础。并可以推广应用于该类药物的临床研究。
Aim
To set a high performance liquid chromatography method with fluorescent detection for quantification of tamoxifen and its major metabolites.Then apply the method to study the pharmacokinetics and relative bioavailability of citric tamoxifen dispersible tablet in Chinese healthy volunteers to validate the method we established.
Methods
In first part,a high performance liquid chromatography method with fluorescent detection for quantification of tamoxifen and its major metabolites has been established.Plasma samples were treated by n-hexane:n-butanol(98:2,v/v),and exposed to a 254-nm UV lamp offline for 10 min.Separation was carried out on the column of Agilent Extend C_(18)(150 mm×4.6 mm,5μm)at 50℃.The mobile phase consisted of methanol:1%triethylamine aqueous solutions(82:18,v/v)pumped at a flow rate of 1 mL·min~(-1).The fluorescence detector was operated at an excitation and emission wavelengths 260 and 375 nm,respectively.
In second part,the pharmacokinetics and relative bioavailability of tamoxifen citrate dispersible tablets in Chinese healthy volunteers was studied.In a randomized two period crossover study,20 healthy volunteers received tested and reference tablets 20 mg.The plasma concentrations of tamoxifen were determined by HPLC method established in the first part.The pharmacokinetics of tamoxifen were estimated by non-compartment model.
Results
In first part,in the conditions described above,TAM,DMT and OHT exhibited good chromatography with baseline resolution of each compound.The method described in our paper was selective and specific. There were no foreign peaks interfered with analytes and internal standard at the retention times.The retention times for internal standard, OHT,DMT,TAM were 2.4,3.9,9.9 and 11 min,respectively.The linear range of TAM,DMT and OHT were 0.5～200 ng·mL~(-1),0.5ng·mL~(-1)～200ng·mL~(-1),0.1ng·mL~(-1)～10ng·mL~(-1).Lower limit of quantification (LLOQ)was 0.5ng·mL~(-1),0.5ng·mL~(-1),0.1ng·mL~(-1).All the inter-day and intra-day precisions were less than 15%.The stability of unprocessed and processed samples stored at 4℃was good.After 4 cycles of freeze and thaw processes,the plasma samples were stable.
In second part,in a randomized two period crossover study,the main pharmacokinetics parameters of tested and reference tablets were as the following:t_(max):(6.3±2.2),(6.7±2.4)h;C_(max):(72±14),(68±16)μg/L; AUC_(0-τ):(4608.43±2054.50),(4586.05±2026.23)ng·h·mL~(-1);t_(1/2):(143±24),(154±33)h,respectively.The relative bioavailability was(101.3±12.9)%.According to the data of t_(max),C_(max),AUG_(0-τ)between two tablets, they have the same bioequivalence.
Conclusions
The method presented here describes a specific,sensitive and reproducible human plasma assay using HPLC with fluorescence detection for the determination and quantification of TAM and its metabolites.And the method was validated by the pharmacokinetics of TAM in a clinical study.It demonstrated that the HPLC-FLU method we set can be applied in the clinical study of TAM kind of drugs.
建立高效液相荧光法检测他莫西芬及其主要代谢物血药浓度,并应用于人体生物利用度研究,为临床合理用药及新药报批提供参考依据。
方法
上部主要内容为他莫西芬及其主要代谢物的血药浓度检测方法的建立。实验方法为血浆样品经样品前处理后,以正己烷-正丁醇(98:2,v/v)提取。应用离线紫外照射(254 nm,10 min)激发其荧光后,再以HPLC进行分离,荧光检测器进行检测。色谱条件为:流动相:甲醇-1%三乙胺水溶液(82:18,v/v);流速:1 ml·min~(-1);色谱柱:Agilent Extend C_(18)(4.6 mm×150 mm,5μm);柱温:50℃。荧光检测波长:激发波长(λ_(ex))260 nm,发射波长(λ_(em))375 nm。
下部为枸橼酸他莫西芬分散片的人体药代动力学及相对生物利用度研究。以市售枸橼酸他莫西芬片为标准对照,进行生物等效性试验。采用上部所建立的高效液相色谱荧光法来测定20名健康受试者随机双交叉口服枸橼酸他莫西芬分散片与标准对照后,他莫西芬的血药浓度变化。应用非房室模型法估算药代动力学参数,统计学软件进行分析,比较两种药物的人体生物等效性。
结果
上部结果:在上述色谱条件下,慢心律(内标)、4-羟基他莫西芬、N-去甲他莫西芬、他莫西芬的出峰时间分别在2.4 min、3.9 min、9.9min、11 min左右。各目标化合物峰形良好,母体化合物和各代谢物能有效分离,无杂峰干扰测定,基线平稳。本方法具有较高的特异性,能准确测定血浆中他莫西芬及代谢物的浓度,灵敏度较高。血浆中杂质不干扰样品的测定。标准曲线线性范围分别为:TAM:0.5ng·mL~(-1)～200ng·mL~(-1),DMT:0.5ng·mL~(-1)～200ng·mL~(-1),OHT:0.1ng·mL~(-1)～10ng·mL~(-1),标准曲线线性关系(r_(TAM)=0.9994,r_(OHT)=0.9994,r_(DMT)=0.9988)良好。定量下限分别为0.5 ng·mL~(-1),0.5 ng·mL~(-1),0.1ng·mL~(-1)。各化合物高、中、低三种浓度的批间、批内变异均小于15%,绝对回收率在92.1%～95.5%范围内,相对回收率在101.1%～113.0%范围内,样品稳定性良好,符合GCP及药物(化学药品)制剂人体生物利用度和生物等效性试验指导原则生物样品分析要求。
下部结果:20名健康受试者口服20mg受试枸橼酸他莫西芬分散片后,估算的他莫西芬的消除半衰期为(143.26±24.52)h,达峰时间和达峰浓度分别为(6.3±2.2)h和(71.82±14.35)ng·mL~(-1),AUC_(0-τ)为(4608.43±2054.50)ng·h·mL~(-1);口服对照枸橼酸他莫西芬片后,估算的他莫西芬的消除半衰期为(153.00±32.91)h,达峰时间和达峰浓度分别为(6.7±2.4)h和(68.54±15.77)ng·mL~(-1),AUC_(0-τ)为(4586.05±2026.23)ng·h·mL~(-1),两种枸橼酸他莫西芬制剂的药代动力学参数相近,相对生物利用度为(101.3±12.9)%,生物利用度符合要求。对参数C_(max)、AUC_(0-∞)进行双单侧t检验,t_(max)进行非参数法检验,其主要药动学参数无显著性差异(P＞0.05),结果表明两种枸橼酸他莫西芬制剂生物等效。
结论
本研究建立的检测他莫西芬及其主要代谢物血药浓度的高效液相色谱-荧光分析法,具有快速、灵敏、专属性强、操作简便等特点,可为该类药物临床血药浓度监测提供科学可行的技术基础。并可以推广应用于该类药物的临床研究。
Aim
To set a high performance liquid chromatography method with fluorescent detection for quantification of tamoxifen and its major metabolites.Then apply the method to study the pharmacokinetics and relative bioavailability of citric tamoxifen dispersible tablet in Chinese healthy volunteers to validate the method we established.
Methods
In first part,a high performance liquid chromatography method with fluorescent detection for quantification of tamoxifen and its major metabolites has been established.Plasma samples were treated by n-hexane:n-butanol(98:2,v/v),and exposed to a 254-nm UV lamp offline for 10 min.Separation was carried out on the column of Agilent Extend C_(18)(150 mm×4.6 mm,5μm)at 50℃.The mobile phase consisted of methanol:1%triethylamine aqueous solutions(82:18,v/v)pumped at a flow rate of 1 mL·min~(-1).The fluorescence detector was operated at an excitation and emission wavelengths 260 and 375 nm,respectively.
In second part,the pharmacokinetics and relative bioavailability of tamoxifen citrate dispersible tablets in Chinese healthy volunteers was studied.In a randomized two period crossover study,20 healthy volunteers received tested and reference tablets 20 mg.The plasma concentrations of tamoxifen were determined by HPLC method established in the first part.The pharmacokinetics of tamoxifen were estimated by non-compartment model.
Results
In first part,in the conditions described above,TAM,DMT and OHT exhibited good chromatography with baseline resolution of each compound.The method described in our paper was selective and specific. There were no foreign peaks interfered with analytes and internal standard at the retention times.The retention times for internal standard, OHT,DMT,TAM were 2.4,3.9,9.9 and 11 min,respectively.The linear range of TAM,DMT and OHT were 0.5～200 ng·mL~(-1),0.5ng·mL~(-1)～200ng·mL~(-1),0.1ng·mL~(-1)～10ng·mL~(-1).Lower limit of quantification (LLOQ)was 0.5ng·mL~(-1),0.5ng·mL~(-1),0.1ng·mL~(-1).All the inter-day and intra-day precisions were less than 15%.The stability of unprocessed and processed samples stored at 4℃was good.After 4 cycles of freeze and thaw processes,the plasma samples were stable.
In second part,in a randomized two period crossover study,the main pharmacokinetics parameters of tested and reference tablets were as the following:t_(max):(6.3±2.2),(6.7±2.4)h;C_(max):(72±14),(68±16)μg/L; AUC_(0-τ):(4608.43±2054.50),(4586.05±2026.23)ng·h·mL~(-1);t_(1/2):(143±24),(154±33)h,respectively.The relative bioavailability was(101.3±12.9)%.According to the data of t_(max),C_(max),AUG_(0-τ)between two tablets, they have the same bioequivalence.
Conclusions
The method presented here describes a specific,sensitive and reproducible human plasma assay using HPLC with fluorescence detection for the determination and quantification of TAM and its metabolites.And the method was validated by the pharmacokinetics of TAM in a clinical study.It demonstrated that the HPLC-FLU method we set can be applied in the clinical study of TAM kind of drugs.
引文
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[5]Gallicchio L,Lord G,Tkaczuk K,et al.Association of tamoxifen (TAM)and TAM metabolite concentrations with self-reported side effects of TAM in women with breast cancer.[J]Breast Cancer Res.Treat.85(2004)1:89-97
[6]汤仲明,刘秀文,柴彪新.蛋白质多肽类药物药代动力学研究的方法学和实验设计.[J].中国药理学与毒理学杂志 10(1996)3;16:1-16
[7]Schild LJ,Phillips DH,Osborne MR,et al.Hepatic DNA adduct dosimetry in rats fed tamoxifen:a comparison of methods.[J].Mutagenesis 20(2005)2:115-124
[8]Sanders J.M, Burka L T, Shelby BT Determination of tamoxifen and metabolites in serum by capillary electrophoresis using a nonaqueous buffer system. [J]. J Chromatogr B Biomed Sci Appl. 695(1997) 18:181-185
[9] Lu W, Poon GK, Carmichael PL, et al. Analysis of tamoxifen and its metabolites by on-line capillary electrophoresis-electrospray ionization mass spectrometry employing nonaqueous media containing surfactants.[J] Anal Chem 68 (1996); 15 (4):668-674
[10]Baez H, Camargo C, Osorio H, et al. Detection of tamoxifen metabolites by GC-MSD.[J].J Chromatogr Sci. 42 ( 2004) 10:551-553
[11]Brown RR, Bain R, Jordan VC. Determination of tamoxifen and metabolites in human serum by high-performance liquid chromatography with post-column fluorescence activation. [J] J Chromatogr. 272(1983)11:351-358
[12]Manns JE, Hanks S, Brown JE. Optimised separation of E- and Z-isomers of tamoxifen, and its principal metabolites using reversed-phase high performance liquid chromatography. [J] J Pharm Biomed Anal. 16(1998)5:847-852
[13]Kisanga ER, Mellgren G, Lien EA. Excretion of hydroxylated metabolites of tamoxifen in human bile and urine. [J] .Anticancer Res. 25(2005)6C:4487-4492
[14]Decensi A, Robertson C, Viale, F et al. A randomized trial of low-dose tamoxifen on breast cancer proliferation and blood estrogenic biomarkers.[J].J Natl Cancer Inst.95(2003)11:779-790
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