【作者】 李宁；

【导师】 李发美；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2011， 博士

【摘要】 本文建立了多个分析降糖药物的方法,并应用于研究工作中。中药非法添加西药是一种危害性很大的造假行为,而降糖类中药是被添加情况最严重的品种之一。本文首先建立了一个超高效液相色谱-串联质谱法(UPLC-MS/MS)同时定性定量地分析降糖类中成药和保健品中可能非法添加的14种西药,包括二甲双胍、苯乙双胍、甲苯磺丁脲、氯磺丙脲、格列吡嗪、格列齐特、格列本脲、格列喹酮、格列美脲、罗格列酮、吡格列酮、瑞格列奈、那格列奈和米格列奈,其中米格列奈的分析尚未见文献报道。液相色谱分离方法采用Waters BEH C18色谱柱(2.1×50 mm I.D,1.7μm),以乙腈-水(均含0.1%甲酸)为流动相梯度洗脱,流速为0.2 mL/min;质谱检测方法采用电喷雾离子源(ESI)正离子模式多反应监测(MRM),同时监测每种降糖西药的两个离子反应。通过比较样品与对照品的保留时间、两个离子反应及两个离子反应色谱峰面积的比值进行定性鉴别,并利用强度更大更稳定的离子反应色谱峰面积进行定量测定。样品用甲醇超声提取,分析时间是5分钟。14种降糖西药的浓度与其定量离子反应色谱峰面积的线性关系良好,相关系数在0.995以上；检测限在0.030-5.45 ng/mL之间,定量限在0.11-10.9ng/mL之间；日内精密度(RSD)小于7.6%,日间精密度(RSD)小于15%,测定的准确度(RE)在-10%-7.8%之间。方法的选择性好、快速、灵敏、可靠,应用于30个中成药和保健品的分析,结果共检测到了9种降糖西药,其中添加格列本脲的情况最多,个别样品同时添加了2种或3种降糖西药,添加的量有的甚至超过了临床最大用药剂量。本文收集、整理中药中可能非法添加的14种降糖西药的基本信息(包括理化性质信息和临床用药信息)、本室建立的检测方法信息(包括薄层色谱法(TLC)、高效液相色谱-二极管阵列检测法(HPLC-DAD)、UPLC-MS/MS法)和相关的文献检测方法信息,采用Microsoft Access数据库管理系统和Visual Basic 6.0系统开发工具建立了一个中药中可能非法添加的降糖西药数据库系统。用户通过访问数据库系统可以方便、快捷地查询中药中可能非法添加的降糖西药的信息,大大地提高检测工作的效率。数据库系统界面友好、操作简便,具有信息维护(增加、修改和删除信息)、用户信息管理和系统维护的功能。格列本脲是被非法添加至降糖类中药中情况最多的一种西药,葛根素是很多降糖中成药和保健品中的组成药味葛根中的有效成分,在临床上这两者可能有意或无意地被患者合用。为研究两者在大鼠体内的药动学相互作用,本文建立了同时测定大鼠血浆中格列本脲和葛根素浓度的UPLC-MS/MS法。血浆样品经磷酸酸化后用乙酸乙酯液液萃取,以甲醇和5 mmol/L醋酸铵水溶液(含0.1%甲酸)为流动相梯度洗脱,在Waters BEH C18色谱柱(2.1×50mm I.D,1.7μm)上进行分离,在ESI正离子模式下进行MRM扫描,定量离子反应分别为m/z 417.1→m/z 297.1(葛根素)、m/z 446.2→m/z 321.1(格列吡嗪,内标)和m/z 494.2→m/z 369.1(格列本脲)。方法的专属性、线性、日内日间精密度和准确度及稳定性均符合相关规定的要求。分别灌胃给予大鼠格列本脲、葛根提取物、格列本脲和葛根提取物后,测定不同时刻血浆中格列本脲和葛根素的浓度,计算格列本脲和葛根素在单用和合用时的主要药动学参数。合用后,格列本脲的药时曲线下面积(AUC0-t、AUC0-∞)显著增加,平均滞留时间(MRT0-t、MRT0-∞)显著延长,清除率(Cl/F)显著减少,其他参数无显著性变化(P>0.05)；葛根素的达峰浓度(Cmax)显著升高,AUC0-t、AUC0-∞显著增加,表观分布容积(Vd/F)、Cl/F显著减少,其他参数无显著性变化,说明两者在合用时存在着药动学的相互影响,Cmax和AUC的增加会使药效增强,而MRT的延长和Cl/F、Vd/F的减少则容易造成毒性的蓄积,提示降糖中药中不能非法添加格列本脲,临床上也应尽量避免葛根素和格列本脲的合用,如要合用时应调整用药的剂量或监测血药浓度。格列本脲的血浆蛋白结合率大于90%,属于高血浆蛋白结合；葛根素属于中等水平血浆蛋白结合,为研究两者与大鼠血浆的蛋白结合率及其相互影响,本文分别建立了大鼠血浆和透析液中测定格列本脲的HPLC-MS/MS方法和测定葛根素的HPLC-UV方法。采用平衡透析法在37℃分别平衡20 h(格列本脲)和8h(葛根素)后,测定透析袋内血浆中药物的浓度(总浓度Dt)和透析外液中的浓度(游离药物浓度Df),计算血浆蛋白结合率。格列本脲在浓度为20.0～400 ng/mL范围内,与大鼠血浆的蛋白结合率为(99.4±0.2)%,加入4.00μg/mL葛根素后为(99.2±0.4)%,无显著性变化,表明葛根素对格列本脲与大鼠血浆的蛋白结合基本无影响。葛根素在浓度为0.200～4.00μg/mL范围内,与大鼠血浆的蛋白结合率为(66.3±4.9)%,加入400 ng/mL格列本脲后为(41.8±4.0)%,下降了36.9%,有非常显著的变化,说明格列本脲会与葛根素竞争与大鼠血浆蛋白的结合,增加葛根素的游离药物浓度,从而加大药效和毒性,进一步说明降糖中药中不能非法添加格列本脲,临床上也应尽量避免葛根素与格列本脲的合用。二甲双胍格列吡嗪片是由两种降糖西药盐酸二甲双胍和格列吡嗪组成的复方制剂,两者相辅相成,具有协同降糖的作用。本文建立了同时测定人血浆中二甲双胍和格列吡嗪浓度的HPLC-MS/MS法,并应用于二甲双胍格列吡嗪片在人体内的药物动力学研究。血浆样品经甲醇沉淀蛋白后,以甲醇和10 mmol/L醋酸铵水溶液(75：25,v/v)为流动相,在Kromasil Phenyl柱(4.6x150mmI.D,5μm)上进行分离,在ESI正离子模式下进行MRM扫描,定量离子反应分别为m/z 129.9→m/z 70.5(二甲双胍)、m/z 446.2→m/z 321.1(格列吡嗪)和m/z 180.0→m/z 109.7(非那西丁,内标)。结果二甲双胍在4.10-656ng/mL、格列吡嗪在2.55～408 ng/mL浓度范围内线性关系良好,日内日间精密度(RSD)均小于10%,测定的准确度(RE)在-7.0%-4.6%之间。健康受试者口服一片二甲双胍格列吡嗪片(盐酸二甲双胍250 mg,格列吡嗪2.5 mg)后,二甲双胍的主要药动学参数Tmax为2.61士0.78 h,Cmax为437±99 ng/mL,t1/2为5.14±1.45 h,AUC0-t为3405±1273ng·h/mL,AUC0-∞为3476±1278 ng·h/mL；格列吡嗪的主要药动学参数Tmax为2.61±0.50h,Clmax为257±20 ng/mL,t1/2为4.70±0.84 h,AUC0-t为1785±239 ng·h/mL,AUC0-∞为1842±252 ng·h/mL.更多还原

【Abstract】 In this thesis, several analysis methods for anti-diabetic drugs were studied and applied. Adulteration of Chinese medicines with synthetic drugs is a very serious problem, so an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was firstly developed for simultaneous qualitative and quantitative analysis of fourteen synthetic anti-diabetic drugs in adulterated Chinese medicines. The fourteen synthetic anti-diabetic drugs are metformin, phenformin, chlorpropamide, tolbutamide, glibenclamide, glipizide, gliclazide, gliquidone, glimepiride, pioglitazone, rosiglitazone, nateglinide, repaglinide and mitiglinide. Among them, mitiglinide was not analyzed by the published methods. The samples were prepared by ultrasonic extraction with methanol and separated on a C18 column with mobile phase consisting of acetonitrile and water (both containing 0.1% formic acid). Gradient elution was applied with a flow rate of 0.20 mL/min. Two transitions from protonated molecule were monitored for each synthetic anti-diabetic drug in positive mode of electrospray ionization (ESI). The two transitions and their peak area ratio, the retention time were used for identification. The more intensive transition was used for quantification. The analysis time was 5 min per sample. Satisfactory linear relationships were estimated between the peak area and the concentration with correlation coefficients higher than 0.995. The limit of detection (LOD) ranged from 0.030 to 5.45 ng/mL and the limit of quantification (LOQ) ranged from 0.11 to 10.9 ng/mL. The relative standard deviation (RSD) of intra-day precision was below 7.6%, the RSD of inter-day precision was below 15% and the relative error (RE) of accuracy was between-10% and 7.8%. The proposed method is rapid, selective, reliable and has been successfully applied to the analysis of 30 real samples. Nine synthetic anti-diabetic drugs were found in 14 samples, and glibenclamide was the most common adulterant. In addition, it was noted that adulteration in dietary supplements was more serious than in Chinese proprietary medicines (CPMs):7 samples were found to contain synthetic anti-diabetic drugs in the 8 examined dietary supplements and 3 synthetic anti-diabetic drugs were simultaneously detected in one dietary supplement.A database system about the information of synthetic anti-diabetic drugs adulterated in Chinese medicines was established. The basic information of synthetic anti-diabetic drugs includes physicochemical properties and clinical administration information; the information on the detection of synthetic anti-diabetic drugs using TLC method, HPLC-DAD method and UPLC-MS/MS method (or HPLC-MS/MS method) is from our studies and related references. The Microsoft Access and Visual Basic 6.0 softwares were employed to establish the database system. Users can inquiry the useful information conveniently after logining in the database system. In addition, the database system has information management fuction (such as addition, modification and deletion), user information management fuction and system maintenance fuction. The database system is user-friendly, easy to operate and can provide reference for the analysis of adulteration in Chinese medicines with synthetic anti-diabetic drugs.Glibenclamide is the most common adulterant in Chinese medicines used for diabetes and puerarin is an active constituent of Pueraria lobata (Willd) Ohwi which is included in many Chinese medicines used for diabetes, so they may be co-administered unwittingly or wittingly. A UPLC-MS/MS method was developed to determine the concentrations of glibenclamide and puerarin in rat plasma for the study of pharmacokinetic interaction between them. Glibenclamide, puerarin and internal standard glipizide were extracted from plasma using liquid-liquid extraction with ethyl acetate. The separation was achieved on a Waters BEH C18 column (2.1×50 mm I.D,1.7μm) with 5 mmol/L ammonium acetate solution (containing 0.1% formic acid) and methanol as mobile phase at a flow rate of 0.2 mL/min with a linear gradient program, column temperature was 40℃. ESI source was applied and operated in the positive mode. Multiple reaction monitoring (MRM) using the precursor→product ion combinations of m/z 417.1→mz 297.1, m/z 446.2→m/z 321.1 and m/z 494.2→m/z 369.1 were used to quantify puerarin, glipizide and glibenclamide, respectively. Method validation was performed in terms of specificity, linearity, accuracy, precision and stability. The proposed method was proved simple, rapid, sensitive and reliable. Glibenclamide, pueraria lobata extract or glibenclamide in combination with pueraria lobata extract were orally administered to rats, respectively. Pharmacokinetic parameters were estimated by Microsoft Excel software and analyzed by SPSS 12.0 software. After glibenclamide and pueraria lobata extract being co-administered, the increased area under the curve (AUC0-t and AUC0-∞), prolonged mean residence time (MRT0-t and MRT0-∞), and decreased clearance (Cl/F) of glibenclamide were observed, and the increased peak concentration (Cmax), AUC0-t and AUC0-∞, decreased clearance (Cl/F) and apparent volume of distribution (Vd/F) of puerarin were observed. These changes could enhance drug efficacy, make drug accumulation and increase adverse effects. It is suggested that glibenclamide should not be added illegally in Chinese medicines used for diabetes and glibenclamide and puerarin should not be co-administered in clinical practice. The dosage should be adjusted or the drug concentration in plasma should be monitored if glibenclamide and puerarin are co-administered.The protein binding rate of glibenclamide with rat plasma was at high level of more than 90% and the rate of puerarin was at moderate level. To study the interaction of rat plasma protein binding between glibenclamide and puerarin, an HPLC-MS/MS method was developed for the determination of glibenclamide in plasma and buffer and an HPLC-UV method was developed for puerarin. The equilibrium dialysis was carried out. After being equilibrated for 20 h, the average plasma protein binding rate of glibenclamide in the concentration of 20.0～400 ng/mL was (99.4±0.2)%, the rate was (99.2±0.4)% when with 4.00μg/mL puerarin. The rat plasma protein binding rate of glibenclamide was not disturbed by puerarin. After being equilibrated for 8 h, the average plasma protein binding rate of puerarin in the concentration of 0.200～4.00μg/mL was (66.3±4.9)%, the rate was (41.8±4.0)% when with 400 ng/mL glibenclamide. The rat plasma protein binding rate of puerarin was reduced by 36.9%, which indicated that glibenclamide could compete for plasma protein binding with puerarin resulting in increased free puerarin concentration. Correspondingly, efficacy and toxicity of puerarin would increase. This further showed that glibenclamide should not be added illegally in Chinese medicines used for diabetes, and glibenclamide and puerarin should not be co-administered in clinical practice.Metformin hydrochloride and glipizide tablet is a compound preparation composed of metformin hydrochloride and glipizide, which can improve glycemic control in patients with type 2 diabetes. An HPLC-MS/MS method was developed to determine the concentrations of metformin and glipizide in human plasma for the pharmacokinetic study of metformin hydrochloride and glipizide tablet in healthy volunteers. After protein precipitation of plasma sample with methanol, metformin, glipizide and internal standard phenacetin were separated on a Kromasil Phenyl column (4.6×150 mm I.D,5μm) at 40℃with an isocratic mobile phase consisting of methanol-ammonium acetate (10 mmol/L)(75:25, v/v) at a flow rate of 0.35 mL/min. ESI source was applied and operated in the positive mode. MRM using the precursor→product ion combinations of m/z 129.9→m/z 70.5, m/z 446.2→m/z 321.1 and m/z 180.0→m/z 109.7 were used to quantify metformin, glipizide and phenacetin, respectively. The linear calibration curves were obtained over the concentration range of 4.10～656 ng/mL for metformin and 2.55～408 ng/mL for glipizide. The RSD of intra-day and inter-day precision was below 10% and the RE of accuracy was between-7.0% and 4.6%. After oral administration of one metformin hydrochloride and glipizide tablet (containing 250 mg metformin hydrochloride and 2.5 mg glipizide), the main pharmacokinetic parameters for metformin and glipizide were as follows:the time to peak concentration (Tmax) of (2.61±0.78) and (2.61±0.50) h, the Cmax of (437±99) and (257±20) ng/mL, the half-life time (t1/2) of (5.14±1.45) and (4.70±0.84) h, the AUC0-t of (3405±1273) and (1785±239) ng·h/mL, the AUC0-∞of (3476±1278) and (1842±252) ng·h/mL, respectively.更多还原