Studies And Applications On Analysis Method For Anti-diabetic Drugs

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 precursorâ†'product ion combinations of m/z 417.1â†'mz 297.1, m/z 446.2â†'m/z 321.1 and m/z 494.2â†'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 precursorâ†'product ion combinations of m/z 129.9â†'m/z 70.5, m/z 446.2â†'m/z 321.1 and m/z 180.0â†'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.