[Abstract] Objective: To establish a method for the determination of the main components of borneol and eugenol in the Chinese medicine Liuying Pill by supercritical fluid extraction (SFE) and capillary gas chromatography (C GC ). The design method investigated the main effects of the factors affecting the extraction of supercritical fluids and the optimal extraction conditions, and quantitative fractionation by capillary gas chromatography. Results: The extraction conditions of SFE were as follows: pressure 48.2MPa, temperature 60 °C. Modified dose 0.2m1. The static extraction time was 10 min. Conclusion: SFE-CGC method is suitable for the determination of borneol and eugenol in Liuying Pills. It is fast, simple, accurate and sensitive.
[Key words] supercritical extraction method; borneol; eugenol; Liuying Pill; capillary gas chromatography; orthogonal design method Liuying Pill consists of borneol, male clove, medlar, pearl, etc., with heat and detoxification, anti-inflammatory and detumescence, Analgesic and other effects, used in chyle, sore sore, pharyngitis and toothache. Supercritical fluid extraction (SFE) technology has the characteristics of high mass transfer rate, high extraction efficiency, non-toxicity, economy, easy operation and good selectivity, which is superior to traditional solvent extraction methods, especially suitable for the international climate with increasing environmental awareness. . However, due to the many factors affecting SFE, it is difficult to explore the extraction conditions of Zui. We used the orthogonal design to experiment with the selection of the extraction conditions of borneol and eugenol in SFE extraction of Liuying Pill for the first time. The main effect and the secondary effect were determined, and the extraction conditions of zui were found out, and the SFE was compared with the traditional solvent extraction method.
1 Instruments and reagents
DM 100 syringe pump, SFX 2-10 extractor (ISCO, USA), gas chromatograph HP-5890 Series II, FID detector.
25 m x 0.32 mm, 0.52 μm, HP-5 column (crosslinked 5% Ph Me Silicone). HP-3365 ChemStation (Hewlett-Packard, USA).
Borneol, eugenol and thymol (China National Institute for the Control of Pharmaceutical and Biological Products); Liuying Pill (Shanghai Traditional Chinese Medicine No. 1); methanol, cyclohexane, chloroform,
Ethyl acetate was of analytical grade.
Standard solution: Weigh accurately the appropriate amount of borneol and eugenol, and prepare a solution of 5.22 mg/ml and 4.87 mg/ml with ethyl acetate. Place in a refrigerator at 4 °C.
Preparation of internal standard solution: accurately weigh the appropriate amount of thymol, and prepare a solution of 8.92 mg/m1 with ethyl acetate, and place it in a refrigerator at 4 °C.
2 sample analysis
2.1 SFE extraction 100 mg of Liuying Pill powder, which has been pulverized and passed through a 100 mesh sieve, is placed in an extraction tank. It is operated according to the extraction conditions of zui, absorbed with an appropriate amount of ethyl acetate, and the internal standard is 0.5 mL, and the volume is adjusted to 10 ml. , spare (extraction 1).
2.2 Ultrasonic solvent extraction 100mg of the above-mentioned powder, add appropriate amount of ethyl acetate, ultrasonic extraction for 60min, after cooling, accurately add 0.5 m1 of internal standard solution, and make up to 1Om1 volumetric flask (extraction 2).
2.3 Soxhlet extraction method Extract 0.5g of the above-mentioned powder, put it into a 50mL Soxhlet extractor, add 75mL of ethyl acetate, heat and extract it in water bath for 6h, filter it in 100ml volumetric flask, wash the residue with ethanol, and take it accurately. 5 ml of the standard solution, and ethyl acetate was added to the scale for use (extract 3).
2.4 Chromatographic conditions Gasification temperature 250 ° C, detector temperature 270 ° C, column temperature 160 ° C, carrier gas N2, column head pressure 55kPa, split ratio 100:1, internal standard thymol, injection volume 1μ1.
2.5 Standard curve preparation Take 0.1, 0.2, 0.4, 0.8, 1.2 ml standard solution in a 10 ml volumetric flask, inject 0.5 ml of the internal standard solution, dilute to the mark, and analyze by gas chromatography. Regression calculation was carried out by the ratio of bee area (Y) to the content (X). The regression equation was obtained: borneol Y=2.115X-9.928×0.001, r=0.9999. Linear range 52.2-626.4 ng, eugenol Y = 2.153X + 1.526 × 0.001. r = 0.9999, linear range 48.7 ~ 584.4 ng.
2.6 Determination of content Extract 1 directly injected 1μl, extract 2, extract 3 due to impurity interference is serious, both samples were purified by alumina column and then injected lμl.
2.7 SFE sample recovery test Quantitatively add 0.8 ml of standard solution to the drug, and the method is the same. The recovery of the sample is 101.93%, RSD=1.56% (n=3), and eugenol 98.43%. , RSD = 2.2696 (n = 3).
2.8 Selection of extraction conditions The orthogonal design method and variance analysis were used to select the orthogonal design table. The four factors were temperature (A), modified dose (B), pressure (C), and static extraction time (D). Orthogonal test results. According to the range analysis, from the extreme difference of borneol and eugenol, the order of influence on the degree of extraction is B, C, A, D, and the results are consistent. In addition, an analysis of variance was performed on the orthogonal design. The results of variance analysis showed that the modified dose had a significant effect on the SFE of borneol and eugenol, which was the main effect of SFE. For borneol, the pressure had a significant effect, and the secondary effect had a certain effect on the temperature. The static extraction time was on the SFE segment. What effect? ​​For eugenol, the effect of pressure is significant, which is a secondary effect, while temperature and static extraction time have a certain effect. Since the amount of modifier added affects the large size of zui, we have made 1 additional extraction conditions: A 6O ° C, B 0.3 ml, C 48.2 MPa. D 10
Min, the results of borneol and eugenol were as follows: A 6O ° C, B 0.2 ml, C 48.2 MPa, D 10min content is similar.
After the modified dose is increased, the impurity peak is also increased in the chromatogram. Our future as a good extraction condition for zui.
2.9 Selection of modifiers The extraction conditions were fixed with chloroform, ethyl acetate, cyclohexane and methanol as the modifiers, and the efficiency obtained by ethyl acetate was high. The experiment selected ethyl acetate.
2.10 Determination of borneol and isoborneol in the borneol reference product The borneol is a mixture of borneol and isoborneol. When the column temperature is 160 °C, the two are a peak. For quick analysis and internal standard Close, the author quantifies the borneol by measuring the total amount of both. The relative content of borneol and isoborneol in the reference substance was determined by the area normalization method. The chromatographic conditions were changed to the above column temperature to 90 °C, and other conditions were unchanged. At this time, the two could reach the baseline separation. The results were: 43.52% for allosaurus, RSD=0.67% (n=3); 54.85% for borneol, RSD=0.9% (n=3)
3 Conclusion
SFE extracts borneol and eugenol, the extraction efficiency is significantly higher than the traditional solvent extraction method, SFE operation is simple and fast, the whole process takes only 10 minutes; CO2 is inexpensive, non-toxic, non-flammable, chemically inert, volatile In addition, SFE has good selectivity, less impurity interference during chromatographic process, and can be directly injected without pretreatment after sample extraction, saving a lot of time and effort. SFE has broad application prospects in quality control of traditional Chinese medicine.
[references]
[1] Zheng Yongxi. Mathematical statistics in analytical chemistry, Beijing: Science Press. 1986.180.
[2] Chang Liping, Ni Kunyi. Wang Xintang. Wait. Capillary gas chromatography analysis of borneol and eugenol in Liuying Pill. Journal of China Pharmaceutical University. 1989, 20 (5): 297
[Key words] supercritical extraction method; borneol; eugenol; Liuying Pill; capillary gas chromatography; orthogonal design method Liuying Pill consists of borneol, male clove, medlar, pearl, etc., with heat and detoxification, anti-inflammatory and detumescence, Analgesic and other effects, used in chyle, sore sore, pharyngitis and toothache. Supercritical fluid extraction (SFE) technology has the characteristics of high mass transfer rate, high extraction efficiency, non-toxicity, economy, easy operation and good selectivity, which is superior to traditional solvent extraction methods, especially suitable for the international climate with increasing environmental awareness. . However, due to the many factors affecting SFE, it is difficult to explore the extraction conditions of Zui. We used the orthogonal design to experiment with the selection of the extraction conditions of borneol and eugenol in SFE extraction of Liuying Pill for the first time. The main effect and the secondary effect were determined, and the extraction conditions of zui were found out, and the SFE was compared with the traditional solvent extraction method.
1 Instruments and reagents
DM 100 syringe pump, SFX 2-10 extractor (ISCO, USA), gas chromatograph HP-5890 Series II, FID detector.
25 m x 0.32 mm, 0.52 μm, HP-5 column (crosslinked 5% Ph Me Silicone). HP-3365 ChemStation (Hewlett-Packard, USA).
Borneol, eugenol and thymol (China National Institute for the Control of Pharmaceutical and Biological Products); Liuying Pill (Shanghai Traditional Chinese Medicine No. 1); methanol, cyclohexane, chloroform,
Ethyl acetate was of analytical grade.
Standard solution: Weigh accurately the appropriate amount of borneol and eugenol, and prepare a solution of 5.22 mg/ml and 4.87 mg/ml with ethyl acetate. Place in a refrigerator at 4 °C.
Preparation of internal standard solution: accurately weigh the appropriate amount of thymol, and prepare a solution of 8.92 mg/m1 with ethyl acetate, and place it in a refrigerator at 4 °C.
2 sample analysis
2.1 SFE extraction 100 mg of Liuying Pill powder, which has been pulverized and passed through a 100 mesh sieve, is placed in an extraction tank. It is operated according to the extraction conditions of zui, absorbed with an appropriate amount of ethyl acetate, and the internal standard is 0.5 mL, and the volume is adjusted to 10 ml. , spare (extraction 1).
2.2 Ultrasonic solvent extraction 100mg of the above-mentioned powder, add appropriate amount of ethyl acetate, ultrasonic extraction for 60min, after cooling, accurately add 0.5 m1 of internal standard solution, and make up to 1Om1 volumetric flask (extraction 2).
2.3 Soxhlet extraction method Extract 0.5g of the above-mentioned powder, put it into a 50mL Soxhlet extractor, add 75mL of ethyl acetate, heat and extract it in water bath for 6h, filter it in 100ml volumetric flask, wash the residue with ethanol, and take it accurately. 5 ml of the standard solution, and ethyl acetate was added to the scale for use (extract 3).
2.4 Chromatographic conditions Gasification temperature 250 ° C, detector temperature 270 ° C, column temperature 160 ° C, carrier gas N2, column head pressure 55kPa, split ratio 100:1, internal standard thymol, injection volume 1μ1.
2.5 Standard curve preparation Take 0.1, 0.2, 0.4, 0.8, 1.2 ml standard solution in a 10 ml volumetric flask, inject 0.5 ml of the internal standard solution, dilute to the mark, and analyze by gas chromatography. Regression calculation was carried out by the ratio of bee area (Y) to the content (X). The regression equation was obtained: borneol Y=2.115X-9.928×0.001, r=0.9999. Linear range 52.2-626.4 ng, eugenol Y = 2.153X + 1.526 × 0.001. r = 0.9999, linear range 48.7 ~ 584.4 ng.
2.6 Determination of content Extract 1 directly injected 1μl, extract 2, extract 3 due to impurity interference is serious, both samples were purified by alumina column and then injected lμl.
2.7 SFE sample recovery test Quantitatively add 0.8 ml of standard solution to the drug, and the method is the same. The recovery of the sample is 101.93%, RSD=1.56% (n=3), and eugenol 98.43%. , RSD = 2.2696 (n = 3).
2.8 Selection of extraction conditions The orthogonal design method and variance analysis were used to select the orthogonal design table. The four factors were temperature (A), modified dose (B), pressure (C), and static extraction time (D). Orthogonal test results. According to the range analysis, from the extreme difference of borneol and eugenol, the order of influence on the degree of extraction is B, C, A, D, and the results are consistent. In addition, an analysis of variance was performed on the orthogonal design. The results of variance analysis showed that the modified dose had a significant effect on the SFE of borneol and eugenol, which was the main effect of SFE. For borneol, the pressure had a significant effect, and the secondary effect had a certain effect on the temperature. The static extraction time was on the SFE segment. What effect? ​​For eugenol, the effect of pressure is significant, which is a secondary effect, while temperature and static extraction time have a certain effect. Since the amount of modifier added affects the large size of zui, we have made 1 additional extraction conditions: A 6O ° C, B 0.3 ml, C 48.2 MPa. D 10
Min, the results of borneol and eugenol were as follows: A 6O ° C, B 0.2 ml, C 48.2 MPa, D 10min content is similar.
After the modified dose is increased, the impurity peak is also increased in the chromatogram. Our future as a good extraction condition for zui.
2.9 Selection of modifiers The extraction conditions were fixed with chloroform, ethyl acetate, cyclohexane and methanol as the modifiers, and the efficiency obtained by ethyl acetate was high. The experiment selected ethyl acetate.
2.10 Determination of borneol and isoborneol in the borneol reference product The borneol is a mixture of borneol and isoborneol. When the column temperature is 160 °C, the two are a peak. For quick analysis and internal standard Close, the author quantifies the borneol by measuring the total amount of both. The relative content of borneol and isoborneol in the reference substance was determined by the area normalization method. The chromatographic conditions were changed to the above column temperature to 90 °C, and other conditions were unchanged. At this time, the two could reach the baseline separation. The results were: 43.52% for allosaurus, RSD=0.67% (n=3); 54.85% for borneol, RSD=0.9% (n=3)
3 Conclusion
SFE extracts borneol and eugenol, the extraction efficiency is significantly higher than the traditional solvent extraction method, SFE operation is simple and fast, the whole process takes only 10 minutes; CO2 is inexpensive, non-toxic, non-flammable, chemically inert, volatile In addition, SFE has good selectivity, less impurity interference during chromatographic process, and can be directly injected without pretreatment after sample extraction, saving a lot of time and effort. SFE has broad application prospects in quality control of traditional Chinese medicine.
[references]
[1] Zheng Yongxi. Mathematical statistics in analytical chemistry, Beijing: Science Press. 1986.180.
[2] Chang Liping, Ni Kunyi. Wang Xintang. Wait. Capillary gas chromatography analysis of borneol and eugenol in Liuying Pill. Journal of China Pharmaceutical University. 1989, 20 (5): 297
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