Method for simultaneously determining seven index components in Fengtongan capsule and constructing specific chromatogram and application

Abstract

The invention relates to a method for simultaneously determining seven index components in Fengtongan capsules and constructing a specific chromatogram and application, and belongs to the technical field of detection and analysis. According to the method, a high performance liquid chromatograph and a gradient elution mode are adopted, the contents of phellodendrine (284nm), loganin (236nm), fangchinoline, tetrandrine (280nm), forsythin (277nm), berberine (265nm) and curcumin (430nm) in a prescription can be simultaneously determined by switching different wavelengths under the same elution condition, and a characteristic spectrum (254nm) is constructed. The tested components are well separated, and the practical purposes of multi-component evaluation by one test, high efficiency, low consumption and easiness in popularization are realized. According to the polarity difference of each substance, a gradient elution procedure is adopted to realize separation and accurate detection. So far, no report about simultaneous determination of the seven components in the Fengtongan capsule is found, and the method makes up the blank of quality control and fingerprint spectrum research of various medicinal materials in the prescription.

Description

Technical Field

[0001] The present invention relates to a method and application for simultaneously determining seven index components in Fengtongan capsules and constructing a characteristic spectrum. That is, by using a high performance liquid chromatograph and gradient elution, the contents of phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine and curcumin in Fengtongan capsules can be simultaneously determined by switching different wavelengths under the same elution condition and constructing a characteristic spectrum. The method belongs to the field of detection and analysis technology. Background Art

[0002] Twenty-five-flavor Pearl Pills has the national medicine standard number Z19983029, and its functions and indications are to clear away heat and dampness, and to activate blood circulation and dredge collaterals. It is used for arthralgia caused by damp-heat blocking collaterals, with symptoms of red, swollen, hot and painful joints, and muscle aches; rheumatoid arthritis with the above symptoms.

[0003] The prescription composition and preparation process are as follows:

[0004] Prescription: 250g of Stephania tetrandra, 167g of Smilax glabra, 125g of cinnamon twig, 167g of turmeric, 500g of gypsum, 333g of coix seed, 250g of papaya, 167g of Pittosporum tobira bark, 333g of honeysuckle vine, 250g of Phellodendron chinense, 250g of talc, and 333g of Forsythia suspensa.

[0005] Preparation process: Take 167g of talcum powder and the other eleven ingredients including Stephania tetrandra, add water and boil three times, the first time for 3 hours, the second time for 2 hours, and the third time for 1 hour. Filter the decoction, combine the filtrate, concentrate to an appropriate amount, add the remaining talcum powder, mix, dry, crush, sieve, mix well, put into capsules, and make 1000 capsules.

[0006] At present, there are few literatures on Fengtongan, and the publication date is very old. In the past five years, there is one article on the determination of aristolochic acid content by ultra-high performance liquid phase tandem mass spectrometry, and the remaining 20 articles are all before 2014. In terms of content determination, single component determination is carried out around loganin, curcumin, forsythiaside, tetrandrine, fangchinoline, and berberine hydrochloride, without characteristic spectra and multiple evaluations of multiple medicinal materials. At present, the current standard of Fengtongan is the 2020 edition of the "Chinese Pharmacopoeia", and the content determination items under it are tetrandrine and fangchinoline.

[0007] This prescription contains twelve medicinal ingredients, and it is difficult to control the intrinsic quality of the preparation by single content determination. In order to ensure the therapeutic effect of Fengtongan Capsules with high quality and strictly control the quality of medicinal materials, it is planned to conduct simultaneous quantitative research on phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin in the capsules and construct a characteristic map.

[0008] According to the literature, the chemical structures of phellodendron pine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin are as follows:

[0009]

[0010]

[0011] From the above structural formula, we know that according to the polarity difference of each substance, a gradient elution program can be used to separate and accurately detect the above components. So far, there is no report on the simultaneous determination of these seven medicinal ingredients (Phellodendron chinense, Loganin, Fangchinoline, Tetrandrine, Forsythin, Berberine, Curcumin) in Fengtongan. Summary of the invention

[0012] The present invention is based on the above background, in order to improve detection efficiency, reduce detection cost and reduce environmental pollution, for the first time, obtains the test solution by a simple and fast pretreatment method, adopts a high performance liquid chromatograph (diode array detector), and uses A (0.1% phosphoric acid)-B (acetonitrile) as the mobile phase for gradient elution, and the elution procedure is as follows: 0-33min 3-8% B, 33-908-14% B, 90-120min 14-19% B, 120-145min 19-30% B, 145-160min 30-40B%, 160-180min 40-65% B, 180-190min 3B%, 190-195min 3% B. The same elution condition can achieve simultaneous determination of seven index components and establishment of characteristic spectrum by switching wavelengths. The detection wavelengths were 254 nm (for the establishment of characteristic spectra), phellodendronine (284 nm), loganin (236 nm), fangchinoline, tetrandrine (280 nm), forsythin (277 nm), berberine (265 nm), and curcumin (430 nm); the column temperature was 28 °C; the flow rate was 0.8 mL min -1 ; Injection volume 10μL. The theoretical plate number of phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin peak calculations are not less than 5000. The peak separation is good, achieving the purpose of one test for multiple evaluations, high efficiency, low consumption, and easy popularization and promotion. The simultaneous determination of seven index components of precious medicinal materials is reported for the first time, demonstrating the novelty, creativity, and practicality of low-cost, double-index detection with half the effort.

[0013] Through methodological investigation, the injection amount of phellodendronine was 100.18-1001.80 ng, the injection amount of loganin was 80.48-804.80 ng, the injection amount of fangchinoline was 100.10-1001.00 ng, the injection amount of tetrandrine was 200.46-2004..60 ng, the injection amount of forsythiaside was 201.56-2015.60 ng, the injection amount of berberine was 200.82-2008.20 ng, and the injection amount of curcumin was 86.88-868.80 ng, which showed a good linear relationship with the peak area (see Tables 1-7, Figures 1 to 7), the regression equations are: Y = 1245.9X + 5192.3, R 2 =0.9999; Y of loganin = 1841.3X-4347.2, R 2 =0.9997; Fangchinoline Y = 915.37X-12575, R 2 =0.9999; Y of tetrandrine = 786.52X-5121.3, R 2 =0.9998; Y of forsythiaside = 785.87X + 2490.4, R 2 =0.9999; Y of berberine = 5030.4X + 28922, R 2 =0.9999; Y of curcumin = 9761.2X-353.83, R 2 =0.9998. The results of the sample recovery experiment showed that the average recovery rate of phellodendronine was 95.09% for 6 determinations, and the RSD was 1.71% (see Table 8); the average recovery rate of loganin was 96.08% for 6 determinations, and the RSD was 1.62% (see Table 9); the average recovery rate of fangchinoline was 97.81% for 6 determinations, and the RSD was 0.73% (see Table 10); the average recovery rate of tetrandrine was 97.71% for 6 determinations, and the RSD was 1.39% (see Table 11); the average recovery rate of forsythin was 96.99% for 6 determinations, and the RSD was 1.31% (see Table 12); the average recovery rate of berberine was 96.69% for 6 determinations, and the RSD was 0.65% (see Table 13); the average recovery rate of curcumin was 96.42% for 6 determinations, and the RSD was 1.01% (see Table 14). Precision (see Table 15), stability (see Table 16), repeatability (see Table 17), UV spectra of the measured components (see Figures 8 to 14 ), reference substance chromatogram (see Figures 15 to 20 ), sample chromatogram (see Figures 21 to 26 ), specificity (see Figures 27 to 31 ), 3 batches of sample determination (see Table 18), ultrasonic time investigation (see Table 19), extraction solvent investigation (Table 20), the experimental method data all meet the methodological requirements. Simple, accurate and efficient, it can be used for the simultaneous quantitative determination of phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine and curcumin in Fengtongan Capsules, and at the same time establish the fingerprint of Fengtongan Capsules (254nm) and investigate the durability and repeatability of the method for better quality control (see Fig.32 ).

[0014] The technical solution adopted by the present invention to solve the technical problem is:

[0015] (1) A. Chromatographic conditions and system suitability test; 0.1% phosphoric acid water was used as mobile phase A and acetonitrile was used as mobile phase B; gradient elution, the elution program was as follows: 0-33 min 3-8% B, 33-90 min 8-14% B, 90-120 min 14-19% B, 120-145 min 19-30% B, 145-160 min 30-40% B, 160-180 min 40-65% B, 180-190 min 3B%, 190-195 min 3% B. The detection wavelengths were 254 nm (for the establishment of characteristic spectra), phellodendronine (284 nm), loganin (236 nm), fangchinoline, tetrandrine (280 nm), forsythin (277 nm), berberine (265 nm), and curcumin (430 nm); the column temperature was 28 °C; the flow rate was 0.8 mL min -1 ; The injection volume was 10 μL. The theoretical plate numbers of phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin peaks were all calculated to be no less than 5000;

[0016] B. Preparation of reference solution Accurately weigh appropriate amount of phellodendronine, loganin, fangchinoline and curcumin reference substances, accurately weigh, add methanol to make a solution containing 100 μg of each per 1 ml, shake well, and obtain;

[0017] Accurately weigh appropriate amounts of tetrandrine, forsythin, berberine, and reference substance, accurately weigh, and add methanol to make a solution containing 50 μg of each per 1 ml, shake well, and obtain;

[0018] C. Preparation of test solution: Take the contents of Fengtong'an capsules, weigh about 0.5 g, accurately weigh, place in a stoppered conical flask, add 20 mL of 90% methanol, stopper, weigh, ultrasonically treat (500 W, frequency 40 Hz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 90% methanol solution, shake well, filter, and take the filtrate to obtain the solution.

[0019] D. Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and calculate the contents of the 7 components respectively.

[0020] The simultaneous determination refers to the use of the same elution condition to switch different wavelengths to simultaneously determine the contents of phellodendronine, loganin, fangchinoline, tetrandrine, forsythiaside, berberine, and curcumin in Fengtong'an Capsules and construct a characteristic spectrum.

[0021] The invention discloses an application of a method for simultaneously determining seven index components in Fengtong'an capsule and constructing a characteristic spectrum in constructing a characteristic spectrum of Fengtong'an capsule.

[0022] The invention discloses an application of a method for simultaneously determining seven index components in Fengtong'an capsules and constructing a characteristic spectrum in the content determination of Fengtong'an capsules.

[0023] The principle of the present invention is as follows:

[0024] Based on the principle of similar-miscible extraction and the difference in dosage forms of substances, gradient elution is used, with polarity from small to large, using the maximum absorption of each substance (see Figures 8 to 14 ), as the detection wavelength, not only makes the size and area of ​​the component peaks suitable, but also greatly reduces the interference peaks at this detection wavelength, presents a stable baseline of the liquid phase spectrum, and has good peak separation, laying the foundation for the simultaneous determination of multiple components. Then, based on the good linear relationship between the injection volume of the multiple components and their peak areas within a certain range, they are used for quantitative determination.

[0025] The innovative features and beneficial effects of the present invention are as follows:

[0026] (1) Gradient elution is adopted, with 0.1% phosphoric acid water as mobile phase A and acetonitrile as mobile phase B; the same elution condition is used to simultaneously determine seven index components and establish characteristic spectra by switching wavelengths. It enables several effective ingredients of traditional Chinese medicine with completely different properties and structures to be quantitatively determined once using the same mobile phase. The quantitative chromatograms of multiple components have a stable baseline and good peak separation. The method has no extraction, no concentration, no evaporation, is simple, fast, accurate, and reproducible, and is easy to popularize and master, effectively improving detection efficiency, reducing detection costs, and reducing environmental pollution.

[0027] (2) According to the literature review, there is little information on Fengtongan, and the publication date is very old. In the past five years, there is one article that measured aristolochic acid content by ultra-high performance liquid phase tandem mass spectrometry, and the remaining 20 articles were all before 2014. In terms of content determination, single component determination was performed on loganin, curcumin, forsythin, tetrandrine, fangchinoline, and berberine hydrochloride, without characteristic spectra or multiple evaluations of multiple medicinal materials. At present, the current standard for Fengtongan is the 2020 edition of the Chinese Pharmacopoeia, and the content determination items under the item are tetrandrine and fangchinoline.

[0028] The method for simultaneously determining these seven components (peppermintine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin) is reported for the first time. In addition, the characteristic spectrum has a total of 40 peaks. In addition to the above-mentioned components, chlorogenic acid and cinnamaldehyde are also identified as characteristic components of the characteristic spectrum. The detection efficiency is greatly improved and the instrument cost is significantly reduced. Not only that, the more critical thing is to upgrade one test and one evaluation to one test and multiple evaluations, achieving twice the result with half the effort.

[0029] (3) The advent of the method of the present invention not only provides a method for the simultaneous detection of multiple medicinal materials (Phellodendron amurense, Lonicera japonica, Stephania tetrandra, Forsythia suspensa, and Curcuma longa) in the prescription of Fengtong'an Capsules, but also provides a reference basis and research ideas for the simultaneous determination of these ingredients in other compound preparations.

[0030] (4) The key technology of the present invention is to make use of the characteristic of acetonitrile in the mobile phase that the peak retention time of various chemical components is the premise, and the phosphoric acid solution can make the acidic components exist as molecules, eliminating the ionic tailing performance. Through the infinite gradient ratio study between acetonitrile and phosphoric acid of multiple concentrations, an elution condition is explored that can make a variety of chemical components with large polarity differences appear in the same mobile phase, and the peak retention time is appropriate, the baseline is stable, and the reproducibility is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Linear relationship diagram of phellodendrine;

[0032] Figure 2 Linear relationship graph of loganin;

[0033] Figure 3 Linear relationship diagram of fangchinoline;

[0034] Figure 4 Linear relationship diagram of tetrandrine;

[0035] Figure 5 Linear relationship diagram of forsythiaside;

[0036] Figure 6 Linear relationship graph of berberine;

[0037] Figure 7 Linear relationship graph of curcumin;

[0038] Figure 8 UV absorption spectrum of phellodendrine;

[0039] Fig. 9 UV absorption spectrum of loganin;

[0040] Fig.10 The ultraviolet absorption spectrum of fangchinoline;

[0041] Fig.11 Ultraviolet absorption spectrum of tetrandrine;

[0042] Fig.12 UV absorption spectrum of Forsythiaside;

[0043] Fig.13 UV absorption spectrum of berberine;

[0044] Fig.14 UV absorption spectrum of curcumin;

[0045] Fig.15 HPLC chromatogram of phellodendrine;

[0046] Fig.16 HPLC chromatogram of loganin;

[0047] Fig.17 HPLC chromatograms of fangchinoline and tetrandrine;

[0048] Fig.18 HPLC chromatogram of forsythiaside;

[0049] Fig.19 HPLC chromatogram of berberine;

[0050] Fig. 20 HPLC chromatogram of curcumin;

[0051] Fig.21 HPLC chromatogram of Fengtongan capsule sample (Phellodendron chinense 284nm);

[0052] Fig. 22 HPLC chromatogram of Fengtongan capsule sample (loganin 236nm);

[0053] Fig.23 HPLC chromatogram of Fengtongan capsule sample (fangchinoline, tetrandrine 280nm);

[0054] Fig.24 HPLC chromatogram of Fengtongan capsule sample (forsythiaside 277nm);

[0055] Fig.25 HPLC chromatogram of Fengtongan capsule sample (berberine 265nm);

[0056] Fig.26 HPLC chromatogram of Fengtongan capsule sample (curcumin 430nm);

[0057] Fig. 27 This is the HPLC chromatogram of a blank sample without Phellodendron chinense;

[0058] Fig.28 This is the HPLC chromatogram of a blank sample without honeysuckle vine;

[0059] Fig.29 This is the HPLC chromatogram of a blank sample without Stephania tetrandra;

[0060] Fig.30 This is the HPLC chromatogram of a blank sample without Forsythia suspensa;

[0061] Fig.31 is the HPLC chromatogram of a blank sample without turmeric;

[0062] Fig.32 Characteristic spectrum of Fengtongan capsule;

[0063] Figures 1 to 7 In the figure, the ordinate is the peak area; the abscissa is the injection amount (ng);

[0064] Figures 8 to 14 In the figure, the ordinate is the absorption intensity (mAU); the abscissa is the absorption wavelength (nm);

[0065] Figures 15 to 32 In the figure, the horizontal axis is the retention time (minutes), and the vertical axis is the response value (mAU);

[0066] Figures 15-26 In the figure, the horizontal axis is the retention time (minutes), the vertical axis is the response value (mAU), and the labels 1 is phellodendrine, 2 is loganin, 3 is fangchinoline, 4 is tetrandrine, 5 is forsythiaside, 6 is berberine, and 7 is curcumin;

[0067] Fig.32 In the figure, the horizontal axis is the retention time (minutes), the vertical axis is the response value (mAU), and the labels 1 is chlorogenic acid, 2 is phellodendronine, 3 is loganin, 4 is fangchinoline, 5 is tetrandrine, 6 is forsythiaside, 7 is berberine, 8 is cinnamaldehyde, and 9 is curcumin. DETAILED DESCRIPTION

[0068] Example

[0069] (1) A. Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel was used as filler; 0.1% phosphoric acid water was used as mobile phase A, acetonitrile was used as mobile phase B; gradient elution, the elution program was as follows: 0.1% phosphoric acid water was used as mobile phase A, acetonitrile was used as mobile phase B; gradient elution, the elution program was as follows: 0-33 min 3-8% B, 33-90 min 8-14% B, 90-120 min 14-19% B, 120-145 min 19-30% B, 145-160 min 30-40% B, 160-180 min 40-65% B, 180-190 min 3B, 190-195 min 3% B. The detection wavelengths were 254 nm (for the establishment of characteristic spectra), phellodendronine (284 nm), loganin (236 nm), fangchinoline, tetrandrine (280 nm), forsythin (277 nm), berberine (265 nm), and curcumin (430 nm); the column temperature was 28 °C; the flow rate was 0.8 mL min -1 ; The injection volume was 10 μL. The theoretical plate numbers of phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin peaks were all calculated to be no less than 5000;

[0070] B. Preparation of reference solution Accurately weigh appropriate amount of phellodendronine, loganin, fangchinoline and curcumin reference substances, accurately weigh, add methanol to make a solution containing 100 μg of each per 1 ml, shake well, and obtain;

[0071] Accurately weigh appropriate amounts of tetrandrine, forsythin, berberine, and reference substance, accurately weigh, and add methanol to make a solution containing 50 μg of each per 1 ml, shake well, and obtain;

[0072] C. Preparation of test solution: Take the contents of Fengtong'an capsules, weigh about 0.5 g, accurately weigh, place in a stoppered conical flask, add 20 mL of 90% methanol, stopper, weigh, ultrasonically treat (500 W, frequency 40 Hz) for 30 minutes, take out, cool, weigh again, make up the lost weight with 90% methanol solution, shake well, filter, and take the filtrate to obtain the solution.

[0073] D. Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and calculate the contents of the 7 components respectively.

[0074] Table 1 Injection amount and peak area of ​​phellodendron chinense

[0075]

[0076] Table 2 Loganin injection amount and peak area

[0077]

[0078] Table 3 Injection amount and peak area of ​​fangchinoline

[0079]

[0080] Table 4 Tetrandrine injection amount and peak area

[0081]

[0082] Table 5 Forsythiaside injection amount and peak area

[0083]

[0084] Table 6 Berberine injection amount and peak area

[0085]

[0086] Table 7 Curcumin injection amount and peak area

[0087]

[0088] Table 8 Test results of recovery rate of phellodendron alkali in samples

[0089]

[0090] Table 9 Loganin recovery test results in samples

[0091]

[0092] Table 10 Test results of recovery rate of fangchinoline in samples

[0093]

[0094] Table 11 Tetrandrine recovery test results in samples

[0095]

[0096]

[0097] Table 12 Results of the recovery rate test of forsythiaside in samples

[0098]

[0099] Table 13 Test results of berberine recovery in samples

[0100]

[0101] Table 14 Curcumin recovery test results in samples

[0102]

[0103] Table 15 Precision test results

[0104]

[0105] Table 16 Stability test results

[0106]

[0107]

[0108] Table 17 Repeatability test results (mg / g)

[0109]

[0110] Table 18 Content determination results of 7 ingredients in 3 batches of Fengtongan capsules (mg / g, n=2)

[0111]

[0112] Table 19 Ultrasonic time investigation (mg / g)

[0113]

[0114] Table 20 Extraction solvent investigation (mg / g)

[0115]

[0116]

Claims

1. A method for simultaneously determining 7 index components in Fengtong'an capsules and constructing a characteristic spectrum, characterized in that: A. Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel was used as filler; 0.1% phosphoric acid water was used as mobile phase A, and acetonitrile was used as mobile phase B; the detection wavelengths were 254 nm for establishing characteristic spectra, 284 nm for determining phellodendrine, 236 nm for determining loganin, 280 nm for determining fangchinoline and tetrandrine, 277 nm for determining forsythin, 265 nm for determining berberine, and 430 nm for determining curcumin; the column temperature was 28°C; the flow rate was 0.8 mL min -1 ; Sample volume: 10 μL; Theoretical plate numbers of phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin peaks were calculated to be no less than 5000; B. Preparation of reference solution Accurately weigh appropriate amount of phellodendronine, loganin, fangchinoline and curcumin reference substances, accurately weigh, add methanol to make a solution containing 100 μg of each per 1 ml, shake well, and obtain; Accurately weigh appropriate amounts of tetrandrine, forsythin, berberine, and reference substance, accurately weigh, and add methanol to make a solution containing 50 μg of each per 1 ml, shake well, and obtain; C. Preparation of test solution Take the contents of Fengtongan capsules, weigh 0.5g, accurately weigh, put in a stoppered conical flask, add 20mL of 90% methanol, stopper, weigh, ultrasonically treat for 30 minutes, ultrasonic power 500W, ultrasonic frequency 40Hz, take out, cool, weigh again, make up the lost weight with 90% methanol solution, shake well, filter, and take the filtrate to obtain; D. Determination method: Accurately pipette 10μl of reference solution and test solution respectively, inject into liquid chromatograph, determine, and calculate the contents of the 7 components respectively.

2. A method for simultaneously determining 7 index components in Fengtong'an Capsules and constructing characteristic spectra according to claim 1, characterized in that: The simultaneous determination refers to the use of the same elution condition to switch different wavelengths to simultaneously determine the contents of phellodendronine, loganin, fangchinoline, tetrandrine, forsythiaside, berberine, and curcumin in Fengtong'an Capsules and construct a characteristic spectrum.

3. Application of the method for simultaneously determining seven index components in Fengtong'an Capsules and constructing a characteristic spectrum according to claim 1 or 2 in constructing a characteristic spectrum of Fengtong'an Capsules.

4. Application of the method for simultaneously determining 7 index components in Fengtong'an Capsules and constructing characteristic graphs according to claim 1 or 2 in the content determination of Fengtong'an Capsules.

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