川芎嗪衍生物TBN的HPLC跟量测定方法.docx

1、川芎嗪衍生物TBN的HPLC跟量测定方法Quantitative HPLC Analytical Method for Tetramethylpyrazine Derivative TBNStudent NameMAK SAU HANGStudent No.2005055160MajorPharmacySupervisorProfessor Wang, YuqiangDate(dd/mm/yyyy)25/05/2009 暨 南 大 学本科生毕业论文论文题目 川芎嗪衍生物TBN的HPLC含量测定方法学 院国际学院学 系药学系专 业药学姓 名 学 号 指导教师 2008年 5月 25日Statem

2、ent of OriginalityI hereby declare that the thesis presented is the result of research performed by me personally, under guidance from my supervisor. This thesis does not contain any content (other than those cited with references) that has been previously published or written by others, nor does it

3、 contain any material previously presented to other educational institutions for degree or certificate purpose to the best of my knowledge. I promise that all facts presented in this thesis are true and creditable. Signed: _ Date：25-05-2009Quantitative HPLC analytical method for tetramethylpyrazine

4、derivative TBNAbstract: A high-performance liquid chromatography (HPLC) method for a new tetramethylpyrazine derivative, TBN, was developed. Analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and limit of quantification (LOQ) were determin

5、ed under the guideline of International Conference on Harmonization Q2B 1. TBN was analyzed by RP-HPLC with C18 column using methanol-water (35:65) as mobile phase. The flow rate is 0.8 ml/min and the detector was set to 295 nm. The linearity of calibration curve is good (r2 0.999) and the LOD and L

6、OQ were 12.096 ng/ml and 40.32 ng/ml respectively. The relative standard deviation (RSD) of precision and accuracy were 0.1245 % and 0.6895 %, respectively, and the sample recovery was 99.08 % (RSD: 0.40 %). This method is reliable and easy for TBN analysis. Key Words: tetramethylpyrazine derivative

7、, TBN, HPLC川芎嗪衍生物TBN的HPLC含量测定方法摘 要：目的：建立川芎嗪衍生物 (TBN) 高效液相含量测定方法。方法：选用 C-18 色谱柱；甲醇-水 (35:65)；流速：0.8 ml/min；检测波长为295 nm。结果：TBN 在12.42- 310.5 g/ml 范围内线性关系良好 (r2 0.999) ；定量限和检测限分别为 12.096 ng/ml 及 40.32 ng/ml；精密度及准确度 RSD 分别为 0.1245 % 及 0.6895 %；加样回收率结果为 99.08 % (RSD: 0.40 %)。结论：本方法简便可靠，可作为TBN之定量分析方法。关键词：

8、川芎嗪衍生物，TBN，高效液相Contents1. Introduction 12. Materials 22.1. Chemicals 22.2. Apparatus 33. Experimental Method 33.1. TBN standard preparation 33.1.1. Column chromatography for TBN purification 43.1.2. Semi-preparative column chromatography 53.2. Development of HPLC method for TBN 53.2.1. max determina

9、tion 53.2.2. Optimization of HPLC conditions for TBN 63.3. Validation of HPLC method 73.3.1. Specificity 73.3.2. Linearity 73.3.3. Precision 73.3.4. Limit of Quantification and Limit of Detection 73.3.5. Range 73.3.6. Accuracy 73.3.7. Stability 73.3.8. Recovery 73.3.9. Content Assay 74. Results and

10、Discussion 75. Conclusion 7Acknowledgement 7References 71. IntroductionTBN is a novel compound developed by the Institute of New Drug Research at the Pharmacy College, Jinan University. TBN has been shown to be antioxidative and thrombolytic, and is under development as a treatment for ischemic stro

11、ke 2. TBN is a derivative of 2,3,5,6-tetramethylpyrazine (TMP), which is the main active ingredient of Ligusticum wallichii Franchat (Chuan Xiong), formed by conjugating TMP and a nitrone moiety (see Figure 1) . TMP is used to treat ischemic stroke in China for many years which was found beneficial

12、in inhibiting platelet aggregation 3, lysing blood clots 4, blocking calcium entry 5 and scavenging reactive oxygen species (ROS) 6. While retaining the thrombolytic activity of TMP, the nitrone added to TMP (i.e. TBN) had been proved to provide a strong antioxidative activity. Nitrones are useful a

13、s therapeutic agents for neural and systemic diseases such as atherosclerosis, septicemia, stroke, and Alzheimers disease 7. (1) (2)(3)Figure 1 Structure of TMP (1); Nitrone (2); TBN (3)As a potential new drug, series of researches like pharmacology, toxicology, pharmacokinetics, pharmacodynamics ar

14、e needed. Therefore, quality analysis and control on TBN is necesary. First of all, a quantitative method of TBN had to be developed. HPLC method for analyzing chemical compound is quick, simple and reliable. Therefore we chose it as the analyzing method for TBN. A reliable HPLC method should be abl

15、e to separate the sample from its impurities completely and can be validated properly, so called the Methodology.The validation of developed HPLC method 1, 8 included several parts which are linearity, precision, range, limit of quantification and limit of detection, accuracy, stability, specificity

16、, and recovery. The content assay would be done after the validation of method. However, reference standard of the substance to be tested is needed in some items of the HPLC method validation.For this case, TBN is a new compound and its reference standard is unavailable. Therefore, in this paper, a

17、relatively pure sample was used as a reference standard. Hence, to obtain a relatively pure TBN sample, a purification of TBN was also done in this research.The target of this research is to establish an easy and reliable HPLC method for TBN analysis, used in routine quality control of its related s

18、tudies. After the establishment of TBN reference standard, this HPLC method can be applied directly to quantify TBN content. In this research, there were three main parts: TBN standard preparation, HPLC method establishment, and Method validation.2. Materials 2.1. Chemicals TBN, prepared by the Inst

19、itute of New Drug Research at Pharmacy College, Jinan University; Petroleum ether, ethyl acetate, acetone and dichloromethane (Analytical reagents) purchased from FUYU Refined Chemical Products Ltd. ; Reagent graded silica gel (200-300 mesh) purchased from Branch of Qingdao Haiyang Chemical Plant; M

20、ethanol (HPLC grade) purchased from Jiangsu Hanbon Sci. & Tech. Co., Ltd. ; Double-distilled water provided by the Institute; Sodium dihydrogenphosphate and disodium hydrogenphosphate (Analytical reagents), purchased from Guangzhou Chemical Reagent Factory. 2.2. Apparatus Glass Chromatographic colum

21、n; Thin layer chromatographic silica gel plates; Rotary Evaporator (EYELA N-1001) and Digital Water Bath (EYELA SB1000) ; Vacuum drying oven (DZF-6050); Ultrasonic Cleaners (KQ-250E); Melting Point Measuring Instrument(SGW X-4); Shimadzu UV-VIS Spectrophotometers (UV-2450) Shimadzu-10AT HPLC and SPD

22、-10AVP UV-VIS Detector; LUBEX Kromasil C18 column (5 100 250 mm4.6 mm); semi preparative HPLC column (VYDAC RP C18 90A PHARMACEUTICAL); Electronic balance (ACCULAB ALC110.4); Other apparatus: volumetric flasks, conical flasks, pipette, micro filters and membranes (0.45 m). 3. Experimental Method3.1.

23、 TBN standard preparationTBN is a white crystal which is water soluble, with molecular weight of 221 g/mol. Due to the difference in purity between each batches of production, the physical properties of TBN samples are varied from one another. In this part, relatively pure TBN sample would be prepar

24、ed and then used in the validation of HPLC method as reference standard.3.1.1. Column chromatography for TBN purification (1) TLC methodUse ethyl acetate as the solvent to dissolve TBN sample, separate the sample components with different mobile phases.A. The continuous separating condition: petrole

25、um ether-ethyl acetate (1:1)B. Modified condition 1: petroleum ether-ethyl acetate (4:1)C. Modified condition 2: petroleum ether-acetone (5:1)Figure 2 TLC of TBN with different mobile phase (from left to right : A, B, C)(2) Column chromatographyFrom the results of TLC, method B and C were implemente

26、d in column. About 300 mg of TBN were used in each method. For method B, yellow semi solid was obtained. Very pale yellow solid was obtained in method C. Hence, method C was chosen.Batch 2008.10.11 TBN (4.26 g) was put in column chromatography with the mobile phase petroleum ether-acetone (5:1) and

27、2.88 g of purified TBN was yielded. Melting point was tested 77 .3.1.2. Semi-preparative column chromatographySince there were trace amount of impurities in the purified TBN from 3.1.1, semi-preparative column chromatography was performed for further purification.One hundred mg of TBN was used this

28、time, and a methanol-water (35:65) system was used as a mobile phase. However, Shimadzu-10AT HPLC is not designed for semi-preparative column, too much solvent was used and too few TBN have been purified. This method is not feasible for producing sufficient purified TBN. And for the TBN purified, si

29、nce there was too much water in the mobile phase that was difficult to dry, only trace amount of TBN was obtained which was not enough for experimental use. With the result, the TBN purified from 3.1.1 was used as reference standard.3.2. Development of HPLC method for TBN3.2.1. max determinationBefo

30、re the procedure of HPLC, the max of TBN should be determined. With the UV-VIS Spectrophotometer, the TBN sample solution was scanned in 200-400 nm for the determination of max.Method:TBN (0.01 g) from 3.1.1 was weighted and was dissolved in 10 ml volumetric flask with methanol. Dilute the solution

31、to 50 g/ml by using pipette and volumetric flasks.Scan the sample solution with the UV-VIS Spectrophotometer and record the result.Figure 3 UV spectrogram for TBNFrom the above result, 295 nm was chose for TBN detection.3.2.2. Optimization of HPLC conditions for TBNLUBEX Kromasil C18 column (5 100 250 mm4.6 mm) was used in this research which is suitable for most organic compound.Different ratio of mobile phase, flow rate, pH, have been used to determine the most suitable HPLC method for TBN. With the aim of separating the TBN from its impurities, TBN sample without purification (Batch 200