Liu Wei Di Huang Wan (Pills) Study on the Determination
【Abstract】 Objective To establish Liu Wei Di Huang Wan (Pills), the determination of paeonol loganin content of high-
performance liquid chromatography. Methods Paeonol: Column:
C18 column (according to Bartlett; 250 mm × 4.6 mm, 5 μm),
mobile phase: methanol - water (6:4), flow rate 1.0 ml * min-
1, column temperature 30 ?, The detection wavelength was 274 nm. Loganin: C18 column (according to Bartlett; 250 mm × 4.6
mm, 5 μm), acetonitrile - water (15:85), flow rate 1.0 ml *
min-1, column temperature 25 ?, detection wavelength was 236
nm. Results paeonol in the range of 54.1 ~ 270.5 ng was linear, r = 0.999 6; The average recovery was 99.42%, RSD = 1.08%. Loganin in the range of 0.223 ~ 4.46 μg was linear, r
= 0.999 6; The average recovery was 99.53%, RSD = 2.15%. Conclusion The method is simple, reproducible, and can be used to Liu Wei Di Huang Wan (Pills), the paeonol loganin by HPLC.
【Key Words】 Liu Wei Di Huang Wan Paeonol loganin high-
performance liquid chromatography
Determination of Liuwei Dihuang Pill (concentrated pill)
Abstract: ObjectiveTo establish an HPLC for determination of paeonol and loganin in Liuwei Dihang Pills (concentrated pills). MethodsPaeonol: HPLC column was C18.The mobile phase was methanol-water (6:4) and the flow rate was 1.0 ml * min -
1.The column temperature was at 30 ? and the detection
wavelength was at 274 nm.Loganin: HPLC column was C18.The mobile phase was acetonitrile-water (15:85) and the flow rate
was 1.0 ml * min-1.The column temperature was at 25 ? and the
detection wavelength was at 236 nm.ResultsThe linear range was from 54.1ng to 270.5 ng (r = 0.999 6) for paeonol and from 0.223 μg to 4.46 μg (r = 0.999 6) for loganin.ConclusionThe
method is convenient and reproducible for determination of paeonol and loganin in Liuwei Dihuang Pills (concentrated pills)
Key words: Liuwei Dihuang Pills; Paeonol; Loganin; HPLC
Liu Wei Di Huang Wan (Pills) quality standards set out in
the ministerial standard closed section 11. The party out of money, B Song, "pediatric drug card straight tactic volume down", the Department of Traditional Chinese Medicine Kidney classic side of the well-known prescription for nourishing Yin kidney. I refer to "Chinese Pharmacopoeia" the product out, in the original ministerial standard, based on the right order to improve quality standards, using HPLC determination of paeonol moutan and Cornus officinalis loganin content, and make the method school inspection. The results now reported as follows.
An apparatus and materials
1.1 Instrument Waters liquid chromatograph, Waters2487 dual wavelength UV detector, 515 pump (USA), 7715 i injector (USA), Empower chromatography workstation (USA), C18 column
(according to Bartlett: 250 mm × 4.6 mm, 5 μm).
1.2 reagent methanol, acetonitrile for chromatography pure (Shanghai, China, Lu Du Reagent Factory), ethyl ether, ethanol, phosphoric acid, tetrahydrofuran, etc. are the analysis of alcohols, water, high pure water.
1.3 Reference Standards Paeonol reference substance (Chinese medicines and biological products ,110708-9704),
loganin reference substance (Chinese medicines and biological products ,111640-200401)
1.4 Sample Liu Wei Di Huang Wan (Pills) for long-A
Pharmaceutical Co., Ltd., Suzhou production.
2 Methods and Results
2.1 Determination of moutan
2.1.1 Chromatographic conditions C18 column (according to Bartlett; 250 mm × 4.6 mm, 5 μm), mobile phase: methanol -
water (6:4), flow rate 1.0 ml * min-1, column temperature
30 ?, detection wavelength 274 nm .
2.1.2 Preparation for the test product solutions
appropriate to take this product, and research fine, precision Weigh 0.5 g, cone-shaped bottle home with Sierra Leone,
precision by adding methanol, 100 ml, Mesa, shake, saying that given the weight, ultrasound treatment 30 min (power 250 W,
frequency 40 kHz), let cool, once again weighing, filled with methanol, reduced weight loss, shaking, filtration, taken for the test items as a renewal of the filtrate solution.
2.1.3 Preparation of reference substance solutions that take precise amount of reference substance, add methanol made from 16 μg per ml of solution, as a reference substance solution.
2.1.4 blank control experiments are made according to a prescription to moutan blank preparation, take blank control samples 0.44 g, precision that set, and placing a plug conical bottle, precision adding methanol 100 ml, with the same approach for the preparation of test materials obtained lack of moutan blank solution. Removed reference substance solution, respectively, samples for the test solution, blank
control solution test sample according to the law. The results showed that in blank control without interference.
2.1.5 The linear relationship of the study, said taking precise amount of paeonol reference substance, add methanol
was diluted to 54.1 μg / ml, shake. Precision drawing
reference substance, respectively paeonol solution 1,2,3,4,5 ml, add methanol diluted to 10 ml, shake, respectively, were concentration 5.41,10.82,16.23,21.64,27.05 μg / ml of paeonol
solution. According to high-performance liquid chromatography,
injection 10 μl, measured peak area. To peak area of the vertical axis in order to sample volume (ng) as the abscissa, the regression treatment, results showed that: Paeonol solution in the range of 54.1 ~ 270.5 ng a good linear
relationship. The regression equation Y = 9 636.508X-545.6.
The correlation coefficient r = 0.999 6.
2.1.6 Experimental precision precision obtained for the test, said goods (lot number 0501102) 0.5 g, made according to 2.1.2 Methods for the solution of test items, test products for precision drawing solution of 10 μl, measured peak area.
RSD = 0.99% (n = 6), showed a good precision.
2.1.7 Stability test for the test solution was obtained precision, respectively, 0,2,4,6,8,12 h sampling,
determination of peak area, RSD = 1.07%, the results in Table Ming Danpi phenol solution at 12 h to stabilize .
2.1.8 Experimental repeatability precision obtained for the test, said goods (lot number 0501102) 6 copies, respectively for the test solution was prepared under
preparation for the test product solution and determination in accordance with law. RSD = 1.32%. Reposted elsewhere in the paper for free download http://
2.1.9 The average recovery obtained experimental precision that is known for the content of 0.25 g of 6 test items were, respectively, adding a certain amount of paeonol reference substance solution, according to the tested materials were prepared under preparation. Determined in accordance with law to calculate the recovery rate. The
results in Table 1.
2.2 Determination of Cornus officinalis
2.2.1 Chromatographic conditions C18 column (according to Bartlett; 250 mm × 4.6 mm, 5 μm), acetonitrile - water
(15:85), flow rate 1.0 ml * min-1, column temperature 25 ?,
detection wavelength was 236 nm .
2.2.2 Preparation for the test product solution  to take this product is about 0.5 g, precision that set, and
placing a plug taper bottle, adding 50% precision of methanol 100 ml, said that given the weight, ultrasound treatment 10 min ( power of 250 W, frequency 40 kHz), let cool, and then said that given the weight of supplement with 50% methanol by weight loss, shaking, filtration, precision volume of 40 ml filtrate continued to take off 5 cm high column of alumina column (diameter 1 cm), with another 50% methanol and 50 ml elution, combined outflow of fluid and eluate, evaporated and the residue dissolved plus 50% methanol, and transferred to 10 ml Liang Ping, add 50% methanol was diluted to scale, shaking, filtration, that is, too. Table 1 The average recovery results (omitted)
2.2.3 Preparation of reference substance solution, said precision reference substance obtained loganin amount, plus 50% methanol made from 20 μg per ml solution, that is, too.
2.2.4 draw a linear relationship study precision 0.233 mg / ml solution of the reference substance loganin 1,2,4,8,16,20 μl were injected into HPLC to measure the peak area to the
inlet loganin sample volume abscissa, the integral value of peak area for the vertical axis regression, r = 0.9996. The results showed that loganin 0.223 ~ 4.46 μg in the context of
2.2.5 blank control experiments are made according to a prescription to dogwood blank preparation, take blank control samples 0.42 g, precision that set, and placing a plug conical bottle, precision adding methanol 100 ml, with the same method for the preparation of test materials obtained lack Cornus
blank solution. Reference substance solution were taken for the test product solution, blank solution of 20 μl, into the
chromatograph, results showed that the blank control without interference.
2.2.6 Stability Test precision experiments obtained for the test solution under continuous repeat injection six times, peak area determination, RSD = 1.28%.
2.2.7 stability test to take this product (batch 0501221) about 0.5 g, precision that will, according to the tested
materials were prepared under preparation for the test solution into. Preparation, respectively, after 0,1,
2,4,8 h according to the determination of loganin peak area RSD = 1.21%. The results showed that for the test product solution at 8 h to stabilize.
2.2.8 repeated experiment to take this product (Lot No.: 0501221) parallel that take six copies, according to test materials for the preparation method 6 were obtained for the test product solutions were measured, respectively, were measured in turn, RSD = 1.20%.
2.2.9 recoveries obtained experimental precision that is known for the content of test items is about 0.5 g (concentration of 0.1120%; lot number 0501221) 6 copies, each
with precision by adding 0.05 mg / ml solution of 9,6 loganin
reference substance , 4 ml, buy a plug conical bottle, respectively for the test materials under preparation method for the test solution was prepared and measured in accordance with law. The results in Table 2. Experimental results show that this law has a good recovery rate. Recovery of
experimental results in Table 2 (omitted)
2.3 The content of the sample determined to take this product 5 batches in accordance with law loganin and paeonol determination, while according to the original ministerial
standard UV spectrophotometry paeonol samples. The results in Table 3. Table 3 Determination results (omitted)
Sample compared Paeonol approach to steam distillation, methanol, ultrasound, reflux, ethanol, ultrasound, reflux of
methanol ultrasonic easy operation, high extraction rate and the impurity less interference. Pairs of ultrasonic time (power 250 W, frequency 40 kHz) to inspect, ultrasound 30 min, the content of the sample that is, without significant change.
Measured using this method is slightly higher than the original content of the sample ministerial sub-sub-standard UV
Reference to "Chinese Pharmacopoeia" experiment investigated loganin sample handling methods, including extraction methods, extraction time and methanol concentration in the choice of removal. Direct ultrasound, ultrasound, after reflux, Soxhlet extraction, the sample showed no significant change, choosing the ultrasonic extraction. Comparison of ultrasound (power 250 W, frequency 40 kHz) 10,20,30 min,
showed no significant change, choosing ultrasound 10 min. Comparison of the concentration of 40% methanol elution, 50%, 70% methanol, 50% extract the best results.
This experiment compared the determination of Strychnos
mobile phase of acetonitrile - water, tetrahydrofuran -
acetonitrile - methanol 0.05% phosphoric acid, the former
theoretical plate number, separation, symmetry are good, while the latter peak shape symmetry somewhat less Therefore, choose the former.
 State Pharmacopoeia Commission. Chinese Pharmacopoeia, ? Department of [S]. Beijing: Chemical Industry Press, 2005:402. Reposted elsewhere in the paper for free download http://