HPLC fingerprint spectrum construction method of Tibetan medicine apium graveolens

Abstract

The invention relates to an HPLC (High Performance Liquid Chromatography) characteristic chromatogram construction method of Tibetan medicine apium graveolens, and belongs to the technical field of detection and analysis. According to the method, a test solution is obtained through a simple and rapid pretreatment method, a high performance liquid chromatograph and a gradient elution mode are adopted, a characteristic spectrum is established, 14 characteristic peaks exist in total, 6 index components of chlorogenic acid, rutin, daidzein, naringin, luteolin and cumin are identified, the relative retention time RSD% of the characteristic peaks is within 3%, and the relative retention time RSD% of the characteristic peaks is within 3%. The wave crest separation of each component is good. According to the method disclosed by the invention, a fingerprint spectrum establishment mode is adopted, so that the medicinal material quality of the apium graveolens can be more effectively ensured, and the blank in the aspect of apium graveolens characteristic spectrum research in the existing standard is filled up.

Description

Technical Field

[0001] The invention relates to a method for constructing an HPLC fingerprint spectrum of a Tibetan medicine celery, and belongs to the technical field of detection and analysis. Background Art

[0002] Cuminum frutescens, also known as cumin, is slightly spicy, flat, light, rough, dull, dry, clears lung heat, calms "Hei Yi", helps digestion, and stimulates appetite. It is used for "Bacon's disease", lung heat cough, sputum and hemoptysis, insomnia, loss of appetite, and indigestion.

[0003] At present, Celery is included in the "Drug Standards of the Ministry of Health of the People's Republic of China" (Volume 1 of Tibetan Medicine) WS3-BC-0072-95. There is only a microscopic identification item under it, and it is difficult to control its quality. There are almost no reports on its content and characteristic spectrum in the literature.

[0004] In order to ensure the quality of Celery and improve the quality standards of the medicinal materials, this paper intends to conduct HPLC fingerprint research on Celery and identify its characteristic components. Summary of the invention

[0005] The invention provides an HPLC fingerprint spectrum of a Tibetan medicine Celery, which is used to solve the deficiency of special spectrum in the existing standards.

[0006] The present invention is achieved through the following technical solutions:

[0007] HPLC fingerprint of a Tibetan medicine, Apium graveolens L., includes 14 characteristic peaks (common peaks), and 6 marker components are identified. The relative retention time RSD% of the characteristic peaks is within 3%. The relative retention times of the characteristic peaks are: Peak 1: 8.902 - 8.960 min, RSD = 0.26%; Peak 2 (chlorogenic acid): 21.700 - 21.793 min, RSD = 0.16%; Peak 3: 31.490 - 31.555 min, RSD = 0.09%; Peak 4 (rutin): 34.943 - 35.018 min, RSD = 0.09%; Peak 5 (glycitin): 36.942 - 37.038 min, RSD = 0.09%; Peak 6: 40.322 - 40.430 min, RSD = 0.10%; Peak 7 (naringin): 43.132 - 43.245 min, RSD = 0.10%; Peak 8: 44.988 - 45.108 min, RSD = 0.11%; Peak 9: 51.402 - 51.540 min, RSD = 0.11%; Peak 10: 52.793 - 52.928 min, RSD = 0.10%; Peak 11 (luteolin): 59.632 - 59.775 min, RSD = 0.10%; Peak 12: 71.190 - 71.328 min, RSD = 0.08%; Peak 13 (cumin aldehyde): 101.057 - 101.125 min, RSD = 0.03%; Peak 14: 104.272 - 104.348 min, RSD = 0.03%.

[0008] To improve the detection efficiency, reduce the detection cost and environmental pollution, for the first time, a simple and rapid pretreatment method is used to obtain the test solution. An Agilent 1260 Infinity II high performance liquid chromatograph (Agilent Technologies Co., Ltd.) is used, and the chromatographic column is Kromasil C18 (250 * 4.6 mm, 5 μm). A (0.08% phosphoric acid) - B (acetonitrile) is used as the mobile phase for gradient elution, the flow rate is 1.0 ml / min, the injection volume is 10 μL, the column temperature is 30 °C, and the detection wavelength is 257 nm.

[0009] Preparation of reference solution

[0010] Accurately weigh appropriate amounts of luteolin, cumin aldehyde, chlorogenic acid, rutin, glycitin, and naringin reference substances, accurately weigh them, and dissolve them in methanol to prepare solutions containing 20 μg each per 1 ml. Shake well to obtain the reference solution.

[0011] Preparation of test solution

[0012] Take 1.0 g of the powder of Apium graveolens L., weigh it precisely, place it in a stoppered conical flask, add 25 mL of an organic solvent, stopper it tightly, weigh it, perform ultrasonic treatment (500 W, frequency 40 Hz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with the same organic solution, shake well, filter, and take the subsequent filtrate to obtain the solution.

[0013] Determination method: Precisely pipette 10 μL each of the reference substance solution and the test solution, inject them into the liquid chromatograph, and perform the determination.

[0014] For the HPLC fingerprint of a Tibetan medicine, Apium graveolens L., as described above, the powder of Apium graveolens L. is passed through a No. 3 sieve and reserved for use.

[0015] For the HPLC fingerprint of a Tibetan medicine, Apium graveolens L., as described above, the organic solvent is methanol.

[0016] For the HPLC fingerprint of a Tibetan medicine, Apium graveolens L., as described above, the specific operation of gradient elution is as follows: 0 - 20 min, 3 - 12% B; 20 - 30 min, 12 - 18% B; 30 - 65 min, 18 - 28% B; 65 - 90 min, 28 - 43% B; 90 - 110 min, 19% B; 110 - 120 min, 49 - 28% B; 120 - 130 min, 28 - 12% B; 130 - 140 min, 12 - 3% B; 140 - 145 min, 3% B.

[0017] Based on the extraction principle of "like dissolves like" and the difference in the dosage forms of substances, gradient elution is adopted, with the polarity increasing from small to large, and 257 nm as the detection wavelength. This not only makes the size and area of the component peaks suitable, but also significantly reduces the interfering peaks at this detection wavelength, presenting a stable baseline and good separation of the peaks in the liquid chromatogram, laying a foundation for the simultaneous determination of multiple components.

[0018] The innovation points and beneficial effects of the present invention are as follows:

[0019] (1) Gradient elution is adopted, with 0.08% phosphoric acid water as mobile phase A and acetonitrile as mobile phase B. Under the same elution conditions, several effective components of traditional Chinese medicines with completely different properties and structures achieve good peak separation and a stable baseline. The method has no extraction, no concentration, no evaporation, is simple, fast, accurate, reproducible, easy to popularize and master, effectively improves the detection efficiency, reduces the detection cost, and reduces environmental pollution.

[0020] (2) It is learned from the literature that at present, Ocimum americanum L. is included in the "Drug Standards of the Ministry of Health of the People's Republic of China" (Tibetan Medicine, Volume 1) WS3-BC-0072-95. There is only a microscopic identification item under it, making it difficult to control its quality. There are almost no reports on its content and characteristic chromatograms in the literature. The established characteristic chromatogram has a total of 14 common peaks. The relative retention time RSD% of the said characteristic peaks is within 3%. Six characteristic index components, namely luteolin, cumin aldehyde, chlorogenic acid, rutin, glycinin, and naringin, have also been identified. The detection efficiency has been greatly improved, and the instrument cost has been significantly reduced. Moreover, more importantly, it is also possible to perform multi-index one-test multi-evaluation, achieving twice the result with half the effort. Its novelty, innovation, resource conservation, improvement of detection efficiency and practical value of detection indicators are obvious to all.

[0021] (3) The advent of the method of the present invention can better reflect the intrinsic quality of Ocimum americanum L. and control the safety and effectiveness of clinical medication. It not only provides a method for the establishment of the fingerprint chromatogram and multi-index content determination of the Tibetan medicinal material Ocimum americanum L., but also provides a reference basis and research idea for the simultaneous determination of this Tibetan medicine in other compound preparations, playing a leading and exemplary role.

[0022] (4) The key technology of the present invention is to utilize the characteristic that acetonitrile in the mobile phase has the property of retaining the peak times of various chemical components in advance, and the phosphoric acid solution can make acidic components exist in the molecular form, eliminating the performance of ionic tailing. Through infinite gradient ratio studies between acetonitrile and phosphoric acid at multiple concentrations, an elution condition is explored that can make a variety of chemical components with large differences in polarity appear in the same mobile phase, and the peak retention time is appropriate, the baseline is stable, and the reproducibility is good. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art.

[0024] Figure 1 It is the HPLC control fingerprint chromatogram of Ocimum americanum L. generated by the "Similarity Evaluation Software System for Traditional Chinese Medicine Chromatographic Fingerprints" of the Pharmacopoeia Commission of the People's Republic of China using the median method in the experimental verification of the present invention.

[0025] Figure 2 It is the superimposed chromatogram of 15 batches of Ocimum americanum L. samples in the experimental verification of the present invention.

[0026] Figure 3 It is the schematic diagram of the calculation result of the precision peak area in the verification test of the present invention.

[0027] Figure 4 It is the schematic diagram of the calculation result of the relative retention time of stability in the verification test of the present invention.

[0028] Figure 5 It is a schematic diagram of the calculation result of the relative retention time in the reproducibility of the verification test of the present invention.

[0029] Figure 6 It is a schematic diagram of the calculation result of the relative retention time at different flow rates in the verification test of the present invention.

[0030] Figure 7 It is a schematic diagram of the calculation result of the relative retention time at different temperatures in the verification test of the present invention.

[0031] Figure 8 It is a schematic diagram of the relative retention time result of the fingerprint spectrum of Ocimum basilicum L. in the verification test of the present invention.

[0032] Figure 9 It is a schematic diagram of the calculation result of the similarity of the fingerprint spectra of 15 batches of Ocimum basilicum L. in the verification test of the present invention. Specific embodiments

[0033] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] The HPLC fingerprint of the Tibetan medicine Apium graveolens L. var. dulce DC. includes 14 characteristic peaks (common peaks), and 6 index components are identified. The relative retention time RSD% of the characteristic peaks is within 3%. The relative retention times of the characteristic peaks are as follows: Peak 1: 8.902 - 8.960 min, RSD = 0.26%; Peak 2 (chlorogenic acid): 21.700 - 21.793 min, RSD = 0.16%; Peak 3: 31.490 - 31.555 min, RSD = 0.09%; Peak 4 (rutin): 34.943 - 35.018 min, RSD = 0.09%; Peak 5 (glycitin): 36.942 - 37.038 min, RSD = 0.09%; Peak 6: 40.322 - 40.430 min, RSD = 0.10%; Peak 7 (naringin): 43.132 - 43.245 min, RSD = 0.10%; Peak 8: 44.988 - 45.108 min, RSD = 0.11%; Peak 9: 51.402 - 51.540 min, RSD = 0.11%; Peak 10: 52.793 - 52.928 min, RSD = 0.10%; Peak 11 (luteolin): 59.632 - 59.775 min, RSD = 0.10%; Peak 12: 71.190 - 71.328 min, RSD = 0.08%; Peak 13 (cuminaldehyde): 101.057 - 101.125 min, RSD = 0.03%; Peak 14: 104.272 - 104.348 min, RSD = 0.03%.

[0035] An Agilent 1260 Infinity II high-performance liquid chromatograph (Agilent Technologies Co., Ltd.) is used. The chromatographic detection conditions are as follows: The chromatographic column is Kromasil C18 (250 * 4.6 mm, 5 μm). A (0.08% phosphoric acid) - B (acetonitrile) is used as the mobile phase for gradient elution. The flow rate is 1.0 ml / min, the injection volume is 10 μL, the column temperature is 30 °C, and the detection wavelength is 257 nm.

[0036] Specifically, the preparation steps of the test solution in this example are as follows: Take 1.0 g of Apium graveolens L. var. dulce DC. powder, accurately weigh it, place it in a stoppered conical flask, add 25 mL of organic solvent, tightly stopper it, weigh it, ultrasonically treat it (500 W, frequency 40 Hz) for 30 minutes, take it out, let it cool, weigh it again, make up the lost weight with the same organic solution, shake well, filter, and take the subsequent filtrate to obtain the test solution.

[0037] The preparation steps of the reference solution in this example are as follows: Accurately weigh appropriate amounts of luteolin, cuminaldehyde, chlorogenic acid, rutin, glycitin, and naringin reference substances, accurately weigh them, and separately make solutions containing 20 μg per 1 ml with methanol, shake well to obtain the reference solutions.

[0038] The celery powder described in this embodiment is passed through a No. 3 sieve and weighed for later use.

[0039] The organic solvent described in this embodiment is methanol.

[0040] The specific operation of the gradient elution described in this embodiment is: 0-20min 3-12% B, 20-30min 12-18% B, 30-65min 18-28% B, 65-90min 28-43% B, 90-110min 19B%, 110-120min49-28% B, 120-130min28-12B%, 130-140min 12-3% B, 140-145min 3% B.

[0041] This embodiment uses the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" of the National Pharmacopoeia Committee to generate a HPLC reference fingerprint of celery consisting of 14 characteristic peaks.

[0042] Verification test

[0043] 1. Instruments and test drugs

[0044] Instruments: High performance liquid chromatograph: Thermo Fisher Vanquish; Chromatographic column: Kromasil C18 (250*4.6 mm, 5 μm); CPA225D electronic balance: Sartorius Scientific Instruments (Beijing) Co., Ltd.; PMK224ZH electronic balance: Ohaus Instruments (Changzhou) Co., Ltd.; KQ5200DV ultrasonic cleaner: Kunshan Ultrasonic Instruments Co., Ltd.

[0045] Reagents: Acetonitrile was a chromatographic-grade reagent from Merck Chemical Technology (Shanghai) Co., Ltd.; methanol was from Concord Chemical Reagent Co., Ltd.; water was purified water produced by a pure water machine from Shanghai Hetai Instrument Co., Ltd.

[0046] 2. Preparation of test solution: Take 1.0 g of celery powder, weigh it accurately, put it in a stoppered conical flask, add 25 mL of organic solvent, stopper it, weigh it, and treat it with ultrasound (500 W, frequency 40 Hz) for 30 minutes. Take it out, cool it, weigh it again, make up the lost weight with the same organic solution, shake it well, filter it, and take the filtrate to obtain the solution.

[0047] 3. Preparation of reference solution: Accurately weigh appropriate amount of luteolin, cuminaldehyde, chlorogenic acid, rutin, glycitin, and naringin reference substances, accurately weigh, and add methanol to make a solution containing 20 μg of each per 1 ml, shake well, and obtain;

[0048] 4. Investigation of chromatographic conditions

[0049] Investigation of chromatographic columns:

[0050] The present invention tested and compared three chromatographic columns, namely Kromasil C18 (250 * 4.6 mm, 5 μm), Acclaim TM 120 C18 (250 mm * 4.6, 5 μm), and Zafex Suppersex AQ-C18 (250 * 4.6 mm, 5 μm). The results showed that the Kromasil C18 (250 * 4.6 mm, 5 μm) chromatographic column had good separation effect and moderate retention time. Considering the separation degree, the number of chromatographic peaks, the peak shape, and the stability of the baseline comprehensively, Kromasil C18 (250 * 4.6 mm, 5 μm) was finally selected.

[0051] Confirmation of the elution program:

[0052] Using the Kromasil C18 (250 * 4.6 mm, 5 μm) chromatographic column, gradient elution was carried out with A (0.08% phosphoric acid)-B (acetonitrile) as the mobile phase, the flow rate was 1.0 ml / min, the injection volume was 10 μL, the column temperature was 30 °C, and the detection wavelength was 257 nm. The present invention screened more than four elution programs (temporarily listed four).

[0053] Elution program 1: 0 - 15 min 8 - 15% B, 15 - 30 min 15 - 25% B, 30 - 45 min 25 - 35% B, 45 - 60 min 35 - 54% B, 60 - 75 min 54 - 35B%, 75 - 90 min 35 - 18% B, 90 - 100 min 18 - 8B%, 100 - 1105 min 8B.

[0054] Elution program 2: 0 - 15 min 8 - 15% B, 15 - 30 min 15 - 25% B, 30 - 45 min 25 - 35% B, 45 - 70 min 35 - 52% B, 70 - 95 min 52 - 58B%, 95 - 100 min 58 - 35% B, 100 - 105 min 35 - 18% B, 105 - 110 min 18 - 8% B, 110 - 115 min 8%.

[0055] Elution program 3: 0 - 20 min 3 - 12% B, 20 - 30 min 12 - 20% B, 30 - 55 min 20 - 30% B, 55 - 80 min 30 - 45% B, 80 - 100 min 45 - 51B%, 100 - 105 min 51 - 12% B, 105 - 110 min 12 - 3% B, 110 - 115 min 3%.

[0056] Elution program 4: 3 - 12% B from 0 to 20 min, 12 - 18% B from 20 to 30 min, 18 - 28% B from 30 to 65 min, 28 - 43% B from 65 to 90 min, 19% B from 90 to 110 min, 49 - 28% B from 110 to 120 min, 28 - 12% B from 120 to 130 min, 12 - 3% B from 130 to 140 min, 3% B from 140 to 145 min.

[0057] After screening through multiple gradient elution programs, the peak elution and separation effect of elution program 4 are the best, and elution program 4 is finally selected. In the following experiments, the calculation of 14 chromatographic peaks is carried out.

[0058] 5. Precision test

[0059] Take an appropriate amount of the cumin aldehyde reference solution of this product and inject it continuously for 6 times. The calculation results of the precision peak area are as Figure 3 shown.

[0060] 6. Stability test

[0061] Take an appropriate amount of the test solution of this product and inject it for determination at 0, 10, 20, 31, and 41 hours after preparation, and calculate its relative retention time as Figure 4 shown. From Figure 4 the data, it can be seen that the relative retention time has little difference within 41 hours, and the RSD of each chromatographic peak is less than 3.0%; the similarity is greater than 0.85, indicating that the test solution has good stability within 41 hours.

[0062] 7. Repeatability test

[0063] Accurately weigh 6 portions of the powder of the test product of the same batch of Ocimum vulgare L., prepare the test solution, and inject the sample. The calculation results of the repeatability relative retention time are as Figure 5 shown. From Figure 5 the data, it can be seen that after calculation, the relative retention time has little difference, and the RSD is less than 3.0%; the similarity is greater than 0.85, indicating good repeatability.

[0064] 8. Robustness test

[0065] Evaluate the robustness of this chromatographic condition for different injection volumes (5 μl, 10 μl, 15 μl), different flow rates (0.9 ml / min, 1.0 ml / min, 1.1 ml / min), and different column temperatures (28 °C, 30 °C, 32 °C), and calculate their similarity.

[0066] After similarity evaluation, the similarity of the fingerprint spectra for different injection volumes is all 1, so the relative retention time is not calculated. The calculation results of the relative retention time for different flow rates are as Figure 6 shown. FromFigure 6 As can be seen from the data, the relative retention time RSD of each chromatographic peak is less than 3.0% under different flow rate conditions, and the chromatogram similarity is greater than 0.85, indicating that the durability of this method under different flow rates is good; the calculation results of the relative retention time under different column temperatures are as Figure 7 shown, and from Figure 7 the data, it can be seen that the RSD values of the relative retention time of each peak are less than 3.0% under different column temperature conditions, and the similarity is greater than 0.85, indicating that the durability of this method under different column temperatures is good.

[0067] 9. Establishment of fingerprint

[0068] Fifteen batches of Ocimum basilicum samples were prepared according to the above method, and were prepared into test solution for testing. Two injections were made for each batch, and two chromatograms of Ocimum basilicum samples were obtained for each batch. The relative retention time of each chromatographic peak is as Figure 8 shown. Import into the software of "Similarity Evaluation System for Traditional Chinese Medicine Chromatographic Fingerprint" for data processing. Through peak matching, calculate the similarity (as Figure 9 shown) to generate an overlay chromatogram (as Figure 2 shown), and generate a reference chromatogram by the median method (as Figure 1 shown). According to the consistency of the relative retention time of each component in 15 batches of samples, 14 common peaks were selected as the characteristic peaks of the fingerprint. The similarity results of the 14 common peaks are all greater than 0.85, and the RSD values of the relative retention time are all less than 3.0%. The common pattern generated by these 15 batches of Ocimum basilicum is used as the reference chromatogram of the fingerprint.

[0069] Through the above verification, the high-performance liquid chromatography fingerprint of Ocimum basilicum established by the present invention is determined for multiple batches of samples, which proves that this method has good precision, repeatability, stability, etc., and can provide a deeper quality evaluation basis than the conventional quality standard.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing an HPLC fingerprint of Tibetan medicine Celery, characterized in that: The invention comprises 14 characteristic peaks, namely common peaks, and identifies 6 index components. The relative retention time RSD% of the characteristic peaks is within 3%. The relative retention time of the characteristic peaks is as follows: Peak 1: 8.902-8.960 min, RSD=0.26%; Peak 2: 21.700-21.793 min, RSD=0.16%; Peak 3: 31.490-31.555 min, RSD=0.09%; Peak 4: 34.943-35.018 min, RSD=0.09%; Peak 5: 36.942-37.038 min, RSD=0.09%; Peak 6: 40.322-40.430 min, RSD=0.10%; Peak 7: 4 3.132~43.245min, RSD=0.10%; Peak 8: 44.988~45.108min, RSD=0.11%; Peak 9: 51.402~51.540min, RSD=0.11%; Peak 10: 52.793~52.928min, RSD=0.10%; Peak 11: 59.632~59.775min, RSD=0.10%; Peak 12: 71.190~71.328min, RSD=0.08%; Peak 13: 101.057~101.125min, RSD=0.03%; Peak 14: 104.272~104.348min, RSD=0.03%; Peak 2 is chlorogenic acid, peak 4 is rutin, peak 5 is glycitin, peak 7 is naringin, peak 11 is luteolin, and peak 13 is cuminaldehyde; A. Chromatographic conditions and system suitability test Octadecylsilane bonded silica gel was used as filler; 0.08% phosphoric acid water was used as mobile phase A, and acetonitrile was used as mobile phase B; the detection wavelength was 257 nm; the column temperature was 30°C; the flow rate was 1.0 mL min -1 ; Injection volume 10 μL; 14 common peaks in total, 6 characteristic components including chlorogenic acid, rutin, glycitin, naringin, luteolin and cuminaldehyde were identified; B. Preparation of reference solution Accurately weigh appropriate amount of luteolin, cuminaldehyde, chlorogenic acid, rutin, glycitin, and naringin reference substances, accurately weigh, and add methanol to make a solution containing 20 μg of each per 1 ml, shake well, and obtain; C. Preparation of test solution: Take celery powder, pass it through a No. 3 sieve, accurately weigh 1.0 g, place it in a stoppered conical flask, add 25 mL of methanol solvent, stopper it, weigh it, treat it with ultrasound for 30 minutes, take it out, cool it, weigh it again, make up the lost weight with the same organic solution, shake it well, filter it, and take the filtrate to obtain the solution.

2. The HPLC fingerprint construction method of a Tibetan medicine Celery according to claim 1, characterized in that: Ultrasonic power 500W, ultrasonic frequency 40Hz.

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