High Performance Liquid Chromatography Method of Tripterygium Tablets
【Abstract】 Objective To improve the quality standards Tripterygium films, and explore the high-performance liquid
chromatography (HPLC) Determination of Tripterygium Tablets
method. Methods HPLC, C18 column, mobile phase was methanol -
water (40:60), detection wavelength 218 nm. The results of the linear range of triptolide 0.047 6 ~ 0.476 0 μg, r = 0.9991,
average recovery was 101.2%, RSD of 2.1% (n = 6). Conclusion
Tripterygium limit is tentatively scheduled for determination of tablets: Each piece contains triptolide to triptolide (C37H22O14) dollars, shall not be less than 0.01 mg / piece.
Key words triptolide Tripterygium chip high-performance
Tripterygium-piece Celastraceae woody vine Tripterygium Tripterygium wilfordii Hook.f, herbs extracted by ethyl acetate tablets made from the original criterion used by the Ministry of Health Drug TLC  Determination of Tripterygium tablets triptolide in the content of the composition due to wilfordii more complex, using thin-layer method difficult to
obtain a better separation efficiency, and expand the color after the highly volatile, fast fading color reagent is difficult to get better determinations. In order to
effectively control the quality standards of the National Drug Plan of Action Group issued quality standards for improving the task of Tripterygium wilfordii tablets. Through experiments, this paper established the HPLC determination of
triptolide triptolide content of the film, after looking into some of triptolide in the selected conditions, good separation, detection sensitivity, determination of specific, can be used as the product The determination of control
An instrument and reagent
HP-1100 High Performance Liquid Chromatography (Ajilent Corporation, USA); Dalian Elite Hypersil C18 liquid chromatography (200 mm × 4.6 mm, 5 μm); triptolide reference
substance (drug and biological products in China Institution,
batch 0857-9601); methanol HPLC pure, water re-distilled
water; and other reagents of analytical pure; Tripterygium film from Huangshi, Hubei Pharmaceutical Co., Ltd. 39.
2 Methods and Results
2.1 Chromatographic conditions
Dalian, according to Bartlett column was Hypersil C18 liquid chromatography (200 mm × 4.6 mm, 5μm); methanol -
water (40:60) as mobile phase; detection wavelength 218 nm; flow rate 1 ml / min; column temperature: 35 ?; theoretical
plate number by triptolide peak count of not less than 3000.
2.2 for the test solution preparation methods and the choice of elution volume
2.2.1 Preparation for the choice of test solution
Tripterygium preparations composition is more complex and difficult to obtain without a refined extract a better separation efficiency, the light often Xue Ling et al  reported in the literature carried out a refined method of extracting the visit. Precision said the powder samples taken at about 1 g, vinegar acid ethyl ester 20 ml, ultrasound
allows the dissolved, add 5% HCl solution, 15 ml washed twice, and abandoned to the acid, ethyl acetate solution plus 10% NaOH solution 20 ml , heated in water bath 15 min, sub-taking
lye, and used 10% NaOH solution 20 ml extraction twice, and
the merger lye, add dilute HCl adjusted pH to 4, to 20 ml ethyl acetate extract three times the combined ethyl acetate fluid, evaporated, add methanol allows the dissolved residue, fixed volume to 10 ml for the test items as a solution.
Precision said the powder samples taken at about 1 g, plus chloroform 10 ml, ultrasound 10 min, plus a neutral alumina 1 g, mixing evenly, evaporated, on the neutral alumina column (inner diameter 12 mm, 100 ~ 200 mesh, 10 g), eluted with ethyl acetate solution, collecting eluate 150 ml,
evaporated and the residue add methanol allows the dissolved, constant volume to 10 ml for the test items as a solution.
Precision said the powder samples taken at about 1 g, plus chloroform 10 ml, ultrasound 10 min, plus a neutral
alumina 1g, mixing evenly, evaporated, on the neutral alumina column (inner diameter 12 mm, 100 ~ 200 mesh, 10 g), with 10% ethyl acetate petroleum ether (60 ~ 90 ?) 50 ml eluted, and
then with 1% ethanol solution of ethyl acetate elution, collecting eluate 100 ml, evaporated and the residue plus methanol allows the dissolved, constant volume to 10 ml for the test items as a solution.
Obtained by the above-mentioned three kinds of solutions
were prepared by injection chromatographic conditions. The
results in Table 1. Table 1 3 kinds of extraction methods of determination results (omitted)
The results showed that: The extraction and separation on a neutral alumina column was better than a good extraction method, Method 3 using 10% ethyl acetate petroleum ether (60 ~ 90 ?) 50 ml elution was mainly to remove impurities, but by comparison this method little impact on the separation, but with 1% ethanol solution of ethyl acetate eluted without much impact on the determination results, but under the more
washing impurities, baseline fluctuations, impurity peaks more, some impact on the separation, so use Method 2 as the assay method.
2.2.2 Precision, said the choice of solvent, to take samples of powder is about 1 g, a total of three copies, each
with chloroform 10 ml, ultrasound 10 min, plus a neutral alumina 1 g, mixing evenly, evaporated, the neutral oxidation Aluminum column (inner diameter 12 mm, 100 ~ 200 mesh, 10 g), eluted with ethyl acetate solution, respectively, to collect
eluate 100, 150, 200 ml, evaporated and the residue add methanol allows the dissolved, will allow to 10 ml for the
test items as a solution. According to the three kinds of solutions proposed chromatographic conditions were injection. The results in Table 2. Table 2 Determination of three kinds of solvent results (omitted) reposted elsewhere in the paper for free download http://
The results showed that ethyl acetate solution eluted, collecting eluate 150 ml a better option.
2.2.3 the choice of mobile phase of acetonitrile - water
(30:70) as mobile phase for a test, triptolide peaks faster, hard to get a better separation of the sample solution in favor of (15:85), ( 20:80), (25:75), etc. are not out of triptolide-feng, the final choice of methanol - water (40:60)
as mobile phase, triptolide peak time is about 11 min, the sample solution than separate Well, no impurity peak interference.
2.3.1 Preparation of reference substance solutions that take precise amount of triptolide reference substance, add methanol made from 0.01 mg per ml of solution, as a reference substance solution.
2.3.2 Preparation for the test product solution
To take this product 30, said that given the weight, crush, precision, said the amount of take 10, add chloroform 10 ml, ultrasound 10 min, plus a neutral alumina 1g, mixing evenly, evaporated, on the neutral alumina column (diameter 12 mm, 100 ~ 200 mesh, 10 g), eluted with ethyl acetate solution, collecting eluate 150 ml, evaporated and the residue add
methanol allows the dissolved, constant volume to 10 ml as a solution for the test items .
2.3.3 linear relationship between the study said that taking precise amount of triptolide reference substance, add methanol made of 0.0238 mg per ml of solution as a reference substance reserve liquid. Precision drawing reference substance reserve liquid 1,2,4,6,8,10 ml, respectively, set 10 ml flask, add methanol was diluted to scale, shake,
respectively, injection 20 μl, measured peak area to peak
area of Ordinate, triptolide reference substance into the sample volume abscissa, drawing the standard curve, calculated regression equation: Y = 2 110.8X 3.7, r = 0.999 1, results showed that triptolide reference substance at 0.047 6 0.476
within the range of 0 μg peak area showed good linearity.
2.3.4 Experimental precision
Precision drawing the same reference substance solution of 20 μl, repeat the injection five times to measure the absorption of triptolide peak area, RSD is 1.84%, indicating
2.3.5 Stability test
Precision drawing the same items for the test solution (batch number 040,311), respectively, 2h intervals were measured in order to triptolide absorption peak area for inspection, sampling six times, the measured RSD = 2.39%, indicating that samples within 12 h in the basic stable.
2.3.6 Repeatability test
Precision that were to take the same batch (batch number 040,311) sample powder five copies, each of about 1 g,
according to preparation methods for the solution of test product preparation, according to the measurement results triptolide samples, the average content of 11.48 μg / g , RSD
1.36%, indicating good repeatability of this Act.
2.3.7 Canadian sample recovery experiment
Precision Weigh a known concentration of sample (batch 040,311) 6 copies, respectively, by adding sophisticated triptolide reference substance solution (0.050 48mg/ml) 1 ml, by adding chloroform 10 ml, according to preparation method
for the solution of test product preparation, determined in accordance with law. 6 times the average recovery was 101.2%, RSD 2.1%. The results in Table 3. Table 3 Canadian-like
recovery of experimental results (omitted)
2.3.8 Determination of content of the sample to take this product 30, said that given the weight, crush, precision, said the amount of take 10, according to solution preparation method for the preparation of test materials. Precision drawing for the test solution and reference substance solution
of the product 20 μl, were measured according to the results in Table 4. Triptolide and triptolide-chip samples by high
performance liquid chromatography shown in Figure 1 ~ 2. Table 4 Sample assay results (omitted)
The experimental results show that, HPLC method can replace the original standard using TLC-scanning Tripterygium
triptolide content of the film. Four batches of samples were triptolide content of 0.01 mg / piece above, but taking into account the differences in Tripterygium piece of the weight, this product content limits can be tentatively scheduled for: Each piece contains triptolide to triptolide (C37H22O14) count shall not be less than 0.01 mg / piece.
 The People's Republic of China Ministry of Health. Drug Standards set prescription of Chinese medicine preparations, the first 16 [M] .1993:113.
 Chang Xue Ling, Chen Huabing, XU Hui-Bi, et al. RP-
HPLC Tripterygium extract a pigment content of [J]. Journal of
Analytical Science, 2003,19 (4): 327. Reposted elsewhere on the free Paper Download Center http://