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Key words: reversed-phase high performance liquid chromatography; Lysimachia quercetin; kaempferol; flavonoid Lysimachia is the whole grass of the perennial herbaceous plant Lysimachia christinae Hance in the primulaceae family; swollen. It is an important medicine for hot shower, stone shower, jaundice, and sores. The decoction has a significant diuretic effect, and can promote the discharge of bile from the bile duct. The antibacterial experiment has an inhibitory effect on Staphylococcus aureus, and the total flavonoids and phenolic acid of Lysimachia have anti-inflammatory effects. The stems and leaves mainly contain phenolic components, sterols, flavonoids, amino acids, tannin, volatile oil, choline, etc .; the roots contain saponins. In addition to the separation of five flavonoids such as quercetin and kaempferol using a polyamide column, the method of determining the content of its single components is rarely reported in the literature. We use RP-HPLC method to determine the content of two flavonoids, which provides a scientific basis for the study of quality standards.
1 Instruments and reagents Swiss Mettler Toledo AG285 electronic balance; DZF-1 vacuum drying oven; ZQ-250 ultrasonic cleaner; American Agilent high-performance liquid chromatograph Agilentl100 Series, equipped with a quaternary pump, automatic sampler (injection range 0.1 ～ 100μ1), ultraviolet visible light detector, column temperature box, online vacuum degassing pump, chromatography workstation, etc.
Quercetin and kaempferol reference materials are provided by the China National Institute for the Control of Pharmaceutical and Biological Products; Lysimachia was purchased from Sichuan and has been identified as the entire yellow grass of the primulaceae, which meets the requirements of the Chinese Pharmacopoeia 2000 edition; Pure; water is ultrapure water; the remaining reagents are analytically pure.
2 Methods and results 2.1 Chromatographic conditions and system suitability Chromatographic column: Alltima Cl8 column (150mmX4.6mm, 5μm); mobile phase: methanol-water-phosphoric acid (500: 500: 1) (0.45μm microporous membrane filter Ultrasonic degassing before use); detection wavelength: 360 nm; column temperature: 30 ° C: flow rate: 1.0 ml / min.
Under this chromatographic condition, the chromatograms of quercetin (Q), kaempferol (K) reference stock solution and test solution were measured.
It can be seen that the retention time of the quercetin (Q) peak is about 9.8 min, and the retention time of the kaempferol (K) peak is about 18.1 min; the resolution of the two peaks in Figures Q and K is greater than 1.5, and the number of theoretical plates 3700 and 6229 respectively.
2.2 Preparation of solution 2.2.1 Preparation of reference solution Precisely weigh the quercetin (Q) and kaempferol (K) reference articles that have been dried over phosphorus pentoxide overnight in the same volumetric flask, add methanol to prepare A mixed solution containing 0.01 mg per ml was used as a stock solution of the reference substance (Q is 1.065 X 0.01 mg / ml, K is 1.084 X 0.01 mg / ml).
2.2.2 Preparation of the solution for the test product Weigh accurately 2g of crushed Lysimachia powder in a Erlenmeyer flask, add 25ml of methanol, sonicate for 30min, let cool to room temperature, filter with medium-speed analysis filter paper, and absorb 20ml of the continuous filtrate Place in a separatory funnel and extract 3 times with petroleum ether (20ml, 15ml, 15ml). After removing the fat-soluble impurities, combine the lower layer liquid and extract 3 times with 25ml of ethyl acetate-water (15:10) mixture , Combine the extracts, recover the ethyl acetate, dissolve the residue in methanol and transfer to a 25ml measuring flask, add methanol to the mark, filter with O.45μm microporous filter membrane, and use it as the test solution.
2.3 Preparation of standard curve Pour the above reference stock solution into the sample bottle, inject in 0.3, 0.5, 1, 2, 4, 6, 8, 10, 15μl volume, and measure according to the above chromatographic conditions, respectively Quercetin (Q) and kaempferol (K) injection volume are plotted on the abscissa, and the peak area integral value is plotted on the ordinate, and the regression equations are:
Quercetin (Q): Y = 37292X, r = 0.9999, linear range: 3.195 × 0.001 ～ 0.15975μg
Kaempferol (K): Y = 39597X, r = 0.9999, linear range: 3.252 × 0.001 ～ 0.1626μg
The results showed that quercetin (Q) and kaempferol (K) were in the range of 3.195 × 0.001 ～ 0.15975μg and 3.252 × 0.001 ～ 0.1626μg, respectively. The injection volume of the reference substance and its peak area integral value had a good linear relationship.
2.4 Precision test Accurately absorb 1OμL of the above reference stock solution, operate according to the method under "Sample determination", repeat the sample injection 6 times, the results determine the integral value of Q and K peak area, RSD are 0.30% and 0.31% ( n = 6).
2.5 Repeatability test Take Lysimachia powder, prepare 5 parts according to the method of "Preparation of test solution", operate according to the method under "Sample determination", and determine the content.
The calculated RSDs for Q and K are 1.12% and 0.31%, respectively (n = 5).
2.6 Stability test Precisely absorb the freshly prepared test solution 10, and measure it every 1 h at 25 ℃ according to the method of “Sample Determinationâ€, and measure 12 times in total, record the peak area, and result 12 times The RSDs of the peak areas of the injected Q and K were 1.91% and 0.44%, respectively, indicating that the test solution was stable within 12h (n = 12).
2.7 Sample recovery rate test Weigh accurately 3 samples of known content, add the control stock solution prepared by the same method (Q is 0.8224 × 0.001mg / ml, K is 1.030 × 0.001 mg / ml) 1.0ml , 1.0ml, 1.5ml, according to the "preparation of test solution" method, according to the above chromatographic conditions.
2.8 Determination of samples According to the method of "2.2.2", prepare the solution of Lysimachia for the test product, according to the above chromatographic conditions, the results of the determination of the three batches of samples.
3 Discussion 3.1 Selection of mobile phase. Because the flavonoids in Lysimachia vulgaris contain phenolic hydroxyl groups and are weakly acidic, an acidic buffer system was chosen. The methanol-water-phosphoric acid (500: 500: 1) system, the acetonitrile-water-phosphoric acid (330: 670: 1) system, and the methanol-acetonitrile-water-phosphoric acid (150: 165: 335: 1) system were compared. Results It shows that the chromatographic peaks of the test products of the latter two systems at different flow rates show different degrees of leading edge or tailing. Using methanol-water-phosphoric acid (500: 500: 1) system can not only obtain good peak shape, but also the target peak and its adjacent chromatographic peaks have good resolution, so this system is selected as the mobile phase.
3.2 Selection of extraction solvent. Comparing methanol, 50% methanol, 60% methanol, 70% methanol and 95% ethanol, 50% ethanol, 60% ethanol, 70% ethanol as extraction solvents, the medicinal powder was treated in the same way. The shape is the best, and the content of the target is high, so methanol is used as the extraction solvent of this method.
3.3 Selection of extraction method. The four methods of extracting Lysimachia powder were compared respectively: â‘ After 30 minutes of ultrasonic extraction without other solvent treatment, the sample was directly filtered; â‘¡ After 30 minutes of ultrasonic extraction, filtered, and the continuous filtrate of 10ml was accurately drawn, and 15ml of 25% hydrochloric acid was added. Reflux extraction for 3O minutes, add methanol to 50ml; â‘¢After ultrasonic extraction for 3O minutes, filter, accurately draw the continuous filtrate 2Oml into a separatory funnel, extract 3 times with petroleum ether (20ml, 15ml, 15ml) to remove fat-soluble impurities After that, the lower layer liquid was combined with ethyl acetate. Water (15: lO) mixed liquid 25 lTl1 was extracted three times, the upper layer liquid was combined, mixed well, 10ml was accurately drawn, 25ml of hydrochloric acid was added, 15ml was refluxed for 30 minutes, and methanol was added to 50ml; â‘£ Same as "2.2" Preparation of test solution. Results â‘ Although the target content of the method is high, but the interference is large; â‘¡, â‘¢ The target content of the method is the highest, but the recovery rate is not high, and considering that the flavonoid glycoside is hydrolyzed and converted into aglycon under acidic conditions, the result involves total flavonoids The actual work is too complicated; â‘£ The method has little interference and the target recovery rate is high, which can reflect the content of flavonoids in quercetin and kaempferol in the crude drug, so the method â‘£ is used for determination.
references
1 National Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China [S]. One, Beijing: Chemical Industry Press, 2000: 176-177.
2 Miao Mingsan, Li Zhenguo. Modern practical quality control technology of traditional Chinese medicine [M]. Beijing: People's Medical Publishing House, 2000: 672.
Determination of two flavonoids in Quercetin and Kaempferol in Lysimachia chinensis by RP-HPLC
Abstract: Objective To establish a reversed-phase high-performance liquid chromatography method for simultaneous determination of quercetin and kaempferol in the contents of Lysimachia for the study of quality standards of Lysimachia. Methods ODS column, methanol-water-phosphoric acid (500: 500: 1) reversed-phase high performance liquid chromatography system. Results Quercetin and kaempferol showed good linear relationship in the range of 3.195 × 0.001 ～ 0.15975μg, 3.252X0.001 ～ 0.1626μg, the correlation coefficients were 0.9999 and 0.9999, the average recoveries were 97.58% and 102.7%, RSD Respectively 1.16% and 1.01%. Conclusion This method is simple, reliable, accurate, and has good resolution. It can be used for quality control of Lysimachia chinensis.