Abstract: The dry whole grass of Sarcandraglabra (Thunb.) Nakai, the genus of Coriolus plantaceae in Jiujie tea family, also known as swollen knot, elderberry, elderberry, elderberry, is a perennial evergreen or subshrub. Jiujie Tea tastes bitter and spicy, calm, mildly poisonous, and has the functions of removing wind and collaterals, promoting blood circulation, removing blood stasis, hemostasis and pain relief, and strengthening bones and tendons.
Objective To optimize the separation conditions of HPLC fingerprint of Jiujiecha. Methods The chromatography column KromasilC18 (250mm × 4.6mm, 5μm) was used. The mobile phase was methanol-0.2% phosphoric acid solution, the column temperature was 25 ℃, the flow rate was 0.7ml / min, the detection wavelength was 340nm, and the optimal gradient of Jiujia tea was investigated. Elution conditions. Results The optimized separation conditions were: 0 ～ 35min, methanol increased linearly from 20% to 40%; 35 ～ 65min methanol increased from 40% to 60%, 65 ～ 75min methanol decreased from 60% to 20%, 75 ～ 78min methanol Maintaining 20% ​​can achieve better separation of the main components. Conclusion By optimizing the separation conditions of Jiujiecha HPLC fingerprint, it is more conducive to controlling the quality of Jiujiecha medicinal materials.
The Chinese Pharmacopoeia 2005 Edition Part I quality control of Jiujiacha medicinal materials is HPLC to determine the content of isopizidine [2], but the determination of the content of a single index component is often difficult to reflect the overall quality of traditional Chinese medicine and cannot be more comprehensively reflected. Chinese medicine quality. It has been reported in the literature that the fingerprints of Jiujiecha's medicinal materials have been studied [3, 4], and it has been found in practical applications that the methods for obtaining the fingerprints mentioned above have poor repeatability. This article intends to optimize the separation conditions of the liquid chromatography fingerprint of Jiujiecha, so as to provide a better method for the quality control of Jiujiecha.
1 Materials and instruments
1.1 Materials Jiujie tea medicinal materials have been identified as the whole grass of Sarcandraglabra (Thunb.) Nakai by the pharmacist Gao Ying of the New Drug Development and Research Center of Guangzhou University of Traditional Chinese Medicine; methanol is chromatographically pure; water is liquid water; other The reagents are of analytical grade.
1.2 Instrument DionexSummit high performance liquid chromatograph (P680HPLCPump, ASI-100AutomatedSampledInjector, PDA-100PhtodiodeArrayDetecter, UVD170U, STH585ColumnOven), Chromeleon data processing system, KromasilC18 column (250mm × 4.6mm, 5μm); ultrasonic cleaner (SB-5200, China Shipbuilding Industry Corporation Seventh Research Institute 726 Research Institute); R-114 Rotary Evaporator (BuCHI, Switzerland).
2 Methods and results
2.1 Preparation of test solution Take 1.0g crude medicinal powder (through a 20 mesh sieve), add twice under reflux with water, add 20ml of water each time, extract for 1h, filter, combine the filtrate, evaporate to dryness, and transfer the residue to a 25ml measuring flask , Add about 20ml of 80% methanol, sonicate for 30min, put to room temperature, dilute to volume with methanol, centrifuge, carefully draw the supernatant, filter with a microporous filter and use.
2.2 Optimization of gradient elution conditions
2.2.1 Optimization Condition â… After experiment, the following initial conditions are obtained:
Conditions of Fig. 1a: 0 ～ 50min, methanol increases linearly from 5% to 70%; 50 ～ 60min methanol increases from 70% to 90%, injection volume is 10μl, column temperature is 25 ℃, flow rate is 0.7ml / min. The chromatogram (see Figure 1a) shows that the chromatographic peaks of the main components are eluted within 10 to 30 minutes, and no chromatographic peaks appear within 30 to 60 minutes, indicating that all components have been eluted.
Figure 1b conditions: 0 ～ 35min, methanol linearly increased from 5% to 25%; 35 ～ 60min methanol increased from 25% to 45%, injection volume was 10μl, column temperature was 25 ℃, flow rate was 0.7ml / min. The chromatogram (see Figure 1b) shows that the chromatographic peaks of the main components are eluted within 20 to 50 minutes, but there are few chromatographic peaks within 0 to 20 minutes, so optimization needs to be continued.
2.2.2 Optimization Conditions â…¡
Figure 2a conditions: 0-40min, methanol linearly increased from 10% to 30%; 40-60min methanol increased from 30% to 45%, injection volume was 10μl, column temperature was 25 ℃, flow rate was 0.7ml / min. The chromatogram (see Figure 2a) shows that the chromatographic peaks of the main components are eluted within 5 to 50 minutes, but the peak-to-peak overlap is large.
Figure 2b conditions: 0 ～ 40min, methanol increases linearly from 15% to 35%; 40 ～ 60min methanol increases from 35% to 50%, 60 ～ 70min methanol decreases from 50% to 15%, 70 ～ 72min methanol keeps 15%, The injection volume was 10 μl, the column temperature was 25 ° C., and the flow rate was 0.7 ml / min. The chromatogram (see Figure 2b) shows that the distribution of chromatographic peaks has improved, but the separation of peaks and peaks is not ideal.
2.2.3 Optimization conditions â…¢
Figure 3a conditions: 0 ～ 35min, methanol increases linearly from 17.5% to 40%; 35 ～ 60min methanol increases from 40% to 60%, 60 ～ 70min methanol decreases from 60% to 17.5%, 70 ～ 72min methanol keeps 17.5%, The injection volume was 10 μl, the column temperature was 25 ° C., and the flow rate was 0.7 ml / min. The chromatogram (see Figure 3a) can be seen that the chromatographic peaks are basically separated, and are evenly distributed within 0 to 60 minutes, but some peaks have a tailing phenomenon.
Figure 3b conditions: 0 ～ 35min, methanol increases linearly from 20% to 40%; 35 ～ 65min methanol increases from 40% to 60%, 65 ～ 75min methanol decreases from 60% to 20%, 75 ～ 78min methanol keeps 20%, The injection volume was 10 μl, the column temperature was 25 ° C., and the flow rate was 0.7 ml / min. The chromatogram (see Figure 3b) can be seen, whether it is the resolution, distribution uniformity and peak shape of the chromatographic peaks are in line with the technical requirements of the fingerprint.
2.3 Methodological investigation In order to investigate the reliability of analytical methods, its stability, instrument precision, and reproducibility of experimental methods were investigated.
2.3.1 Precision experiment Take the sample solution for 5 consecutive injections and record the fingerprint. Taking the peak with a retention time of 49.5 min as the reference peak, the RSD values ​​of the relative retention time and relative peak area of ​​the common peak were calculated to be between 0.48% and 0.92% and 1.15% and 1.97%, respectively, which met the fingerprint requirements.
2.3.2 Reproducibility experiment Weigh 5 parts of medicinal materials accurately, prepare 5 sample solutions according to the method of sample solution preparation, inject samples and record fingerprints respectively. Taking the peak with a retention time of 49.5 min as the reference peak, the RSD values ​​of the relative retention time and the relative peak area of ​​the common peak were between 0.45% and 1.03% and 1.41% and 2.06%, respectively, which basically met the fingerprint requirements.
2.3.3 Stability experiment Sample solutions were taken for injection analysis at 0, 2, 6, 12, and 24h, and the color spectrum was recorded. The peak with a retention time of 49.5min was taken as the reference peak, and the relative retention time of the common peak was calculated. The RSD values ​​of the relative peak areas were between 0.36% and 0.96% and 1.20% and 2.33%, respectively, indicating that the sample solution was stable within 24 hours.
3 Discussion
3.1 Selection of mobile phase The chemical components in Jiujiecha are sesquiterpenes and their glycosides, flavonoids and their glycosides, organic acids, and coumarins, and these components mostly have certain polarity and acidity [5]. This experiment compared acetonitrile-0.1% phosphoric acid, acetonitrile-0.2% phosphoric acid, acetonitrile-0.1% acetic acid, acetonitrile-1% acetic acid, methanol-0.1% phosphoric acid, methanol-0.2% phosphoric acid, methanol-0.1% acetic acid, methanol-1% The separation effect of acetic acid and other mobile phases. Results Under the same conditions, methanol was better than acetonitrile in separating the extract of Jiujia tea, and 0.2% phosphoric acid solution was better than other acid solutions in separating the extract of Jiujia tea. Therefore, methanol and 0.2% phosphoric acid were selected as mobile phase units. .
3.2 Selection of column temperature During the experiment, it was found that the column temperature had a greater influence on the separation effect. Under the same conditions, the effect of 20, 25, 30, 35 ℃ column temperature on the separation effect of Jiujia tea extract was investigated, and the result was 25 ℃ Better.
3.3 Selection of flow rate The effects of 0.6, 0.7, 0.8, and 1.0 ml / min flow rate on the separation effect of Jiujia tea extract were investigated under the same conditions. The result is that the separation effect of 0.7 ml / min flow rate is better.
3.4 Selection of detection wavelength Select 340nm as the detection wavelength [3].
In isocratic elution, if the elution capacity of the solvent is too strong, the chromatographic peaks of the main components cannot be separated; when the elution capacity of the solvent is weak, although it can be separated, the retention time is too long and the peak shape is not ideal. In order to obtain a chromatographic peak with good resolution, suitable retention time and ideal peak shape, only gradient elution is used.
The column temperature and flow rate in liquid chromatography conditions have little effect on the chromatographic peaks, so the composition of the mobile phase, the ratio of the solvent and the gradient steepness should be adjusted mainly to achieve better separation between the chromatographic peaks. When examining the composition of the mobile phase, in reversed-phase chromatography, the elution intensity of methanol is lower than that of acetonitrile, so the peak shape and resolution of the chromatographic peaks of the methanolic acid water system are better than those of the acetonitrile water system. At the same time, phosphoric acid is more acidic than acetic acid, and it has a strong inhibitory effect on the ionization of acidic components in medicinal materials. The use of phosphoric acid can ensure that the chromatographic peaks do not trail.
Through experiments, the resolution, distribution uniformity and peak shape of Jiujiacha liquid chromatograms obtained by optimizing the conditions III can be used to evaluate the quality differences of the fingerprints of Jiujiacha of different origins and varieties.
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