A method for simultaneous determination of seven index components in Fengtongan capsules and construction of characteristic maps and its application

Abstract

The present invention relates to a method for simultaneously determining seven index components in Fengtong'an capsules and constructing a characteristic spectrum and its application, belonging to the field of detection and analysis technology. The present invention adopts a high performance liquid chromatograph and a gradient elution method. By switching different wavelengths under the same elution conditions, the contents of phellodendronine (284nm), loganin (236nm), fangchinoline, tetrandrine (280nm), forsythin (277nm), berberine (265nm), and curcumin (430nm) in a prescription can be simultaneously determined and a characteristic spectrum (254nm) can be constructed. The measured components are well separated, achieving the practical purpose of one measurement and multiple evaluations, high efficiency, low consumption and easy popularization. According to the polarity differences of each substance, a gradient elution program is adopted to achieve separation and accurate detection. To date, no report on the simultaneous determination of these seven components in Fengtong'an capsules has been found. This method fills the gap in the quality control of multiple medicinal materials in prescriptions and the research on fingerprints.

Description

Technical Field

[0001] The present invention relates to a method and application for simultaneously determining seven index components in Fengtong'an capsules and constructing a characteristic spectrum. Specifically, using a high-performance liquid chromatograph with gradient elution, switching between different wavelengths under the same elution conditions, the contents of phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin in Fengtong'an capsules can be simultaneously determined and a characteristic spectrum constructed. This method belongs to the field of detection and analysis technology. Background Art

[0002] Twenty-five Ingredients Pearl Pills, with National Medical Approval No. Z19983029, are indicated for clearing heat and dampness, promoting blood circulation and unblocking the meridians. It is used for arthritis caused by damp-heat blocking the meridians, with symptoms including red, swollen, hot, painful joints, and muscle aches; and for rheumatoid arthritis with the above symptoms.

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

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

[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 filtrates, concentrate to an appropriate amount, add the remaining talcum powder, mix, dry, crush, sieve, mix well, put into capsules, and make 1000 capsules.

[0006] Currently, there is little literature on Fengtongan, and the publication dates are long. One article published in the past five years measured aristolochic acid content using ultra-high performance liquid chromatography-tandem mass spectrometry, and the remaining 20-plus articles all date from before 2014. Content determination focused on single-component assays for loganin, curcumin, forsythin, tetrandrine, fangchinoline, and berberine hydrochloride, with no characteristic spectra or multiple evaluations across multiple medicinal materials. The current standard for Fengtongan is the 2020 edition of the Chinese Pharmacopoeia, under which the assay items are tetrandrine and fangchinoline.

[0007] This prescription contains twelve medicinal ingredients, and single content determination alone makes it difficult to control the intrinsic quality of the preparation. To ensure the high-quality therapeutic effect of Fengtong'an Capsules and strictly control the quality of the medicinal ingredients, we conducted a simultaneous quantitative analysis and constructed a characteristic profile for phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin in the capsules.

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

[0009]

[0010]

[0011] Based on the above structural formula, a gradient elution procedure can separate and accurately detect these components based on their polarity differences. To date, no reports have been found on the simultaneous determination of these seven components (phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin) in Fengtongan. Summary of the Invention

[0012] Based on the above background, the present invention, for the first time, uses a simple and rapid pretreatment method to obtain a test solution in order to improve detection efficiency, reduce detection costs, and minimize environmental pollution. High performance liquid chromatography (diode array detector) is used with A (0.1% phosphoric acid)-B (acetonitrile) as the mobile phase for gradient elution. The elution program is as follows: 0-33 min 3-8% B, 33-90 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 same elution condition is achieved by switching wavelengths to simultaneously determine seven index components and establish a characteristic spectrum. The detection wavelengths were 254 nm (for establishing characteristic spectra), phellodendrine (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; and the flow rate was 0.8 mL min. -1 The injection volume was 10 μL. The calculated theoretical plate counts for phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin were all no less than 5000. Excellent peak separation achieved a single test for multiple evaluations, achieving high efficiency, low cost, and easy popularization. The simultaneous determination of seven index components in a precious medicinal material is a first reported method, demonstrating the novelty, creativity, and practicality of this low-cost, dual-index assay.

[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 forsythin 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 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 of phellodendrine was 95.09% with an RSD of 1.71% (see Table 8); the average recovery of loganin was 96.08% with an RSD of 1.62% (see Table 9); the average recovery of fangchinoline was 97.81% with an RSD of 0.73% (see Table 10); the average recovery of tetrandrine was 97.71% with an RSD of 1.39% (see Table 11); the average recovery of forsythin was 96.99% with an RSD of 1.31% (see Table 12); the average recovery of berberine was 96.69% with an RSD of 0.65% (see Table 13); and the average recovery of curcumin was 96.42% with an RSD of 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 ), chromatogram of reference substance (see Figures 15-20 ), sample chromatogram (see Figures 21-26 ), specificity (see Figures 27-31 ), 3 batches of sample determination (see Table 18), ultrasonic time investigation (see Table 19), extraction solvent investigation (Table 20), the experimental method data are in line with 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 the fingerprint of Fengtongan capsules (254nm) was established and the durability and repeatability of the method were investigated for better quality control (see Figure 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 was performed with the following procedure: 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 3% B, 190-195 min 3% B. The detection wavelengths were 254 nm (for establishing characteristic spectra), phellodendrine (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; and the flow rate was 0.8 mL min. -1 The injection volume was 10 μL. The theoretical plate number for the peaks of phellodendrine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin was no less than 5000.

[0016] B. Preparation of Reference Solution: Accurately weigh appropriate amounts of phellodendrine, loganin, fangchinoline, and curcumin reference substances, add methanol to each solution to make a solution containing 100 μg of each per 1 ml, and shake well.

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

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

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

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

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

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

[0023] The principles of the present invention are 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, taking advantage of the maximum absorption of each substance (see Figures 8 to 14 ) as the detection wavelength, not only does it optimize the size and area of ​​component peaks, but it also significantly reduces interfering peaks at this detection wavelength, resulting in a stable chromatographic baseline and excellent peak separation, paving the way for simultaneous multi-component determination. Furthermore, quantitative determination can be performed based on the good linear relationship between the peak areas of the injected components within a certain range.

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

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

[0027] (2) According to the literature review, there is currently very little information on Fengtongan, and the publication date is very old. In the past five years, there is one article that used ultra-high performance liquid chromatography-tandem mass spectrometry to determine the content of aristolochic acid, and the remaining 20 or so articles were all published before 2014. In terms of content determination, single-component determination was performed on loganin, curcumin, forsythin, tetrandrine, fangchinoline, and berberine hydrochloride. There were no characteristic spectra and multiple evaluations of multiple medicinal materials. The current standard for Fengtongan is the 2020 edition of the Chinese Pharmacopoeia, and the content determination items under it are tetrandrine and fangchinoline.

[0028] This method for the simultaneous determination of these seven components (phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin) is reported for the first time. Furthermore, the characteristic spectrum contains 40 peaks. In addition to the components determined above, chlorogenic acid and cinnamaldehyde were also identified as characteristic components of the characteristic spectrum. This significantly improves detection efficiency and reduces instrument costs. Furthermore, the key point is the upgrade from 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 Fengtongan 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 it has the premise of the peak retention time of various chemical components. When combined with phosphoric acid solution, it can make the acidic components exist as molecules and eliminate the tailing of ions. Through the study of the gradient ratio between acetonitrile and phosphoric acid at 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] Figure 9 UV absorption spectrum of loganin;

[0040] Figure 10 Ultraviolet absorption spectrum of fangchinoline;

[0041] Figure 11 Ultraviolet absorption spectrum of tetrandrine;

[0042] Figure 12 Ultraviolet absorption spectrum of forsythiaside;

[0043] Figure 13 UV absorption spectrum of berberine;

[0044] Figure 14 UV absorption spectrum of curcumin;

[0045] Figure 15 HPLC chromatogram of phellodendrine;

[0046] Figure 16 HPLC chromatogram of loganin;

[0047] Figure 17 HPLC chromatograms of fangchinoline and tetrandrine;

[0048] Figure 18 HPLC chromatogram of forsythiaside;

[0049] Figure 19 HPLC chromatogram of berberine;

[0050] Figure 20 HPLC chromatogram of curcumin;

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

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

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

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

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

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

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

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

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

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

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

[0062] Figure 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 vertical axis is the absorption intensity (mAU); the horizontal axis is the absorption wavelength (nm);

[0065] Figures 15-32 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 forsythin, 6 is berberine, and 7 is curcumin;

[0067] Figure 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 phellodendrine, 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 the filler; 0.1% phosphoric acid water was used as the mobile phase A, and acetonitrile was used as the mobile phase B; gradient elution was performed with the following procedure: 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 3% B, 190-195 min 3% B. The detection wavelengths were 254 nm (for establishing characteristic spectra), phellodendrine (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; and the flow rate was 0.8 mL min. -1 The injection volume was 10 μL. The theoretical plate number for the peaks of phellodendrine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin was no less than 5000.

[0070] B. Preparation of Reference Solution: Accurately weigh appropriate amounts of phellodendrine, loganin, fangchinoline, and curcumin reference substances, add methanol to each solution to make a solution containing 100 μg of each per 1 ml, and shake well.

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

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

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

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

[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 Forsythin 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 of phellodendron alkali in samples

[0089]

[0090] Table 9 Loganin recovery test results in samples

[0091]

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

[0093]

[0094] Table 11 Tetrandrine recovery test results in samples

[0095]

[0096]

[0097] Table 12 Test results of recovery rate 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 seven index components in Fengtongan Capsules and constructing a characteristic spectrum, characterized by: A. Chromatographic Conditions and System Suitability Test: Octadecylsilane bonded silica gel was used as the filler; 0.1% phosphoric acid aqueous solution was used as mobile phase A, and acetonitrile was used as mobile phase B. Detection wavelengths were 254 nm for establishing characteristic spectra, 284 nm for determination of phellodendronine, 236 nm for determination of loganin, 280 nm for determination of fangchinoline and tetrandrine, 277 nm for determination of forsythin, 265 nm for determination of berberine, and 430 nm for determination of curcumin. Theoretical plate numbers calculated for the phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin peaks were not less than 5000. Gradient elution with the following program: 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 3% B, 190-195 min 3% B; column temperature 28°C; flow rate 0.8 mL min -1 ;Injection volume 10µL; B. Preparation of Reference Solution: Accurately weigh appropriate amounts of phellodendrine, loganin, fangchinoline, and curcumin reference substances, add methanol to each solution to make a solution containing 100 μg of each per 1 ml, and shake well. Accurately weigh appropriate amounts of tetrandrine, forsythin, berberine, and reference substance, add methanol to each to make a solution containing 50 μg of each per ml, shake well, and obtain; C. Preparation of Test Solution: Weigh 0.5 g of the contents of Fengtong'an Capsules accurately, place in a stoppered conical flask, add 20 mL of 90% methanol, stopper tightly, weigh the sample, and sonicate for 30 minutes at a power of 500 W and a frequency of 40 Hz. Remove the sample, cool, and weigh the sample again. Make up the lost weight with 90% methanol solution, shake well, filter, and collect the filtrate. D. Determination Method: Accurately pipette 10 μl of each reference solution and test solution, inject into liquid chromatography, and determine and calculate the contents of the seven components respectively; The simultaneous determination refers to the use of the same elution conditions to switch different wavelengths to simultaneously determine the contents of phellodendronine, loganin, fangchinoline, tetrandrine, forsythin, berberine, and curcumin in Fengtongan Capsules and construct a characteristic spectrum.

2. Application of the method for simultaneously determining seven index components in Fengtongan Capsules and constructing a characteristic spectrum according to claim 1 in constructing a characteristic spectrum of Fengtongan Capsules.

3. Application of the method for simultaneously determining 7 index components in Fengtongan Capsules and constructing characteristic maps according to claim 1 in the content determination of Fengtongan Capsules.

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