Anshenbuxin diazepam Pill Identification
【Abstract】 Objective To thin layer chromatography and high performance liquid chromatography (HPLC) Identification of whether Anshenbuxin pill diazepam added. Methods The thin-
layer chromatography, HPLC method identification. The results for the test materials were not detected in diazepam. Conclusion The method is simple and reliable TLC, HPLC method is more sensitive and accurate, can be two kinds of identification methods to combat in the Traditional Chinese
Medicine in Anshenbuxin diazepam without permission to add ingredients to provide a strong basis for the offense.
Key words Anshenbuxin pills diazepam thin-layer
Anshenbuxin pills  is a red sage root, Schisandra
(steam), Shichangpu, Albizia, Dodder, Eclipta, Rehmannia composed of 10 Chinese herbal medicines as a medicine pill, it has raised soothe the nerves of the heart efficacy, is widely used in clinical Yinxue palpitations caused by lack of
insomnia, dizziness, ringing in the ears. This drug in particular by a number of insomnia patients of all ages, people are usually more trust in traditional Chinese medicine preparations, would take safety, toxic side effects, but the attitude of Chinese medicinal preparations are often held at arm's length, can not do not have to. Some lawless elements in order to fully expand the market, profits, by whatever means, in Traditional Chinese Medicine in the artificially added to
diazepam. Of diazepam  belongs to benzodiazepine class
anti-anxiety drugs, anxiety, insomnia effects on excellent, but the long-term use can cause tolerance and dependence, the sudden withdrawal with withdrawal symptoms. Newborns, breast-
feeding women, pregnant women hanged. Therefore, this offense
there is a serious risk. I sampled a Beijing production of a pharmaceutical Anshenbuxin pills, after tests found the drug diazepam is not added, the majority of patients can safely take, will now be established by testing results and determination method described below.
An apparatus and materials
R200D type electronic analytical balance (Sartorius Inc., Germany); UV-1 type three ultraviolet analyzer (Shanghai Gu Village, Electro-Optical Instrument Factory); Agilent 1100 High Performance Liquid Chromatography (Agilent, USA); UV2450-
type purple Foreign / VIS spectrophotometer (Shimadzu Corporation); silica gel GF254 thin-layer plate (Qingdao
Marine Chemical Factory); cyclohexane, ethyl acetate, chloroform, acetone (AR, both Beijing Chemical Reagent
Company); methanol (chromatography alcohol); diazepam reference substance (China Pharmaceutical and Biological Products Institution, Lot :115-9302); Anshenbuxin pill; water as purified water.
2 Methods and Results
2.1 Thin-Layer Chromatography
2.1.1 Preparation for the test product solution
Be Anshenbuxin pill 15 pills, crush, take powder plus moderate amount of chloroform dissolved, dubbed the concentration of 2 mg / ml of the solution, Zhen Yao, filtration, the filtrate as a solution for the test items.
2.1.2 Preparation of reference substance solution
Weigh an appropriate reference substance diazepam, Chi 2 ml flask, add chloroform dissolved and diluted to the scale, made of 2 mg per ml of solution, as a reference substance solution.
2.1.3 Preparation of mixed solution for the test product solution + reference substance solution to take appropriate Anshenbuxin pill powder (about 5 pills), diazepam reference substance amount (about 20 mg), plus chloroform 10 ml, super -
sound processing 15 min, filtration, the filtrate as a mixture.
Lessons for the test product solution, reference substance solution of 5 μl, mixed solution of 2 μl,
respectively, points on the same silica gel GF254 thin-layer
board with cyclohexane: ethyl acetate: chloroform (6:3:1 ) as the agent to start to dry out after the home UV lamp
observation, in the mixed solution at 254nm and diazepam in the location of the corresponding reference substances show purple spots, for the corresponding location of test materials no such spots. Figure 1.
2.2 HPLC Method
2.2.1 Chromatographic conditions
Column ZORBAX C18 (4.6 mm × 250 mm, 5 μm); mobile
phase: methanol - water (50:50); a flow rate of 1.0 ml / min; detection wavelength was 254 nm; injection volume of 20 μl.
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2.2.2 Preparation of reference substance solution
Weigh an appropriate reference substance diazepam and placing 25 ml flask, add methanol dissolved and diluted to the scale, dubbed the concentration of 0.02 mg / ml solution, as a
reference substance solution.
2.2.3 Preparation for the test product solution
Anshenbuxin pill taking 5 pills, crush, add 10 ml of acetone ultrasonic treatment 10 min, filtration, take filtrate Hui dry, add 10 ml of methanol dissolved with a microporous
membrane (0.45 μm) filtration derived.
2.2.4 Preparation of mixed solution (for the test product solution + reference substance solution)
To take appropriate Anshenbuxin pill powder (about 5 pills), diazepam reference substance amount (about 20 mg), plus 10 ml acetone ultrasonic treatment 10 min, filtration, take filtrate Hui dry, add 10 ml of methanol dissolved in with a microporous membrane (0.45 μm) filtration derived.
2.2.5 HPLC identification of
Reference substance solution, diazepam to a peak time of 20.1min, mixed solution at the same time they also found a peak of peaks, and this peak and other impurities can also get a better peak separation, while the solution for the test materials same time not found out the peak of peaks. Figure 2.
If you use silica gel G thin-layer plate, can use the
following: the silica gel G thin-layer plate at 105 ? for
activated 30 min, respectively, points for the test product solution, reference substance solution of 5 μl, mixed
solution of 2 μl, with the ring hexane: ethyl acetate:
chloroform (6:3:1) as the agent to start first in the chromatography tank saturated 5min, and then start from the exhibition of about 15 cm. Out, waving to make developing agent, followed by spraying dilute sulfuric acid solution, bismuth potassium iodide test solution color. Mixed solution and diazepam in the corresponding reference substances show the location of the brick-red spots, the corresponding
location of the tested products had no such spots. With the use of silica gel G thin-layer board, compared with GF254
thin-layer plate method is simple and the color spots clearer.
HPLC method for the proportion of the mobile phase conducted a number of screening, respectively, of methanol -
water 70:30 for 30:70,40:60,50:50,60:40 and experiment results
show that the choice of methanol - water (50:50)
chromatography Fengfeng good shape and separation.
Thin-layer chromatography experiments by comparing the test product and reference substances for fluorescence spots, to ensure the accuracy of the qualitative results, the entire analysis of the characteristics method is simple, and spots were clear, specific and reproducible; HPLC method than the thinner layer chromatography is more sensitive, accurate, reliable and, therefore, the use of thin-layer chromatography
to detect the presence of a certain component, may be further confirmed using the HPLC method.
Thin-layer chromatography and HPLC method could serve as Anshenbuxin category in traditional Chinese medicines diazepam
without permission to add an effective identification method for the investigation and handling of such counterfeit drugs provide strong technical frame of reference.
 State Pharmacopoeia Commission. Chinese Pharmacopoeia, ? Department of [S]. Beijing: Chemical Industry Press, 2005:452.
 Chen Xin Qian, Kim You-yu, TANG Guang. Society for
Pharmacology, 15th edition [M]. Beijing: People's Health Press, 2001:253. Reposted elsewhere in the paper for free download http://www.hi138. com