川芎嗪衍生物TBN的HPLC含量测定方法.docx

Quantitative HPLC Analytical Method for Tetramethylpyrazine Derivative TBN

Student Name
MAK SAU HANG
Student No.
2005055160
Major
Pharmacy
Supervisor
Professor Wang, Yuqiang
Date(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

Statement of Originality

I 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 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：25-05-2009

Quantitative HPLC analytical method for tetramethylpyrazine
derivative TBN

Abstract:
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 determined 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 LOQ 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, 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 mg/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：川芎嗪衍生物，TBN，高效液相

Contents
1. Introduction 1
2. Materials 2
2.1. Chemicals 2
2.2. Apparatus 3
3. Experimental Method 3
3.1. TBN standard preparation 3
3.1.1. Column chromatography for TBN purification 4
3.1.2. Semi-preparative column chromatography 5
3.2. Development of HPLC method for TBN 5
3.2.1. lmax determination 5
3.2.2. Optimization of HPLC conditions for TBN 6
3.3. Validation of HPLC method 7
3.3.1. Specificity 7
3.3.2. Linearity 7
3.3.3. Precision 7
3.3.4. Limit of Quantification and Limit of Detection 7
3.3.5. Range 7
3.3.6. Accuracy 7
3.3.7. Stability 7
3.3.8. Recovery 7
3.3.9. Content Assay 7
4. Results and Discussion 7
5. Conclusion 7
Acknowledgement 7
References 7

1. Introduction
TBN 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 stroke [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 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 as therapeutic agents for neural and systemic diseases such as atherosclerosis, septicemia, stroke, and Alzheimer’s 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 are 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 able 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, 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 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 studies. 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
l TBN, prepared by the Institute of New Drug Research at Pharmacy College, Jinan University;
l Petroleum ether, ethyl acetate, acetone and dichloromethane (Analytical reagents) purchased from FUYU Refined Chemical Products Ltd. ;
l Reagent graded silica gel (200-300 mesh) purchased from Branch of Qingdao Haiyang Chemical Plant;
l Methanol (HPLC grade) purchased from Jiangsu Hanbon Sci. & Tech. Co., Ltd. ;
l Double-distilled water provided by the Institute;
l Sodium dihydrogenphosphate and disodium hydrogenphosphate (Analytical reagents), purchased from Guangzhou Chemical Reagent Factory.
2.2. Apparatus
l Glass Chromatographic column;
l Thin layer chromatographic silica gel plates;
l Rotary Evaporator (EYELA N-1001) and Digital Water Bath (EYELA SB1000) ;
l Vacuum drying oven (DZF-6050);
l Ultrasonic Cleaners (KQ-250E);
l Melting Point Measuring Instrument(SGW X-4);
l Shimadzu UV-VIS Spectrophotometers (UV-2450)
l Shimadzu-10AT HPLC and SPD-10AVP UV-VIS Detector;
l LUBEX Kromasil C18 column (5 m 100Å 250 mm´4.6 mm);
l semi preparative HPLC column (VYDAC RP C18 90A PHARMACEUTICAL);
l Electronic balance (ACCULAB ALC110.4);
l Other apparatus: volumetric flasks, conical flasks, pipette, micro filters and membranes (0.45 mm).
3. Experimental Method
3.1. TBN standard preparation
TBN 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 prepared and then used in the validation of HPLC method as reference standard.

3.1.1. Column chromatography for TBN purification
(1) TLC method
Use ethyl acetate as the solvent to dissolve TBN sample, separate the sample components with different mobile phases.
A. The continuous separating condition: petroleum 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 chromatography
From the results of TLC, method B and C were implemented 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 2.88 g of purified TBN was yielded. Melting point was tested 77 ℃.

3.1.2. Semi-preparative column chromatography
Since 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 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, since 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 TBN
3.2.1. lmax determination
Before the procedure of HPLC, the lmax of TBN should be determined. With the UV-VIS Spectrophotometer, the TBN sample solution was scanned in 200-400 nm for the determination of lmax.
Method:
TBN (0.01 g) from 3.1.1 was weighted and was dissolved in 10 ml volumetric flask with methanol. Dilute the solution to 50 mg/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 TBN
From the above result, 295 nm was chose for TBN detection.
3.2.2. Optimization of HPLC conditions for TBN
LUBEX Kromasil C18 column (5 m 100Å 250 mm´4.6 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 20081019) were used. The mobile phase were prepared daily and degassed by the ultrasonic cleanser for not less than 20 minutes.

Table 1 Different HPLC conditions tested for TBN
Methanol
Water
Detector l
Flow rate
pH
70
30
295 nm
0.9-1.0 ml/min
No added
60
40
0.8-0.8 ml/min
No added
50
50
0.8-1.0 ml/min
No added, 7.0-7.6
45
55
0.8-1.0 ml/min
No added, 7.2
40
60
0.8-1.0 ml/min
No added
35
65
0.8 ml/min
No added, 6.9-7.2

3.3. Validation of HPLC method
The reference standard used in this part was purified in 3.1, which was assumed 98.5 % pure. The HPLC chromatogram of TBN reference standard showed about 99 % purity of TBN, but since the UV detector in HPLC cannot detector every component in the sample, therefore the percentage content of TBN is still unknown.
3.3.1. Specificity
Two methods have been used to evaluate the specificity of the established HPLC method: 1) Raw material; 2) Degraded sample [1].
Method 1
Weight precisely about 10 mg of TMP raw material, diluted in 10 ml volumetric flask, takes 2 ml into a 5 ml volumetric flask and diluted to the mark, take 1 ml of the solution into a 10 ml volumetric flask, add 0.5 ml of the TBN standard solution and diluted to mark. The dilutions were done with mobile phase. Inject and record the chromatogram.
Method 2
Use the TBN standard solution stayed for 48 hours. Inject and record the chromatogram.
3.3.2. Linearity
The method used in linearity here had been used in lots of literature [9-17].
Weight 0.0630 g of TBN in 50 ml volumetric flask and dilute to mark. Purity of TBN was assumed 98.5 %, thus there were 0.0621 g of TBN in the standard. Concentration was equal to 1.242 mg/ml, thus 1242 mg/ml. (The standard solution of TBN)
Measure precisely 0.1 ml, 0.5 ml, 1.0 ml, 1.5 ml, and 2.0 ml individually into five 10 ml volumetric flasks and dilute to marks. All of dilutions were done by mobile phase.
Inject the solution and record he peak areas. Plot a standard curve and calculate the r2.
3.3.3. Precision
Measure 2.2 ml from the TBN standard solution (1242 mg/ml) into a 10 ml volumetric flask, diluted to the mark with mobile phase.
Inject the TBN solution for 6 times continually and record the peak areas for evaluating the precision and repeatability [1, 8-18].
3.3.4. Limit of Quantification and Limit of Detection
Dilute the TBN standard solution (1242 mg/ml) gradually until the peak height equal to 10:1 and 3:1 to the baseline noise respectively [1].
3.3.5. Range
The standard curve obtained from linearity would give the range [1, 8-18].
3.3.6. Accuracy
Six portions of 1.5 ml TBN standard solution (1242 mg/ml) were measured and diluted in six 10 ml volumetric flasks, named 1-6. Inject the solutions and record the peak areas [15-18].
3.3.7. Stability
1.5 ml TBN standard solution (1242 mg/ml) was measured and diluted in 10 ml volumetric flask. The solution was injected at 0, 2, 4, 6, 8 hours respectively after preparation, record the peak areas [15-18].
3.3.8. Recovery
(1) Content assay (for sample recovery [1, 9-17])
Use the batch 20081107 as sample.
About 10 mg TBN was precisely weighted and dissolved in 10 ml volumetric flask with mobile phase solution to the mark. Measure 1 ml from the sample solution and diluted in 10 ml volumetric flask with mobile phase. Inject the solution for 3 times and record the peak area. Apply to the standard curve and calculate the percentage content of the TBN sample.
(2) Recovery test
TBN batch 20081107 was used in this part as sample.
Method
Measure 3 ml from the TBN sample solution (concentration: 109 mg/ml, 86.15 % TBN content) to nine 10 ml volumetric flasks respectively.
Measure the standard solution (concentration: 1242 mg/ml) which was equivalent to 80 %, 100 % and 120 % of the TBN content in the sample solution respectively, and transfer to the volumetric flasks with TBN sample solution, 3 portions for each were prepared.
Dilute the solution to the marks with mobile phase. Inject and record the peak areas.
3.3.9. Content Assay
About 10 mg TBN was weighted precisely and dissolved in 10 ml volumetric flask with mobile phase solution to the mark.
Repeat step 1 for total 4 batches of TBN.
Measure 1 ml TBN solution from each of flasks and dilute in another 4 volumetric flasks with mobile phase solution to the marks.
Inject the 4 batches of diluted solution to HPLC for 3 times each and record the peak area.
Apply the result to the standard curve and calculate the TBN content in each batch [15-18].
4. Results and Discussion
Optimization of HPLC conditions for TBN

Figure 4 Methanol-water (35:65) flow rate: 0.8 ml/min (solution stayed for 48 hours)
This HPLC method was performed in two Shimadzu-10AT HPLC machines of Pharmacy College and had similar results. I decided to start the