Determination of osthole in compound snake bed tincture by high performance liquid chromatography

Abstract: The main active ingredients of the Cnidium sibiricum extract include pinene, camphene, bornyl isovalarate, isoborneol, methoxy parsley phenol (osthole), and osthol ( cindimine), cnidiadin, isopimpinelline, etc.

Objective To establish an HPLC method for determining the content of osthole in the compound tincture tincture. Methods A HypersilODS column (150mm × 4.6mm, 5μm) was used; the mobile phase was methanol-water (70:30), the flow rate was 1.0mL / min; the measurement wavelength was 322nm; the column temperature was room temperature. Results The osthole in the range of 0.018 ~ 0.126μg showed a good linear relationship with the peak area (r = 0.9999), the average recovery rate was 98.55%, RSD = 1.45%. Conclusion The method is simple, rapid, accurate and reliable, and can be used for the determination of osthole in compound snake bed tincture.

Compound snake bed tincture is a external tincture developed by the School of Pharmacy, Hunan University of Traditional Chinese Medicine. It is composed of Chinese medicines such as snake bed, earth radix bark, and yellow cedar. Gynecological inflammation, itchy skin and eczema. The osthole is the main active ingredient of the sperm scrophulariae, which is determined by HPLC in this experiment. Now it is reported as follows.

1 Instruments and reagents

Waters1525 / 2487 high performance liquid chromatograph, Breeze chromatography workstation; MICROLITER # 702 micro sampler (HAMILTON company). Ophiopogon standard product (provided by China National Institute for the Control of Pharmaceutical and Biological Products, batch number 110822-200406); compound snake bed tincture (self-made); experimental Chinese medicinal materials were purchased from Changsha Medicinal Materials Company. Methanol is chromatographically pure; ethanol is analytically pure; water is double distilled water.

2 Methods and results

2.1 Chromatographic conditions

HypersilODS chromatographic column (150mm × 4.6mm, 5μm, Dalian Elite Analytical Instrument Co., Ltd.); mobile phase: methanol-water (70:30); flow rate: 1.0mL / min; measurement wavelength: 322nm; column temperature: room temperature; The sample volume is 5 μL.

2.2 Preparation of reference solution

Precisely weigh the appropriate amount of osthole reference substance, add ethanol to make a 9.0μg / mL solution, and you get it.

2.3 Preparation of test solution

Precisely draw 0.2mL of compound snake bed tincture, place it in a 10mL volumetric flask, add absolute ethanol to the mark, shake well, filter with 0.45μm microporous filter membrane, and take the continuous filtrate as the test solution.

2.4 Preparation of negative control solution

With the prescription of the compound snake bed tincture without the snake bed, the negative solution was prepared according to the production process of the compound snake bed tincture, and then the negative control solution was prepared according to the method under "2.3".

2.5 Interference test

According to the above chromatographic conditions, take 5 μL of the reference solution, the test solution and the negative control solution, and inject them into the chromatograph. The chromatogram of the test product has the same chromatographic peak at the corresponding position as the chromatogram of the reference product, while the chromatogram of the negative control solution does not interfere here (see Figure 1).

2.6 Investigation of linear relationship

Accurately draw 2, 4, 6, 8, 12, and 14 μL of the reference solution, and perform linear regression on the injection volume (μg) based on the peak area. The regression equation is obtained: Y = 4.392 × 106X-6036.38, r = 0.9999. It shows that osthole has a good linear relationship with the peak area from 0.018 to 0.126 μg.

2.7 Precision test

The same sample solution was used to repeat the injection measurement 5 times, based on the peak area, RSD = 1.06%.

2.8 Repeatability test

Take the same batch of compound snake bed tincture, according to the method under "2.3" to prepare 5 test product solutions respectively, measured according to the above chromatographic conditions, the average content was 0.0203mg / mL, RSD = 0.94%.

2.9 Stability test

Take the same test solution, inject at 0, 2, 4, 6, 12, 16, 24h after preparation, determine the peak area, calculate, RSD = 1.21% (n = 7). It indicates that the test solution is stable within 24h.

2.10 Sample recovery test

Accurately draw 6 portions of compound ostracin tincture 0.2mL with known osthole content (21.74μg / mL), prepared according to the method under item "2.3", place them in 6 10mL measuring flasks, and add the appropriate amount of reference solution , Dilute the volume to the mark with absolute ethanol, shake well, measure according to the above chromatographic conditions, calculate the recovery rate, the results are shown in Table 1. Table 1 Sample recovery test results (omitted)

2.11 Sample determination

Accurately draw 5μL of each test product solution (4 batches of 3 parts each), and measure according to the above chromatographic conditions. The results are shown in Table 2. Table 2 Determination results of osthole in compound snake bed tincture (omitted)

3 Discussion

The ultraviolet spectrophotometer was used to scan the wavelength of the osthole reference solution under the test conditions in the article, and the maximum absorption wavelength was found to be 321.5nm. After reference [1-2], 322nm was selected as the detection wavelength.

The author has tested several different mobile phase systems. Under the chromatographic conditions determined in this experiment, using methanol as the mobile phase is simpler and more economical than acetonitrile as specified in the 2005 edition of the Pharmacopoeia of the People ’s Republic of China (Part 1) [3]. The measurement time is relatively short (peak at about 7.5 min), osthole can be separated from other components at baseline, and the theoretical plate number is greater than 3,000 in terms of osthole.

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