反相高效液相法测定温胃舒片中补骨脂素和异补骨脂素的含量

amy54321 · 发表于 2014-8-18 18:28:05

  作者：蔡皓，张科卫，李伟，崔小兵

【摘要】  目的建立一种测定温胃舒片中补骨脂素和异补骨脂素含量的反相高效液相法。方法采用Kromasil C18柱，以甲醇水(55∶45)为流动相，紫外检测波长246 nm，流速1 ml·min-1。结果在所使用的色谱条件下，补骨脂素和异补骨脂素出峰位置未见干扰，补骨脂素和异补骨脂素分别在5.06～30.36 μg·ml-1和5.02～30.12 μg·ml-1浓度范围内线性关系良好(r=0.999 9)。温胃舒片中补骨脂素和异补骨脂素的平均加样回收率分别为97.64%和99.42%，相对标准偏差分别为1.16% (n=6)和0.86% (n=6)。结论该法可信度高，操作简便，重现性好，可用于温胃舒片生产上的质量控制。

【关键词】  温胃舒片；补骨脂素；异补骨脂素；反相高效液相法；定量分析

Determination of the Contents of Psoralen and Isopsoralen Contained in Wenweishu Tablet by RPHPLC

　　Abstract:ObjectiveTo establish a RPHPLC method for determination of the contents of Psoralen and Isopsoralen contained in Wenweishu Tablet. MethodsThe analysis was carried out by using a Kromasil C18 analytical column with methanol-water (55∶45, v/v) as mobile phase. The UV detection wavelength was 246 nm and the flow rate was 1 ml·min-1.ResultsUnder the chromatographic conditions used, there were no interferences found in the positions of Psoralen peak and Isopsoralen peak. Psoralen and Isopsoralen had fine linear responses (r=0.999 9) in the concentration ranges of 5.06～30.36 μg·ml-1 and 5.02～30.12μg·ml-1 respectively, the average recoveries of Psoralen and Isopsoralen in Wenweishu Tablet were 97.64% with the relative standard deviation of 1.16% (n=6) and 99.42% with the relative standard deviation of 0.86% (n=6) respectively. ConclusionThe method employed in this analysis is reliable and convenient with good repetition and can be used for quality control in the production of Wenweishu tablet.

　　Key wordsWenweishu Tablet；  Psoralen；  Isopsoralen；  RP-HPLC； Quantitative Analysis

　　Wenweishu Tablet is a pure formulated preparation of traditional Chinese medicine made of twelve crude herbal materials Radix Codonopsis, Radix Aconiti Lateralis Preparata, Radix Astragali Preparata, Cortex Cinnamomi, Rhizoma Dioscoreae, Herba Cistanches, Rhizoma Atractylodis Macrocephalae, Fructus Crataegi, Fructus Mume, Fructus Amomi, Pericarpium Citri Reticulatae and Fructus Psoraleae by the method of boiling with water, concentration, dry granulation, tabletting and coating. Wenweishu Tablet has the following functions: strengthening healthy "qi", warming and nourishing stomach，promoting "qi" circulation to relieve pain. It can mainly be used to treat patients and symptoms for chronic atrophic gastritis and gastric abscess, abdominal distension, belching, anorexia, chilly, acratia etc. caused by chronic gastritis in clinical application. Psoralen and Isopsoralen are the major active constituents of Fructus Psoraleae and also the main effective contents in Wenweishu Tablet. The Pharmacopoeia of China requires the total content of Psoralen and Isopsoralen in Fructus Psoraleae to be no less than 0.7%［1］. Psoralen and Isopsoralen are furocoumarin compounds and have been reported to have anti-tumor activity against BGC823 cancer cell by Morphological and MTT assays in vitro［2］. The chemical structures of Psoralen and Isopsoralen are shown in Figure 1.

　　For determination of the contents of Psoralen and Isopsoralen, the major analytical methods reported in the literatures are UV-spectrophotometry, TLCScanning, GC, CGC, SFECGC and HPLC［3～8］. Since HPLC has the advantage of convenient and good repetition, thus in the study of quality control of Wenweishu Tablet, we choose the indexes of Psoralen and Isopsoralen as quality control criteria and establish a RP-HPLC method for determining the contents of Psoralen and Isopsoralen in Wenweishu Tablet. The analytical results are satisfying and will provide a quantitative evaluation for quality control in the production of Wenweishu Tablet.

　　Fig 1  The chemical structures of Psoralen (A) and Isopsoralen (B)（略）

　　1  Apparatus and chemicals

　　Chromatographic analysis was performed on a 2690 Separations Module high performance liquid chromatograph (Waters, USA) equipped with a 996 Photodiode Array detector and connected to a Millennium 32 data processing system. A Model BP211D electronic balance (Sartorius, Germany) was used for weighing samples.

　　Psoralen (Batch number: 110709200310) and Isopsoralen (Batch number: 110738200309) were supplied from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, P.R. China) and their purities were 99.37% and 99.36% respectively by the identification from RP-HPLC. Wenweishu Tablet (Batch number: 050323, 050324, 050325, 050326, 050329, 050330, 050331, 050406, 050407, 050408) was supplied from Zhejiang Yixin Pharmaceutical Co., Ltd. The reagent used for the preparation and for the RP-HPLC analysis was of chromatographic grade (methanol). The water used for the preparation and for the RP-HPLC analysis was freshly deionized and redistilled.

　　2  Experimental methods and results

　　2.1  Chromatographic conditions and systematic compatibility testSeparations were obtained on a Kromasil C18 analytical column（250 mm×4.6 mm，φ5 μm）(Dalian chemical and physical Institute of China Academy of Science, P.R. China) with methanolwater (55∶45, v/v) as mobile phase. The UV detection wavelength was 246 nm and the flow rate was 1 ml·min-1. The column temperature was 30℃ and injection volume was 10μl. Under the chromatographic conditions used, the number of theoretical plates calculated on the peak of Psoralen was 4341 and there was a complete baseline separation among Psoralen, Isopsoralen and other components with a good degree of resolution (Rs = 2.26).

　　2.2  The preparation of standard solution, sample solution and blank solution

　　2.2.1  The preparation of standard solution The suitable amounts of Psoralen and Isopsoralen were accurately weighed and dispersed together in methanol, mixed, a 20 μg per milliliter mixing standard solution was obtained.

　　2.2.2 The preparation of sample solution About 1 g of the sample (comminuted powder) was accurately weighed and dispersed in 20ml of methanol, extracted ultrasonically for 30min. After cooling, methanol was added to this mixture till the total volume 25ml, mixed, the filtrate was obtained as a sample solution by filtration.

　　2.2.3 The preparation of blank solutionAccording to the proportion of prescription, other crude herbal materials in the prescription were weighed in the suitable amounts except Fructus Psoraleae. Using preparation technology of Wenweishu Tablet and preparation method of sample solution, a blank solution was obtained.

　　2.3 Interference test of blank solution10μl of standard solution, sample solution and blank solution were injected respectively into the RP-HPLC column under the chromatographic conditions described above. The results (Figure 2) showed that there were the same peaks of Psoralen and Isopsoralen appeared in the same retention times between standard solution and sample solution, whereas there were no peaks of Psoralen and Isopsoralen appeared in these retention times in blank solution, thus there were no interferences for the determination of the contents of Psoralen and Isopsoralen in sample solution.

　　(A) chromatogram of the standard solution

　　(B) chromatogram of the sample solution  (C) chromatogram of the blank solution

　　Fig 2 Typical chromatograms for the determination of the contents of Psoralen and Isopsoralen（略）

　　2.4  Determination of the contents of Psoralen and Isopsoralen in Wenweishu Tablet

　　2.4.1  Calibration curve and linear rangeAbout 5.06mg of Psoralen and 5.02 mg of Isopsoralen were accurately weighed and dispersed together in 100ml of methanol, mixed, a 50.60μg per milliliter and 50.20 μg per milliliter mixing standard stock solution was obtained. Then 1.0，2.0，3.0，4.0，5.0，6.0 ml of this mixing standard stock solution were taken respectively in six 10ml volumetric flasks, diluted with methanol to a constant-volume of 10 ml, mixed, a series of standard solutions were obtained. Under the chromatographic conditions described above, 10μl of each standard solution was taken for measuring and the chromatograms were recorded. After regression analysis of linearity, the calibration curves were described by regression equations A1=7126 2C1-3828 (r=0.999 9, for Psoralen) and A2=71 840C2-17 823 (r=0.999 9, for Isopsoralen), where A1 and A2 were the peak areas of Psoralen and Isopsoralen (as longitudinal coordinate), C1 and C2 were the concentrations of Psoralen and Isopsoralen (as horizontal coordinate), and r was the correlation coefficient. The results showed that the peak area of Psoralen (Isopsoralen) and the concentration of psoralen (Isopsoralen) had fine linear responses in the concentration range of 5.06～30.36 μg·ml-1 (5.02～30.12 μg·ml-1).

　　2.4.2  Chromatographic test of precision10μl of a same concentration of Psoralen and Isopsoralen mixing standard solution was taken to continuously analyze for six times, the RSD of peak area of Psoralen (Isopsoralen) was 0.27% (0.62%). The results showed that the chromatographic method was quite precise.　　2.4.3  Chromatographic test of stabilityAccording to the method for the determination of the contents of Psoralen and Isopsoralen, a sample of the same batch number was accurately weighed and the sample solution was prepared, and 10 μl of this solution was measured in 0, 2, 4, 6, 8, 10, 12 h within a day, the RSD of peak area of Psoralen (Isopsoralen) was 0.33% (0.49%). The results showed that the sample solution was stable within 12 h.

　　2.4.4  Chromatographic test of reproducibilitySix of 1g of sample (comminuted powder, Batch number: 050407) were accurately weighed, and the contents of Psoralen and Isopsoralen were determined after treated like the method of determination of the contents of samples, the average content of Psoralen (Isopsoralen) was 0.166 mg·tablet-1 (0.319 mg·tablet-1), and the RSD was 1.39% (1.67%). The results showed that the chromatographic method had a good reproducibility.

　　2.4.5 Recovery of the chromatographic methodSix of 0.5g of samples (comminuted powder, Batch number: 050407) were accurately weighed and put into six of 25ml volumetric flasks respectively. Then two of 4, 5, 6 ml of the mixing standard stock solution of Psoralen (30.40 μg per milliliter) and Isopsoralen (63.60 μg per milliliter) and two of 16, 15, 14 ml of methanol were added respectively, extracted ultrasonically for 30 min. After cooling, these mixtures were diluted with methanol to a constant-volume of 25ml, mixed, the filtrates were obtained by filtration, 10μl of each filtrate was taken for measuring and the recovery was calculated, the average recovery of Psoralen (Isopsoralen) was 97.64% (99.42%) and the RSD was 1.16% (0.86%) (n=6). The results were shown in Table 1 and Table 2.

　　Tab 1  Recovery test of Psoralen（略）

　　Tab 2  Recovery test of Isopsoralen （略）

　　2.4.6  Determination of the contents of samplesThree of 1g of samples (comminuted powder, Batch number: 050323, 050324, 050325, 050326, 050329, 050330, 050331, 050406, 050407, 050408) were accurately weighed, and treated like the item “2.2.2”, thirty of sample solutions were obtained. 10μl of each sample solution was taken for measuring and the contents of Psoralen and Isopsoralen were calculated based on each calibration curve. The results were shown in Table 3.

　　Tab 3  Result of sample analysis （略）

　　3  Discussion

　　3.1  Selection of detection wavelengthThe UV spectrum showed that Psoralen and Isopsoralen had obvious absorbent peaks at the wavelength 244 nm and 245 nm respectively after UV-scanning via a 996 Photodiode Array detector, thus 246 nm was selected as detection wavelength.

　　3.2  Selection of the methods of sample pre-treatmentThe experiments investigated three kinds of extraction methods - ultrasonic extraction, refluxing extraction and Soxhlet's extraction. The results showed that the sample solution treated by ultrasonic extraction had minimum impurities that disturbed the determination of the contents of Psoralen and Isopsoralen, and the effect of extraction was the best. On further investigation to the time of ultrasonic extraction, we found that Psoralen and Isopsoralen could be extracted at the maximum after the sample being extracted ultrasonically for 30 min. In addition, the experiments compared the effect of different extraction solvents (methanol, ethanol, chloroform, mobile phase and redistilled water) and different number of extractions on the results, which revealed that methanol as extraction solvent would give better separation effect and Psoralen and Isopsoralen would be extracted completely from the sample only one time.

　　3.3  Comparison of the methods of the content determinationWenweishu tablet is a product derived from Wenweishu capsule with dosage form changed. The criterion for content determination of Wenweishu capsule is not sufficient since only TLC-scanning is used to determine the content of Psoralen in the course of quality control (WS3-B-2629-97). However, in the study of quality control of Wenweishu Tablet, we chose indexes of Psoralen and Isopsoralen as quality control criteria and adopted RP-HPLC method for determining both contents at the same time. After examination of methodology, the established method satisfies the requirements of content determination and is convenient, and the results are reliable with good repetition.

　　

【参考文献】
  　　［1］ Committee of National Pharmacopoeia, "Pharmacopoeia of People's Republic of China" ［S］. Press of Chemical Industry, Beijing, 2005, Vol. 1：129..

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　　［5］ YAO Santao, YANG Bin, XU Zhiling. Determination of effective compounds in Psoralea corylifolia by GC［J］. Chinese Pharmaceutical Journal, 1996, 31(7)：394.

　　［6］ Xing Wangxing, CHEN Bin, MI Heming, WU Yutian, CHEN Shijing. Determination of the Psoralen and Isopsoralen in Gubenkechuan Tablets by CGC method ［J］. The Journal of Pharmaceutical Practice, 2002, 20(2)：96.

　　［7］ XING Wangxing, WANG Xiaoming, CHEN Bin, CHEN Shijing, WU yutian. SFE-CGC applied to determination of the Psoralen and Isopsoralen in Capsule Qibaomeiran［J］. Journal of Southeast China National Defence Medical Science, 2003, 5(2)：85.

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