WO2024087945A1 - 一种夏桑菊颗粒定性和定量检测方法 - Google Patents

一种夏桑菊颗粒定性和定量检测方法 Download PDF

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WO2024087945A1
WO2024087945A1 PCT/CN2023/119685 CN2023119685W WO2024087945A1 WO 2024087945 A1 WO2024087945 A1 WO 2024087945A1 CN 2023119685 W CN2023119685 W CN 2023119685W WO 2024087945 A1 WO2024087945 A1 WO 2024087945A1
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acid
xiasangju
isochlorogenic
qualitative
solution
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French (fr)
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孙维广
黄志云
杨琪
王彬彬
陈肖家
赵永华
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广州白云山星群(药业)股份有限公司
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G01N30/94—Development
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of quality detection of traditional Chinese medicines, and in particular to a qualitative and quantitative detection method for Xiasangju granules.
  • Xiasangju Granules are a traditional Chinese medicine preparation composed of three herbs, Prunella vulgaris, mulberry leaves, and wild chrysanthemum, in a ratio of 500:175:80 (w/w/w). It is included in the Pharmacopoeia of the People's Republic of China (2020 Edition) and is also often used as a cold tea beverage. According to records, Xiasangju Granules have the effects of clearing the liver and improving eyesight, dispersing wind and heat, removing dampness and arthritis, and relieving sores and toxins. It is used for symptoms such as wind-heat colds, red eyes and headaches, dizziness and tinnitus, sore throat, and furuncles and swellings. Modern pharmacological studies have shown that it also has antioxidant, anti-inflammatory, antiviral, anti-cancer, immunomodulatory activity, and cardiovascular protection.
  • the Chinese Pharmacopoeia In terms of qualitative identification, in addition to reference medicinal materials, the Chinese Pharmacopoeia also uses rosmarinic acid and monanthus glycosides as indicator components to identify Prunella vulgaris and wild chrysanthemum, but the identification of mulberry leaves only uses reference medicinal materials and lacks specific indicator components. In addition, the pharmacopoeia uses three development systems to identify Prunella vulgaris, mulberry leaves, and wild chrysanthemums, respectively, and the identification method is relatively complicated.
  • the patent number CN 103018391 B involves "A quality control method for Xiasangju granules", which simplifies the identification conditions and selects reference medicinal materials and rosmarinic acid as reference substances to simultaneously detect the three medicinal materials on a thin layer plate, but the method lacks specific indicator components for mulberry leaves and wild chrysanthemums, and chloroform is used in the developing agent, which is highly toxic.
  • the one-test-multiple-evaluation method is a multi-index quality control method first proposed by Wang Zhimin et al. in 2006. This method uses the intrinsic functional relationship and proportional relationship of the effective ingredients of traditional Chinese medicine, and uses a relatively easy-to-obtain and inexpensive reference substance as an internal reference to achieve simultaneous determination of multiple components.
  • the one-test-multiple-evaluation method has the advantages of low detection cost and high analysis efficiency. It has been applied to the quality standards of Chinese herbal medicines and traditional Chinese medicine preparations such as Danshen, Epimedium, Ginkgo Leaf Capsules, and Ketaling Tablets in the Chinese Pharmacopoeia. However, there has been no report on the simultaneous determination of the content of multiple components in Xiasangju Granules using the one-test-multiple-evaluation method.
  • the present invention is committed to simplifying the qualitative identification experiment, optimizing the identification conditions, achieving simultaneous identification of the three medicinal materials in Xiasangju granules, and identifying their specific index components.
  • the present invention also adopts a double internal reference one-test multiple evaluation method to simultaneously determine multiple active ingredients in Xiasangju granules, thereby establishing a simple and comprehensive quality evaluation method.
  • the present invention aims to provide a qualitative and quantitative detection method for Xiasangju granules.
  • the qualitative detection method adopts thin layer chromatography to achieve simultaneous identification of three Chinese medicinal herbs, namely, herba prunellae, mulberry leaves and wild chrysanthemum, on the same thin layer plate, and identify their specific index components, namely, rosmarinic acid, scopoletin and hyoscyamine, thereby simplifying the identification steps and improving the identification efficiency.
  • the quantitative detection method adopts ultra-high performance liquid chromatography technology, combined with a double internal reference one-test multiple evaluation method, with chlorogenic acid and rosmarinic acid as internal references, and simultaneously determines the contents of 9 components in Xiasangju granules, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isorosmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A, rosmarinic acid, and montmorillonine, saving detection costs.
  • the method is accurate and reliable, with good repeatability, and has no significant difference in the results with the external standard method, providing a reference for quality control of Xiasangju granules products with varying quality on the market.
  • a qualitative and quantitative detection method for Xiasangju granules comprising qualitative identification by thin layer chromatography and quantitative determination by ultra-high performance liquid chromatography;
  • the thin layer chromatography qualitative determination of Xiasangju granules is to use rosmarinic acid, scopoletin, and hyoscyamine as reference substances, and to apply reference medicinal material solutions, reference substance solutions, and test solution of Prunella vulgaris, mulberry leaves, and wild chrysanthemum to the same silica gel thin layer plate in strips, and to obtain a thin layer chromatogram after development and color development to achieve qualitative identification of Xiasangju granules.
  • the specific steps are as follows:
  • Thin layer chromatography identification aspirate the test sample solution, reference medicinal material solution and reference substance solution and spot them on the same silica gel.
  • the GF 254 high-efficiency thin layer plate is in strips and developed with toluene-ethyl acetate-formic acid-water in a volume ratio of 3:15:2:1. After being taken out and dried, it is inspected under a UV lamp at 366nm. Then, the thin layer silica gel plate is heated at 100-105°C for 3-5 minutes, first sprayed with a 1% 2-aminoethyl biphenyl borate methanol solution, and then sprayed with a 5% polyethylene glycol ethanol solution. After drying, it is inspected under a UV lamp at 366nm.
  • the preparation method of the test solution described in the above step (1) is as follows: 10 g of Xiasangju granules are taken, ground into powder, 30 mL of anhydrous ethanol is added, ultrasonic treatment is performed for 30 minutes, filtered, the filtrate is spin-dried, and the residue is dissolved in 2 mL of anhydrous ethanol as the test solution;
  • the preparation method of the control medicinal material solution described in the above step (2) is as follows: 0.5 g of Prunella vulgaris control medicinal material, 1.0 g of mulberry leaf control medicinal material, and 1.0 g of wild chrysanthemum control medicinal material are taken, and 15 mL of anhydrous ethanol is added to each of them for ultrasonic treatment for 30 minutes, filtered, the filtrate is spin-dried, and the residue is dissolved in 2 mL of anhydrous ethanol as the control medicinal material solution;
  • the preparation method of the reference solution described in the above step (3) is: take rosmarinic acid, scopoletin, and hyoscyamine reference substances, add methanol to prepare a mixed solution containing 0.3-0.5 mg of each of the above reference substances per 1 mL, as the reference solution.
  • the ultra-high performance liquid chromatography content determination of Xiasangju granules uses chlorogenic acid as an internal reference to calculate the relative retention time and relative correction factor of neochlorogenic acid, cryptochlorogenic acid, and caffeic acid; uses rosmarinic acid as an internal reference to calculate the relative retention time and relative correction factor of isoresmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A, and montanaglycosides, and adopts a double internal reference one-measurement multiple evaluation method to simultaneously determine the contents of 9 components in Xiasangju granules, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isoresmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A, rosmarinic acid, and montanaglycosides, thereby realizing quantitative analysis of Xiasangju granules.
  • the preparation method of the reference solution in the above step (a) is: respectively taking neochlorogenic acid, chlorogenic acid, Accurately weigh appropriate amounts of cryptochlorogenic acid, caffeic acid, isorosmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A, rosmarinic acid, and monanthus glycosides, add methanol to dissolve, and obtain a reference solution;
  • the mass concentrations of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isorosmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A, rosmarinic acid and montmorillonine in the reference solution are 0.192 mg/mL, 0.192 mg/mL, 0.185 mg/mL, 0.131 mg/mL, 0.160 mg/mL, 0.162 mg/mL, 0.135 mg/mL, 0.262 mg/mL and 0.198 mg/mL, respectively.
  • the preparation method of the test solution described in the above step (b) is: take 1.25 g of Xiasangju granules and grind them into fine powder, add 25 mL of 50% ethanol solution, ultrasonically treat for 30 minutes, cool to room temperature, make up for weight loss, shake well, centrifuge at 3500 rpm for 10 minutes, take the supernatant, filter with a 0.22 ⁇ m filter membrane, and take the filtrate as the test solution.
  • the C18 reverse phase chromatography column described in the above step (c) is one of Waters Acquity UPLC HSS T3 (100mm ⁇ 2.1mm, 1.8 ⁇ m), Waters Acquity UPLC CORTECS T3 (100mm ⁇ 2.1mm, 1.6 ⁇ m), Waters Acquity UPLC BEH C18 (100mm ⁇ 2.1mm, 1.7 ⁇ m) or Thermo scientific Hypersil GOLD (100mm ⁇ 2.1mm, 1.9 ⁇ m); preferably Waters Acquity UPLC HSS T3 (100mm ⁇ 2.1mm, 1.8 ⁇ m).
  • the relative retention times of the chlorogenic acid in the above step (d) to neochlorogenic acid, cryptochlorogenic acid, and caffeic acid are 0.561-0.635, 1.120-1.343, and 1.301-1.355, respectively; preferably 0.561, 1.153, and 1.309;
  • the relative retention times of rosmarinic acid to isorosmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A and montmorillonine are 0.779-0.866, 0.815-0.888, 0.855-0.906 and 1.522-1.699, respectively; preferably 0.787, 0.815, 0.855 and 1.547.
  • the relative correction factors of chlorogenic acid for neochlorogenic acid, cryptochlorogenic acid, and caffeic acid are 0.788-0.884, 0.894-0.927, and 0.462-0.517, respectively, preferably 0.804, 0.904, and 0.499;
  • the relative correction factors of rosmarinic acid for isosmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A, and montmorillonine are 1.269-1.403, 1.276-1.444, 0.659-0.747, and 1.075-1.272, respectively, and are preferably 1.352, 1.321, 0.671, and 1.152.
  • the present invention has the following beneficial effects:
  • the present invention optimizes the development conditions and identifies the specific indicator components of Prunella vulgaris, mulberry leaves, and wild chrysanthemum, namely rosmarinic acid, scopoletin, and hyoscyamine, thereby achieving simultaneous identification of the three Chinese medicinal materials in Xiasangju Granules on a thin layer plate, simplifying the identification steps, thereby improving the identification efficiency and promoting the overall quality control of Xiasangju Granules.
  • the present invention adopts ultra-high performance liquid chromatography combined with double internal reference one-test multiple evaluation method to simultaneously determine the contents of 9 active ingredients in Xiasangju granules, including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isorosmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A, rosmarinic acid, and monanthranilic acid.
  • the method is accurate and reliable, with good repeatability, and there is no significant difference in the results with the external standard method, which provides a comprehensive method for the quality evaluation of Xiasangju granules. And a comprehensive measurement method.
  • FIG1 is a thin layer chromatogram of mixed reference substances (MS1 and MS2) and Xiasangju granule sample (S16) using different development systems;
  • A toluene-ethyl acetate-formic acid-acetic acid-water (1:15:1:1:2, v/v/v/v/v); B: toluene-ethyl acetate-formic acid-water (3:15:2:1, v/v/v/v).
  • 1 rutin; 2: scopoletin; 3: montanol; 4: chlorogenic acid; 5: hyperoside; 6: luteolin; 7: isoquercetin; 8: astragaloside; 9: isochlorogenic acid C; 10: isochlorogenic acid B; 11: isochlorogenic acid A; 12: rosmarinic acid; 13: scopoletin; 14: caffeic acid.
  • FIG2 is a thin layer chromatogram of mixed reference substances (MS1 and MS2) and Xiasangju granule sample (S16) using different color development methods;
  • 1 rutin; 2: scopoletin; 3: montanol; 4: chlorogenic acid; 5: hyperoside; 6: luteolin; 7: isoquercetin; 8: astragaloside; 9: isochlorogenic acid C; 10: isochlorogenic acid B; 11: isochlorogenic acid A; 12: rosmarinic acid; 13: scopoletin; 14: caffeic acid.
  • FIG3 is a thin layer chromatogram of the mixed reference substances (MS1 and MS2), reference medicinal materials (RD1-RD3) and Xiasangju granule samples (S1-S16).
  • RD1 Prunella vulgaris control medicinal material
  • RD2 mulberry leaf control medicinal material
  • RD3 Chrysanthemum wild chrysanthemum control medicinal material
  • 1 rutin
  • 2 scopoletin
  • 3 monanthus glycoside
  • 4 chlorogenic acid
  • 5 hyperoside
  • 6 oleoside
  • 7 isoquercetin
  • 8 astragaloside
  • 9 isochlorogenic acid C
  • 10 isochlorogenic acid B
  • 11 isochlorogenic acid A
  • 12 rosmarinic acid
  • 13 scopoletin
  • 14 caffeic acid.
  • FIG4 is a thin layer chromatogram of the reference substance (R1-R3), single medicinal material (D1-D3) and Xiasangju granule sample (S9).
  • D1 mulberry leaf medicinal material
  • D2 wild chrysanthemum medicinal material
  • D3 selfheal medicinal material
  • 1 rutin
  • 2 montana glycoside
  • 3 chlorogenic acid
  • 4 isoquercetin
  • 5 astragaloside
  • 6 luteolin
  • 7 isochlorogenic acid A
  • 8 isochlorogenic acid B
  • 9 isochlorogenic acid C
  • 10 rosmarinic acid.
  • FIG5 is an ultra-high performance liquid chromatogram of a mixed reference solution (A) and a Xiasangju granule test solution (B).
  • 1 neochlorogenic acid
  • 2 chlorogenic acid
  • 3 cryptochlorogenic acid
  • 4 caffeic acid
  • 5 isorosmarinic acid glycoside
  • 6 isochlorogenic acid B
  • 7 isochlorogenic acid A
  • 8 rosmarinic acid
  • 9 montmorillonine.
  • Figure 6 is a high performance liquid chromatogram of the Xiasangju granule test solution.
  • Neochlorogenic acid 2. Chlorogenic acid; 3. Cryptochlorogenic acid; 4. Caffeic acid; 5. Isorosmarinic acid glycoside; 6. Isorosmarinic acid B; 7. Isorosmarinic acid A; 8. Rosmarinic acid; 9. Menhua glycoside.
  • Example 1 Thin layer chromatography identification of Xiasangju granules
  • Thin layer chromatography automatic sampling system AUTOMATIC TLC SAMPLER 4 (CAMAG, Switzerland), thin layer chromatography automatic development system ADC2 (CAMAG, Switzerland), thin layer chromatography digital imager TLC VISUALIZER 2 (CAMAG, Switzerland), thin layer chromatography heating plate TLC PLATE HEATER III (CAMAG, Switzerland); Mettler Toledo XS105 electronic analytical balance (one hundred thousandth, Mettler-Toledo International Trading (Shanghai) Co., Ltd.), Branson 8510 ultrasonic cleaning machine (Branson Ultrasonic Technology Co., Ltd., USA).
  • High-efficiency silica gel thin layer plate GF 254 (20 ⁇ 10 cm) was purchased from Merck KGaA, Germany. All experimental reagents were of analytical grade. 2-Aminoethyldiphenylborate and polyethylene glycol 400 were purchased from Düsseldorf, Germany, acetic acid (Glacial, 100%) was purchased from Merck KGaA, Germany (Darmstadt, Germany), toluene (GR, ACS 99.5%) was purchased from American International Laboratories (South San Francisco, CA, USA), ferric chloride and aluminum chloride crystals were purchased from Tianjin Damao Chemical Reagent Factory, China, and other reagents such as formic acid, ethyl acetate, methanol, and ethanol were purchased from Guangdong Xilong Science Co., Ltd. Water was prepared by Millipore Milli Q-Plus ultrapure water system (Millipore, Billerica, MA, USA).
  • the Chinese medicine reference substances were all HPLC grade, and the purity was above 98%.
  • Chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, caffeic acid, rosmarinic acid, mongoside, rutin, hyperoside, scopoletin, and scopoletin were purchased from Chengdu Mansite Biotechnology Co., Ltd., China.
  • Astragaloside and luteolin were purchased from Chengdu Pufeide Biotechnology Co., Ltd., and isoquercitrin was purchased from Chengdu Purifa Biotechnology Co., Ltd.
  • the reference medicinal materials of Prunella vulgaris (batch number 120993-202007), mulberry leaf reference medicinal materials (batch number 121123-201806), and wild chrysanthemum reference medicinal materials (batch number 120995-201707) were all purchased from China Food and Drug Administration (Beijing).
  • the plate is in strip shape on a 254 high-efficiency thin layer plate, and is developed with toluene-ethyl acetate-formic acid-water in a volume ratio of 3:15:2:1 as a developing agent. After being taken out and dried, it is examined under an ultraviolet lamp at 366 nm.
  • a main fluorescent band of the same color is shown at a position corresponding to the chromatogram of the reference medicinal material; at a position corresponding to the chromatogram of the rosmarinic acid and scopolamine reference substances, the same blue fluorescent band is shown; then the thin layer silica gel plate is heated at 100-105° C. for 3-5 minutes, first sprayed with a 1% 2-aminoethyl biphenyl borate methanol solution, and then sprayed with a 5% polyethylene glycol ethanol solution (NP/PEG), and after drying, it is examined under an ultraviolet lamp at 366 nm.
  • NP/PEG polyethylene glycol ethanol solution
  • a main fluorescent band of the same color is shown at a position corresponding to the chromatogram of the reference medicinal material; at a position corresponding to the chromatogram of the hyoscyamine reference substance, the same yellow fluorescent band is shown; at a position corresponding to the chromatogram of the rosmarinic acid reference substance, the same cyan fluorescent band is shown.
  • Prunella vulgaris contains rutin, chlorogenic acid, hyperoside, isoquercitrin, luteolin, rosmarinic acid, and caffeic acid
  • mulberry leaves contain rutin, scopoletin, chlorogenic acid, isoquercitrin, astragalus glycoside, and scopoletin
  • wild chrysanthemum contains rutin, mongoside, chlorogenic acid, hyperoside, isoquercitrin, luteolin, astragalus glycoside, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, and caffeic acid.
  • scopolamine, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, rosmarinic acid, scopolamine, and caffeic acid showed blue fluorescence, and other reference substances did not show color.
  • the final choice was to first directly inspect under ultraviolet light at 366nm to identify rosmarinic acid and scopolamine, and then spray with NP/PEG color developer, inspect under ultraviolet light at 366nm, identify hyoscyamine, and confirm rosmarinic acid at the same time.
  • the optimized TLC identification method was used to perform TLC identification analysis on 16 batches of Xiasangju granules collected (Figure 3). The results showed that clear rosmarinic acid and scopolamine bands were observed in all samples, but no hyoscyamine band was observed in one batch of samples (S14). From the TLC chart, the quality of Xiasangju granules circulating on the market varies significantly.
  • the detection was carried out according to the thin layer chromatography identification method provided in the "Quality Control Method of Xiasangju Granules" disclosed in Chinese patent application CN103018391B:
  • Color developer 1% aluminum chloride ethanol solution, heated at 105°C for 5 minutes.
  • this method can clearly identify herb summer cypress, but the identification effect of mulberry leaves and wild chrysanthemum is not good.
  • the Rf values of all reference substances are between 0.1-0.9, the separation effect is good, and rosmarinic acid, scopolamine and scopolamine are identified as the specific index components of herb summer cypress, mulberry leaves and wild chrysanthemum, respectively, and the three constituent medicinal materials of Xiasangju granules can be identified at the same time.
  • Example 2 Double internal reference, one test, multiple evaluations content determination of Xiasangju granules
  • Thermo Fisher Ultimate TM 3000 ultra-high performance liquid chromatography system (Thermo Fisher Scientific Inc., Waltham, MA, USA); Waters ACQUITY UPLC I-Class ultra-high performance liquid chromatograph (Waters, Milford, MA, USA); Mettler Toledo XS105 electronic analytical balance (1:100,000, Mettler Toledo International Trading (Shanghai) Co., Ltd.); Branson 8510 ultrasonic cleaner (Branson Ultrasonic Technology Co., Ltd., USA); Thermo Fisher Heraeus Multifuge X3R centrifuge (Thermo Fisher Scientific Inc., Waltham, MA, USA).
  • Acetic acid (Glacial, 100%) was purchased from Merck, Darmstadt, Germany, and analytical grade methanol and ethanol were purchased from Tianjin Damao Chemical Reagent Factory, China. Water was prepared by Millipore Milli Q-Plus Ultrapure Water System (Millipore, Billerica, MA, USA).
  • the Chinese medicine reference substances were all HPLC grade, and the purity was above 98%.
  • Cryptochlorogenic acid, chlorogenic acid, neochlorogenic acid, isorosmarinic acid glycosides, caffeic acid, isochlorogenic acid A, isochlorogenic acid B, rosmarinic acid, and mongoside were purchased from Chengdu Mansite Biotechnology Co., Ltd., China.
  • Xiasangju granules from different manufacturers were purchased from pharmacies in various places, and the detailed information is shown in Table 1.
  • Chromatographic column Waters ACQUITY UPLC HSS T3 column (2.1mm ⁇ 100mm,1.8 ⁇ m); acetonitrile (A) and 1% acetic acid aqueous solution (B) were used as mobile phases, and gradient elution was performed according to the following elution program: 0-4min, 8%-10% A; 4-6min, 10%-19.5% A; 6-13min, 19.5% A; 13-18min, 19.5%-35% A; 18-20min, 35%-90% A; flow rate, 0.4mL/min; column temperature, 30°C; detection wavelength, 325nm; injection volume, 2 ⁇ L.
  • neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isorosmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A, rosmarinic acid, and mongoside accurately weigh them, add methanol to dissolve them, and prepare the mixture containing neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isorosmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A, rosmarinic acid, and mongoside with mass concentrations of 0.192 mg/mL, 0.192 mg/mL, 0.185 mg/mL, 0.131 mg/mL, 0.160 mg/mL, 0.162 mg/mL, Mixed reference solutions of 0.135 mg/mL, 0.262 mg/mL and 0.198 mg/mL.
  • the mixed reference solution of three concentrations within the linear range was accurately pipetted and injected continuously for 6 times in one day and twice a day for 3 days.
  • the peak area was recorded and the RSD value of the peak area was used to evaluate the intra-day and inter-day precision.
  • the results showed that the instrument had good precision (Table 3).
  • components in Chinese medicine preparations that are relatively easy to obtain, have high content, and have relatively clear pharmacological effects should be selected as internal reference substances.
  • Rosmarinic acid and chlorogenic acid are phenolic acid components with high content in Xiasangju Granules. They have good stability, and the reference substances are cheap and easy to obtain. Therefore, this experiment selected rosmarinic acid and chlorogenic acid as internal reference substances, and simultaneously determined the contents of the 9 components in Xiasangju Granules.
  • the mixed reference solution of different concentrations in "2.4.1” was injected according to the chromatographic conditions of "2.1” and the peak area was recorded.
  • Chlorogenic acid and rosmarinic acid were used as internal references and the relative correction factors of each compound were calculated according to formula (1).
  • Results The relative correction factors of chlorogenic acid for neochlorogenic acid, cryptochlorogenic acid and caffeic acid were 0.804, 0.904 and 0.499, respectively; the relative correction factors of rosmarinic acid for isorosmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A and monanthin were 1.352, 1.321, 0.671 and 1.152, respectively.
  • f si is the relative correction factor of the internal reference (s) for a certain component to be measured (i);
  • Ai is the peak area of the reference substance i of a component to be tested
  • Cs is the concentration of internal reference substance s; Ci is the concentration of reference substance i of a component to be tested.
  • This experiment investigated the effects of different column temperatures (25, 30, 35°C), different flow rates (0.35, 0.40, 0.45 mL/min) and different detection wavelengths (320, 325, 330 nm) on the relative correction factor.
  • the results showed that the RSD values of the relative correction factors of each component to be measured were all less than 4%.
  • the results showed that there was no significant difference in the relative correction factors of each component measured at different flow rates, different column temperatures and different detection wavelengths (Table 7).
  • the premise for the application of the one-measurement-multiple-evaluation method to the simultaneous quantification of multiple index components is the accurate positioning of the chromatographic peaks of each component to be measured.
  • the methods widely used in the literature are relative retention time positioning method or retention time difference positioning method.
  • the collected 16 batches of Xiasangju granules were used to prepare the test solution according to the method in item "2.3". Each batch of samples was paralleled for 3 times and injected according to the chromatographic conditions in item "2.1". The retention time and peak area of each component to be tested were recorded. The content of each component was calculated by the external standard method (ESM) and the established double internal reference one measurement multiple evaluation method (QAMS). The results of the two methods were compared, and the relative standard deviation RSD value was used to evaluate the difference in the content determination results of the two methods. The RSD values of the two methods were less than 5%. The results are shown in Tables 9-1 and 9-2.
  • the ultra-high performance liquid chromatograms of the mixed reference solution and the representative sample (S8) are shown in Figure 5.
  • the results show that the double internal reference one measurement multiple evaluation method is feasible for the simultaneous determination of 9 components in Xiasangju granules. The method is accurate and reliable, and can save detection costs.
  • Chromatographic column Agilent ZORBAX SB-C18 (4.6mm ⁇ 150mm, 5 ⁇ m); acetonitrile (A) and 1.0% acetic acid aqueous solution (B) were used as mobile phases, and gradient elution was performed according to the following elution program: 0-50min, 8%-33% A; 50-51min, 33%-8% A; 51-60min, 8% A; flow rate: 0.9mL/min; column temperature: 35°C; detection wavelength: 320nm; injection volume: 10 ⁇ L.
  • the present invention optimizes the development conditions, identifies the specific indicator components of Prunella vulgaris, mulberry leaves, and wild chrysanthemum, namely rosmarinic acid, scopoletin, and hyoscyamine, and realizes the simultaneous identification of the three Chinese medicinal materials in Xiasangju granules on a thin layer plate, simplifies the identification steps, thereby improving the identification efficiency and promoting the overall quality control of Xiasangju granules; in terms of quantitative analysis, in order to save detection costs and improve analysis efficiency, the present invention adopts ultra-high performance liquid chromatography combined with a double internal reference one-test multiple-evaluation method to simultaneously determine the contents of 9 active ingredients in Xiasangju granules, namely neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, caffeic acid, isorosmarinic acid glycosides, isochlorogenic acid B, isochlorogenic acid A, ros

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Abstract

本发明涉及一种夏桑菊颗粒定性和定量检测方法，涉及中药质量检测技术领域。所述的定性检测方法采用薄层色谱法，在同一薄层板上实现了夏枯草、桑叶和野菊花三味中药的同时检测，并识别其专属性指标成分，简化了鉴别步骤，提高了鉴别效率；定量检测方法采用超高效液相色谱技术，结合双内参一测多评法，以绿原酸和迷迭香酸为内参物，同时测定夏桑菊颗粒中新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷9种成分的含量，节省了检测成本，具有简便高效、准确稳定的特点，应用前景广阔。

Description

一种夏桑菊颗粒定性和定量检测方法
本发明要求于2022年10月27日提交中国专利局、申请号为202211326382.8、发明名称为“一种夏桑菊颗粒定性和定量检测方法”的中国专利申请的优先权，其全部内容通过引用结合在申请中。
技术领域
本发明涉及中药质量检测技术领域，具体是一种夏桑菊颗粒定性和定量检测方法。
背景技术
夏桑菊颗粒是由夏枯草、桑叶、野菊花三味药材按500:175:80(w/w/w)的比例配伍组成的一种中药制剂，收录于中华人民共和国药典(2020版)一部，也常作为凉茶饮料。据记载，夏桑菊颗粒具有清肝明目、疏风散热、除湿痹、解疮毒的功效，用于风热感冒、目赤头痛、头晕耳鸣、咽喉肿痛、疔疮肿毒等症状。现代药理研究表明，它还具有抗氧化、抗炎、抗病毒、抗癌、免疫调节活性和心血管保护等作用。
近年来，有诸多夏桑菊颗粒质量控制的相关研究。在定性鉴别方面，中国药典除对照药材外，还采用迷迭香酸和蒙花苷作为指标成分鉴别夏枯草和野菊花，但桑叶的鉴别仅采用对照药材，缺乏专属性指标成分。另外，药典中采用3个展开系统分别对夏枯草、桑叶、野菊花进行鉴别，鉴别方法相对复杂。专利号为CN 103018391 B的专利涉及“一种夏桑菊颗粒的质量控制方法”简化了鉴别条件，选择对照药材和迷迭香酸作为对照品在一块薄层板上同时检测三味药材，但该法缺乏桑叶和野菊花的专属性指标成分，且展开剂中使用了三氯甲烷，毒性较大。
在含量测定方面，中国药典中仅测定迷迭香酸的含量。然而考虑到中药制剂成分的复杂性，单一成分的定量分析无法全面、准确、有效地控制该制剂的质量。林丽美等人采用《RP-HPLC法同时测定夏桑菊颗粒中绿原酸、异迷迭香酸苷、迷迭香酸和蒙花苷》(中成药，2013年11期)；孙鹏等人在文献《夏桑菊凉茶中多成分含量测定方法的研究》(食品工业，2016年4期)中采用高效液相色谱法对夏桑菊凉茶中的绿原酸、迷迭香酸、木犀草苷、芦丁、槲皮素和蒙花苷6种活性成分进行了定量分析；桑岚在文献《夏桑菊颗粒不同干燥方式对指标成分含量的影响》(中医学报，2019年34期)中通过测定绿原酸，迷迭香酸和蒙花苷的含量探究不同干燥方式对制剂的质量影响。多成分含量测定虽然可以较好地反映制剂的质量，但外标法需要对照品种类多，对照品消耗量大，检测经济成本较高，限制了其在实际工作中 的应用。
一测多评法是由王智民等人在2006年率先提出的多指标质量控制方法，该方法是利用中药有效成分内在的函数关系和比例关系，以一种相对易得、廉价的对照品为内参物，实现对多个成分的同时测定。一测多评法具有检测成本低，分析效率高的优点，目前已经应用于丹参、淫羊藿、银杏叶胶囊、咳特灵片等中药材和中药制剂的中国药典质量标准中。但是，尚未见用一测多评法同时测定夏桑菊颗粒中多种成分含量的报道。
因此，本发明致力于简化定性鉴别实验，优化鉴别条件，实现同时鉴别夏桑菊颗粒中的三味药材，并识别其专属性指标成分。同时为了节省检测成本，提高定量分析效率，本发明还采用双内参一测多评法同时测定夏桑菊颗粒中多种活性成分，从而建立一种简易且全面的质量评价方法。
发明内容
本发明目的在于提供一种夏桑菊颗粒定性和定量检测方法。
所述的定性检测方法采用薄层色谱法，在同一薄层板上实现了夏枯草、桑叶和野菊花三味中药的同时鉴别，并识别其专属性指标成分迷迭香酸、东莨菪苷和蒙花酸，简化了鉴别步骤，提高了鉴别效率。
所述的定量检测方法采用超高效液相色谱技术，结合双内参一测多评法，以绿原酸和迷迭香酸为内参物，同时测定夏桑菊颗粒中新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷9种成分的含量，节省了检测成本，方法准确可靠，重复性良好，且与外标法测定结果无显著性差异，为市面上质量参差不齐的夏桑菊颗粒产品的质量控制提供参考。
为实现上述目的，本发明是通过以下技术方案实现的：
一种夏桑菊颗粒定性和定量检测方法，所述得检测方法包括薄层色谱定性鉴别和超高效液相色谱定量测定；
所述夏桑菊颗粒薄层色谱定性测定是以迷迭香酸、东莨菪苷、蒙花苷为对照品，将夏枯草、桑叶、野菊花的对照药材溶液、对照品溶液、供试品溶液在同一硅胶薄层板上成条带状点样，经展开和显色后得到薄层色谱图，实现对夏桑菊颗粒的定性鉴别，具体步骤如下：
(1)供试品溶液的制备；
(2)对照药材溶液的制备；
(3)对照品溶液的制备；
(4)薄层色谱鉴别：吸取供试品溶液，对照药材溶液和对照品溶液，分别点于同一硅 胶GF254高效薄层板上成条带状，以体积比为3:15:2:1的甲苯-乙酸乙酯-甲酸-水为展开剂展开，取出晾干后，置紫外光灯366nm下检视；之后100-105℃加热薄层硅胶板3-5分钟，先喷以浓度为1％的2-氨基乙基联苯基硼酸酯甲醇溶液，再喷以5％的聚乙二醇乙醇溶液，晾干后，置于紫外光灯366nm下检视。
进一步地，上述步骤(1)中所述的供试品溶液的制备方法为：取夏桑菊颗粒10g，研细，加无水乙醇30mL，超声处理30分钟，滤过，滤液旋干，残渣溶解于2mL无水乙醇，作为供试品溶液；
进一步地，上述步骤(2)中所述的对照药材溶液的制备方法为：取夏枯草对照药材0.5g、桑叶对照药材1.0g、野菊花对照药材1.0g，分别加15mL无水乙醇超声处理30分钟，滤过，滤液旋干，残渣溶解于2mL无水乙醇，作为对照药材溶液；
进一步地，上述步骤(3)中所述的对照品溶液的制备方法为：取迷迭香酸、东莨菪苷、蒙花苷对照品，加甲醇制成每1mL含以上对照品各0.3-0.5mg的混合溶液，作为对照品溶液。
所述夏桑菊颗粒超高效液相色谱含量测定是以绿原酸内参物，计算新绿原酸、隐绿原酸、咖啡酸的相对保留时间和相对校正因子；以迷迭香酸为内参物，计算异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷的相对保留时间和相对校正因子，采用双内参一测多评法同时测定夏桑菊颗粒中新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷9种成分的含量，从而实现对夏桑菊颗粒的定量分析。
具体内容如下：
(a)对照品溶液的制备；
(b)供试品溶液的制备；
(c)色谱条件：采用C18反相色谱柱，其规格为2.1mm×100mm，粒径小于2μm；以乙腈(A)和1％乙酸水溶液(B)作为流动相，按如下洗脱程序进行梯度洗脱：0-4min，8％-10％A；4-6min，10％-19.5％A；6-13min，19.5％A；13-18min，19.5％-35％A；18-20min，35％-90％A；流速为0.4mL/min，柱温为30℃，检测波长为325nm，进样量为2μL；
(d)相对校正因子和相对保留时间的计算：以绿原酸内参物，计算新绿原酸、隐绿原酸、咖啡酸的相对保留时间和相对校正因子；以迷迭香酸为内参物，计算异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷的相对保留时间和相对校正因子；
(e)测定：测定夏桑菊颗粒中绿原酸和迷迭香酸的含量，依据相对校正因子计算新绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷7种成分的含量。
进一步地，上述步骤(a)中所述的对照品溶液的制备方法为：分别取新绿原酸、绿原酸、 隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷适量，精密称定，加甲醇溶解，得到对照品溶液；
再进一步地，所述的对照品溶液中新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷的质量浓度分别为0.192mg/mL、0.192mg/mL、0.185mg/mL、0.131mg/mL、0.160mg/mL、0.162mg/mL、0.135mg/mL、0.262mg/mL和0.198mg/mL。
进一步地，上述步骤(b)中所述的供试品溶液的制备方法为：取夏桑菊颗粒研细粉末1.25g，加50％乙醇溶液25mL，超声处理30分钟，冷却至室温，补足失重，摇匀，3500rpm离心10分钟，取上清液，用0.22μm滤膜过滤，取续滤液作为供试品溶液。
进一步地，上述步骤(c)中所述的C18反相色谱柱为Waters Acquity UPLC HSS T3(100mm×2.1mm,1.8μm)、Waters Acquity UPLC CORTECS T3(100mm×2.1mm,1.6μm)、Waters Acquity UPLC BEH C18(100mm×2.1mm,1.7μm)或Thermo scientific Hypersil GOLD(100mm×2.1mm,1.9μm)中的一种；优选为Waters Acquity UPLC HSS T3(100mm×2.1mm，1.8μm)。
进一步地，上述步骤(d)中所述的绿原酸对于新绿原酸、隐绿原酸、咖啡酸的相对保留时间分别为0.561-0.635、1.120-1.343、1.301-1.355；优选为0.561、1.153、1.309；
所述的迷迭香酸对于异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷的相对保留时间分别为0.779-0.866、0.815-0.888、0.855-0.906、1.522-1.699；优选为0.787、0.815、0.855、1.547。
进一步地，所述绿原酸对于新绿原酸、隐绿原酸、咖啡酸的相对校正因子分别为0.788-0.884、0.894-0.927、0.462-0.517，优选为0.804、0.904、0.499；
所述的迷迭香酸对于异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷的相对校正因子分别为1.269-1.403、1.276-1.444、0.659-0.747、1.075-1.272，优选为1.352、1.321、0.671、1.152。
与现有技术相比本发明的有益效果在于：
(1)在定性方面，本发明优化了展开条件，识别了夏枯草、桑叶、野菊花的专属性指标成分迷迭香酸、东莨菪苷和蒙花苷，在一块薄层板上实现了夏桑菊颗粒中三味中药材的同时鉴别，简化了鉴别步骤，从而提高了鉴别效率，促进了夏桑菊颗粒的整体质量控制。
(2)在定量方面，为节省检测成本和提高分析效率，本发明采用了超高效液相色谱法结合双内参一测多评法同时测定夏桑菊颗粒中新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷9种活性成分的含量，方法准确可靠，重复性良好，且与外标法测定结果无显著性差异，为夏桑菊颗粒的质量评价提供了一种全面 且综合的测定方法。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为混合对照品(MS1和MS2)和夏桑菊颗粒样品(S16)采用不同展开系统的薄层色谱图；
A：甲苯-乙酸乙酯-甲酸-乙酸-水(1:15:1:1:2,v/v/v/v/v)；B：甲苯-乙酸乙酯-甲酸-水(3:15:2:1,v/v/v/v)。
其中1：芦丁；2：东莨菪苷；3：蒙花苷；4：绿原酸；5：金丝桃苷；6：木犀草苷；7：异槲皮苷；8：紫云英苷；9：异绿原酸C；10：异绿原酸B；11：异绿原酸A；12：迷迭香酸；13：东莨菪素；14：咖啡酸。
图2为混合对照品(MS1和MS2)和夏桑菊颗粒样品(S16)采用不同显色方式的薄层色谱图；
A：直接在紫外光灯254nm下检视；B：直接在紫外光灯366nm下检视；C：喷以5％三氯化铁乙醇溶液，105℃加热3分钟后在白光下检视；D：喷以1％三氯化铝乙醇溶液，105℃加热5分钟后在紫外光灯366nm下检视；E：100-105℃加热薄层硅胶板3-5分钟，喷以浓度为1％的2-氨基乙基联苯基硼酸酯甲醇溶液，再喷以5％的聚乙二醇乙醇溶液，晾干后，在紫外灯366nm下检视。
其中1：芦丁；2：东莨菪苷；3：蒙花苷；4：绿原酸；5：金丝桃苷；6：木犀草苷；7：异槲皮苷；8：紫云英苷；9：异绿原酸C；10：异绿原酸B；11：异绿原酸A；12：迷迭香酸；13：东莨菪素；14：咖啡酸。
图3为混合对照品(MS1和MS2)、对照药材(RD1-RD3)和夏桑菊颗粒样品(S1-S16)的薄层色谱图。
其中，RD1：夏枯草对照药材；RD2：桑叶对照药材；RD3：野菊花对照药材；1：芦丁；2：东莨菪苷；3：蒙花苷；4：绿原酸；5：金丝桃苷；6：木犀草苷；7：异槲皮苷；8：紫云英苷；9：异绿原酸C；10：异绿原酸B；11：异绿原酸A；12：迷迭香酸；13：东莨菪素；14：咖啡酸。
图4为对照品(R1-R3)、单味药材(D1-D3)和夏桑菊颗粒样品(S9)的薄层色谱图，
其中，D1：桑叶药材；D2：野菊花药材；D3：夏枯草药材；1：芦丁；2：蒙花苷；3：绿原酸；4：异槲皮苷；5：紫云英苷；6：木犀草苷；7：异绿原酸A；8：异绿原酸B；9：异绿原酸C；10：迷迭香酸。
图5为混合对照品溶液(A)和夏桑菊颗粒供试品溶液(B)的超高效液相色谱图，
其中，1：新绿原酸；2：绿原酸；3：隐绿原酸；4：咖啡酸；5：异迷迭香酸苷；6：异绿原酸B；7：异绿原酸A；8：迷迭香酸；9：蒙花苷。
图6为夏桑菊颗粒供试品溶液的高效液相色谱图，
其中，1.新绿原酸；2.绿原酸；3.隐绿原酸；4.咖啡酸；5.异迷迭香酸苷；6.异绿原酸B；7.异绿原酸A；8.迷迭香酸；9.蒙花苷。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1：夏桑菊颗粒的薄层色谱鉴别
1.仪器与试药
1.1仪器
薄层色谱自动点样系统AUTOMATIC TLC SAMPLER 4(CAMAG,Switzerland)、薄层色谱自动展开系统ADC2(CAMAG,Switzerland)、薄层色谱数码成像仪TLC VISUALIZER 2(CAMAG,Switzerland)、薄层色谱加热板TLC PLATE HEATERⅢ(CAMAG,Switzerland)；梅特勒托利多XS105型电子分析天平(十万分之一，梅特勒-托利多国际贸易(上海)有限公司)，必能信8510超声波清洗机(美国Branson超声波科技有限公司)。
1.2材料和试剂
高效硅胶薄层板GF254(20×10cm)购自于德国默克股份有限公司。实验试剂均为分析纯。2-氨基乙基二苯硼酸盐和聚乙二醇400购自德国卡尔罗斯公司(Karlsruhe,Germany)，醋酸(Glacial,100％)购自德国默克公司(Darmstadt,Germany)，甲苯(GR,ACS 99.5％)购自美国国际实验室(South San Francisco,CA,USA)，氯化铁、氯化铝晶体购于中国天津大茂化学试剂厂，其他试剂如甲酸、乙酸乙酯、甲醇和乙醇购于广东西陇科学股份有限公司。水为Millipore Milli Q-Plus超纯水系统(Millipore,Billerica,MA,USA)制备所得。
1.3对照品
中药对照品均为HPLC级，纯度均在98％以上。绿原酸、异绿原酸A、异绿原酸B、异绿原酸C、咖啡酸、迷迭香酸、蒙花苷、芦丁、金丝桃苷、东莨菪苷、东莨菪素购于中国成都曼思特生物科技有限公司。紫云英苷、木犀草苷购自成都普菲德生物技术有限公司，异槲皮苷购自成都普瑞法生物技术有限公司。夏枯草对照药材(批号120993-202007)、桑叶对照药材(批号121123-201806)和野菊花对照药材(批号120995-201707)均购自中国食品药品检定研究院(北京)。
1.4样品
不同厂家夏桑菊颗粒采购于各地药房，详细信息见表1。
表1夏桑菊颗粒样品信息
2.方法与结果
2.1供试品溶液的制备
取夏桑菊颗粒10g，研细，加无水乙醇30mL，超声处理30分钟，滤过，滤液旋干，残渣溶解于2mL无水乙醇，作为供试品溶液；
2.2对照药材溶液的制备
取夏枯草对照药材0.5g、桑叶对照药材1.0g、野菊花对照药材1.0g，分别加15mL无水乙醇超声处理30分钟，滤过，滤液旋干，残渣溶解于2mL无水乙醇，作为对照药材溶液；
2.3对照品溶液的制备
取芦丁、东莨菪苷、蒙花苷、绿原酸、金丝桃苷、异槲皮苷、木犀草苷、紫云英苷、异绿原酸A、异绿原酸B、异绿原酸C、迷迭香酸、东莨菪素和咖啡酸对照品，加甲醇制成每1mL含以上对照品各0.3-0.5mg的混合溶液，作为对照品溶液；
2.4薄层色谱鉴别
吸取供试品溶液，对照药材溶液和对照品溶液2-8μL，分别点于同一硅胶GF254高效薄层板上成条带状，以体积比为3:15:2:1的甲苯-乙酸乙酯-甲酸-水为展开剂，展开，取出晾干后，置紫外光灯366nm下检视，供试品色谱中，在与对照药材色谱相应的位置上，显相同颜色的荧光主条带；在与迷迭香酸、东莨菪苷对照品色谱相应的位置上，显相同的蓝色荧光条带；之后100-105℃加热薄层硅胶板3-5分钟，先喷以浓度为1％的2-氨基乙基联苯基硼酸酯甲醇溶液，再喷以5％的聚乙二醇乙醇溶液(NP/PEG)，晾干后，置于紫外光灯366nm下检视，供试品色谱中，在与对照药材色谱相应的位置上，显相同颜色的荧光主条带；在与蒙花苷对照品色谱相应的位置上，显相同的黄色荧光条带；在与迷迭香酸对照品色谱相应的位置上，显相同的青绿色荧光条带。
2.5对照品的选择
为选择合适的专属性指标成分，我们选择了14种对照品进行考察。根据文献报道以及与对照药材的比对，夏枯草中含有芦丁、绿原酸、金丝桃苷、异槲皮苷、木犀草苷、迷迭香酸、咖啡酸，桑叶中含有芦丁、东莨菪苷、绿原酸、异槲皮苷、紫云英苷、东莨菪素，野菊花中含有芦丁、蒙花苷、绿原酸、金丝桃苷、异槲皮苷、木犀草苷、紫云英苷、异绿原酸A、异绿原酸B、异绿原酸C、咖啡酸。综合考虑对照品的专属性、薄层色谱的分离情况(图1-3)、各成分在夏桑菊颗粒中的含量以及实际应用中的可行性和对照品的成本，最终选择迷迭香酸、东莨菪苷和蒙花苷分别为夏枯草、桑叶和野菊花的专属性指标成分。
2.6展开系统的考察
我们分别考察了甲苯-乙酸乙酯-甲酸-乙酸-水(1:15:1:1:2,v/v/v/v/v)和甲苯-乙酸乙酯-甲酸-水(3:15:2:1,v/v/v/v)，两种展开剂分离情况类似，但后者为四相体系，比前者少用一种试剂，故选择后者作为展开系统。在此条件下，东莨菪苷和蒙花苷；异槲皮苷和木犀草苷；异绿原酸B和异绿原酸C；东莨菪素和咖啡酸未能分离，为便于比对，14种对照品分为两组配制混合对照品溶液(图1)。
2.7显色方式的考察
我们还考察了直接在紫外光灯254nm和366nm下检视；喷以5％三氯化铁乙醇溶液，105℃加热3分钟后在白光下检视；喷以1％三氯化铝乙醇溶液，105℃加热5分钟后在紫外光灯 366nm下检视；100-105℃加热薄层硅胶板3-5分钟，喷以NP/PEG显色剂，在紫外灯366nm下检视几种检视方式，结果见图2。在254nm下，东莨菪素和东莨菪苷显蓝色荧光，其它对照品均为暗斑，无法根据颜色区分不同结构类型的化合物。在366nm下，东莨菪苷、绿原酸、异绿原酸A、异绿原酸B、异绿原酸C、迷迭香酸、东莨菪素、咖啡酸显蓝色荧光，其它对照品不显色。喷显色剂5％三氯化铁乙醇溶液后，除东莨菪素和东莨菪苷外，其它对照品均显棕褐色，但灵敏度较低，样品中只有蒙花苷、迷迭香酸和咖啡酸的条带较清晰，其它条带难以观察到。喷显色剂1％三氯化铝乙醇溶液和NP/PEG后均能使所有对照品显示不同颜色的荧光条带，但后者比前者颜色更丰富，且灵敏度更高，故最终选择了NP/PEG显色剂。由于东莨菪苷和蒙花苷未能分开，显色前在366nm下蒙花苷不显色而东莨菪苷显蓝色荧光，显色后蒙花苷显黄色荧光而东莨菪苷条带在样品中很微弱几乎被掩盖，可据此辨认这两个成分。因此，最终选择先在紫外光灯366nm下直接检视，辨认迷迭香酸和东莨菪苷，再喷以NP/PEG显色剂，在紫外光灯366nm下检视，辨认蒙花苷，同时确认迷迭香酸。
2.8样品测定
采用优化后的薄层色谱鉴别方法，对收集到的16批夏桑菊颗粒进行薄层色谱鉴别分析(图3)。结果显示，所有样品中均能观察到清晰的迷迭香酸和东莨菪苷条带，但有一批样品中观察不到蒙花苷条带(S14)。从薄层色谱图来看，市面上流通的夏桑菊颗粒质量差异明显。
对比例1
按照中国专利申请CN103018391B中公开的“一种夏桑菊颗粒的质量控制方法”中提供的薄层色谱鉴别方法进行检测：
展开剂：氯仿：乙酸乙酯：丙酮：甲酸(25：15：10：2)
显色剂：1％三氯化铝乙醇溶液，105℃加热5分钟。
检测结果见附图4。
根据图4的检测结果可以看出：采用现有的展开剂进行有效成分检测时，迷迭香酸的Rf值为0.41，位置合适，分离度佳。但其它对照品的Rf值均小于0.2，且蒙花苷、绿原酸和异槲皮苷无法分离，异绿原酸A、B、C也无法分离。与单味药材比对时，在夏桑菊颗粒的薄层图谱中虽可检测出夏枯草药材中主要条带，但与桑叶和野菊花药材相比无明显相同条带。因此，该方法可清晰地鉴别夏枯草，但对桑叶和野菊花的鉴别效果不佳。而在本申请的方法中，所有对照品的Rf值均在0.1-0.9之间，分离效果较好，且指认了迷迭香酸、东莨菪苷和蒙花苷分别为夏枯草、桑叶和野菊花的专属性指标成分，可同时鉴别夏桑菊颗粒的三味组成药材。
实施例2：夏桑菊颗粒的双内参一测多评含量测定
1.仪器与试药
1.1仪器
赛默飞UltimateTM 3000型超高效液相色谱系统(Thermo Fisher Scientific Inc.,Waltham,MA,USA)；沃特世ACQUITY UPLC I-Class超高效液相色谱仪(Waters,Milford,MA,USA)；梅特勒托利多XS105型电子分析天平(十万分之一，梅特勒-托利多国际贸易(上海)有限公司)；必能信8510超声波清洗机(美国Branson超声波科技有限公司)；赛默飞Heraeus Multifuge X3R型离心机(Thermo Fisher Scientific Inc.，Waltham，MA，USA)。
1.2材料和试剂
色谱柱：Waters Acquity UPLC HSS T3(100mm×2.1mm,1.8μm)、Waters Acquity UPLC CORTECS T3(100mm×2.1mm,1.6μm)、Waters Acquity UPLC BEH C18(100mm×2.1mm,1.7μm)和Thermo scientific Hypersil GOLD(100mm×2.1mm,1.9μm)。HPLC级乙腈和甲醇购自ACI Labscan有限公司(Bangkok,Thailand)。醋酸(Glacial,100％)购自德国默克公司(Darmstadt,Germany)，分析纯甲醇和乙醇购于中国天津大茂化学试剂厂。水为Millipore Milli Q-Plus超纯水系统(Millipore,Billerica,MA,USA)制备所得。
1.3对照品和样品
中药对照品均为HPLC级，纯度均在98％以上。隐绿原酸、绿原酸、新绿原酸、异迷迭香酸苷、咖啡酸、异绿原酸A、异绿原酸B、迷迭香酸和蒙花苷购于中国成都曼思特生物科技有限公司。不同厂家夏桑菊颗粒采购于各地药房，详细信息见表1。
2.方法与结果
2.1色谱条件
色谱柱：Waters ACQUITY UPLC HSS T3 column(2.1mm×100mm,1.8μm)；以乙腈(A)和1％乙酸水溶液(B)作为流动相，按如下洗脱程序进行梯度洗脱：0-4min，8％-10％A；4-6min，10％-19.5％A；6-13min，19.5％A；13-18min，19.5％-35％A；18-20min，35％-90％A；流速0.4mL/min；柱温30℃；检测波长为325nm；进样量2μL。
2.2对照品溶液制备
分别取新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷适量，精密称定，加甲醇溶解，制备成含新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷的质量浓度分别为0.192mg/mL、0.192mg/mL、0.185mg/mL、0.131mg/mL、0.160mg/mL、0.162mg/mL、 0.135mg/mL、0.262mg/mL和0.198mg/mL的混合对照品溶液。
2.3供试品溶液的制备
取夏桑菊颗粒研细粉末1.25g，精密称定，加入50％乙醇25mL，超声处理30分钟，冷却至室温，补足失重，摇匀，3500rpm离心10分钟，取上清液，用0.22μm滤膜过滤，取续滤液作为供试品溶液。
2.4方法学考察
2.4.1线性范围、检测限和定量限
精密吸取“2.2”项混合对照品溶液1.5mL、1.2mL、1.0mL、0.8mL、0.6mL、0.4mL、0.2mL、0.1mL分别置于2mL容量瓶中，用甲醇稀释至刻度，摇匀，按“2.1”项色谱条件进样测定，分别以各对照品质量浓度为横坐标(x)，峰面积为纵坐标(y)，绘制标准曲线，得到回归方程、线性范围和相关系数。将混合对照品溶液逐级稀释，以信噪比为3:1时为检测限，以信噪比为10:1时为定量限。新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷的线性回归方程、相关系数、线性范围、检测限和定
量限结果见表2。表2标准曲线、检测限和定量限
2.4.2精密度
精密吸取线性范围内的三个浓度的混合对照品溶液，在一天内连续进样6次，以及在3天每天进样2次，记录峰面积，用峰面积的RSD值来评价日内、日间精密度，结果表明仪器精密度良好(表3)。
表3日间和日内精密度结果
2.4.3重复性
精密称取夏桑菊颗粒(S8)研细粉末1.00g、1.25g、1.50g，按“2.3”项下方法制备供试品溶液，每个浓度水平平行3份，按“2.1”项色谱条件进样测定，记录峰面积，计算各峰峰面积的RSD值，结果表明方法的重复性良好(表4)。
2.4.4稳定性
精密称取夏桑菊颗粒(S8)研细粉末1.25g，按“2.3”项下方法制备供试品溶液，按“2.1”项色谱条件，分别在0、2、4、8、12、24、48h进样，记录峰面积，计算各峰峰面积的RSD值，结果表明供试品溶液在48h内稳定(表4)。
表4重复性和稳定性结果

注：+：低于定量限。
2.4.5加样回收率试验
精密称量夏桑菊颗粒(S8)研细粉末0.65g，分别按照各成分已知含量的50％、100％、150％加入各对照品适量，按“2.3”项下方法制备供试品溶液，每个浓度水平平行3份，按“2.1”项色谱条件进样测定，计算加样回收率，结果见表5，各成分的加样回收率在95.03％-105.71％内，表明方法的准确性良好。
表5加样回收率结果


2.5双内参一测多评法
参考一测多评法指南，应选择中药制剂中相对易得、含量较高、药理作用相对明确的成分作为内参物。根据实验经验以及文献检索，采用一测多评法进行多组分含量定量分析时，选择的内参物与待测成分的结构越接近，与待测成分的保留时间越接近，计算结果更为准确， 也更具有参考意义。考虑到夏桑菊颗粒中9种待测成分结构差异较大，只采用一个内参物无法准确评估各成分，故本实验采用两个内参物进行多成分含量测定，以保证一测多评结果的准确性。迷迭香酸、绿原酸作为夏桑菊颗粒中含量较高的酚酸类成分，稳定性较好，对照品价格便宜易得，因此本实验选择迷迭香酸和绿原酸作为内参物，同时测定夏桑菊颗粒中9种成分的含量。
2.5.1相对校正因子的确定
将“2.4.1”项下不同浓度系列混合对照品溶液按“2.1”项色谱条件进样，记录峰面积。以绿原酸和迷迭香酸为内参物，按公式(1)分别计算各化合物的相对校正因子。结果绿原酸对于新绿原酸、隐绿原酸、咖啡酸的相对校正因子分别为0.804、0.904、0.499；迷迭香酸对于异迷迭香酸苷、异绿原酸B、异绿原酸A以及蒙花苷的相对校正因子分别为1.352、1.321、0.671和1.152。
式中：fsi为内参物(s)对于某待测成分(i)的相对校正因子；
As为内参物对照品s峰面积；Ai为某待测成分对照品i峰面积；
Cs为内参物对照品s浓度；Ci为某待测成分对照品i浓度。
2.5.2相对校正因子的耐用性考察
2.5.2.1不同仪器和色谱柱对相对校正因子的影响
本实验分别考察了Waters Acquity UPLC HSS T3(100mm×2.1mm,1.8μm)、Waters Acquity UPLC CORTECS T3(100mm×2.1mm,1.6μm)、Waters Acquity UPLC BEH C18(100mm×2.1mm,1.7μm)和Thermo scientific Hypersil GOLD(100mm×2.1mm,1.9μm)4根UPLC色谱柱以及Thermo U3000型和Waters I-Class型超高效液相色谱仪对各成分的相对校正因子的影响，以绿原酸和迷迭香酸为内参物，分别计算各待测组分的相对校正因子，结果显示各待测成分的相对校正因子的RSD值均小于5％(表6)，不同仪器和不同色谱柱对相对校正因子的影响不大。
表6不同仪器和色谱柱对相对校正因子的影响

注：C1:新绿原酸；C2:绿原酸；C3:隐绿原酸；C4:咖啡酸；C5:异迷迭香酸苷；C6:异绿原酸B；C7:异绿原酸A；
C8:迷迭香酸；C9:蒙花苷。
2.5.2.2不同流速、柱温及检测波长对相对校正因子的影响
本实验考察了不同柱温(25、30、35℃)、不同流速(0.35、0.40、0.45mL/min)以及不同检测波长(320、325、330nm)对相对校正因子的影响，结果显示各待测成分的相对校正因子RSD值均小于4％，结果表明在不同流速、不同柱温及不同检测波长下测得的各组分相对校正因子无显著性差异(表7)。
表7不同检测波长、流速及柱温对相对校正因子的影响

注：C1:新绿原酸；C2:绿原酸；C3:隐绿原酸；C4:咖啡酸；C5:异迷迭香酸苷；C6:异绿原酸B；C7:异绿原酸A；
C8:迷迭香酸；C9:蒙花苷。
2.5.3色谱峰定位
一测多评法应用于多指标成分同时定量可行的前提是对各待测成分色谱峰的准确定位。 目前文献中较多采用的方法为相对保留时间定位法或保留时间差值定位法。本实验在Thermo U3000型和Waters I-Class型超高效液相色谱仪上分别用Waters Acquity UPLC HSS T3(100mm×2.1mm,1.8μm)、Waters Acquity UPLC CORTECS T3(100mm×2.1mm,1.6μm)、Waters Acquity UPLC BEH C18(100mm×2.1mm,1.7μm)和Thermo scientific Hypersil GOLD(100mm×2.1mm,1.9μm)4根不同UPLC色谱柱考察了各待测组分色谱峰的相对保留时间的重现性；实验结果表明，7个待测成分的相对保留时间的RSD值小于5％，结果见表8。
表8不同液相仪器和色谱柱对色谱峰定位的影响

注：C1:新绿原酸；C2:绿原酸；C3:隐绿原酸；C4:咖啡酸；C5:异迷迭香酸苷；C6:异绿原酸B；C7:异绿原酸A；
C8:迷迭香酸；C9:蒙花苷。
2.6样品含量测定
将收集的16批夏桑菊颗粒按“2.3”项方法制备供试品溶液，每批样品平行3份，按“2.1”项下色谱条件进样，记录各待测成分保留时间及峰面积，分别用外标法(ESM)和所建立的双内参一测多评法(QAMS)计算各成分的含量，并将两种方法的测定结果进行比较，用相对标准偏差RSD值评价两种方法含量测定结果的差异，结果两种方法RSD值小于5％，结果详见表9-1、9-2，混合对照品溶液和代表性样品(S8)的超高效液相色谱图见图5。结果表明双内参一测多评法应用于同时测定夏桑菊颗粒中9种成分具有可行性，方法准确可靠，且能节省检测成本。
表9-1外标法和双内标一测多评法测定夏桑菊颗粒中4种成分含量结果(mg/g,n＝3)

注：ESM：外标法；QAMS：一测多评法；+：低于定量限；-：低于检测限
表9-2外标法和双内标一测多评法测定夏桑菊颗粒中5种成分含量结果(mg/g,n＝3)

注：ESM：外标法；QAMS：一测多评法；+：低于定量限；-：低于检测限
对比例2
按照中国药典2020版“夏桑菊颗粒”标准中【指纹图谱】项下提供的高效液相色谱法进行检测，与实施例2的区别在于：
2.1色谱条件
色谱柱：Agilent ZORBAX SB-C18(4.6mm×150mm,5μm)；以乙腈(A)和1.0％醋酸水溶液(B)作为流动相，按如下洗脱程序进行梯度洗脱：0-50min，8％-33％A；50-51min，33％-8％A；51-60min，8％A；流速：0.9mL/min；柱温：35℃；检测波长：320nm；进样量10μL。
检测结果见附图6。
根据图6可以看出：在此条件下，绿原酸(2)和隐绿原酸(3)未能完全分开，异绿原酸B(6)和异绿原酸A(7)的峰形不好。且采用高效液相色谱柱，分析时间较长，需要约40分钟。而在本申请的方法中，采用超高效液相色谱柱，仅需不到20分钟，且各对照品分离良好。
综上，在定性方面，本发明优化了展开条件，识别了夏枯草、桑叶、野菊花的专属性指标成分迷迭香酸、东莨菪苷和蒙花苷，在一块薄层板上实现了夏桑菊颗粒中三味中药材的同时鉴别，简化了鉴别步骤，从而提高了鉴别效率，促进了夏桑菊颗粒的整体质量控制；在定量方面，为节省检测成本和提高分析效率，本发明采用了超高效液相色谱法结合双内参一测多评法同时测定夏桑菊颗粒中新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷9种活性成分的含量，方法准确可靠，重复性良好，且与外标法测定结果无显著性差异，为夏桑菊颗粒的质量评价提供了一种全面且综合的测定方法。
以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (15)

  1. 一种夏桑菊颗粒定性和定量检测方法，其特征在于，所述检测方法包括薄层色谱定性鉴别和超高效液相色谱含量测定；
    所述的超高效液相色谱含量测定为采用双内参一测多评法同时测定夏桑菊颗粒中新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸和蒙花苷9种成分的含量。
  2. 根据权利要求1所述的夏桑菊颗粒定性和定量检测方法，其特征在于，所述的薄层色谱定性鉴别的具体步骤为：
    (1)供试品溶液的制备；
    (2)对照药材溶液的制备；
    (3)对照品溶液的制备；
    (4)薄层色谱鉴别：吸取供试品溶液，对照药材溶液和对照品溶液，分别点于同一硅胶薄层板上，展开剂展开，取出晾干后，置紫外光灯366nm下检视；之后100-105℃加热薄层硅胶板3-5分钟，先喷以浓度为1％的2-氨基乙基联苯基硼酸酯甲醇溶液，再喷以5％的聚乙二醇乙醇溶液，晾干后，置于紫外光灯366nm下检视。
  3. 根据权利要求2所述的夏桑菊颗粒定性和定量检测方法，其特征在于，所述的展开剂为甲苯、乙酸乙酯、甲酸、乙酸和水的混合液或甲苯、乙酸乙酯、甲酸和水的混合液；所述的甲苯、乙酸乙酯、甲酸、乙酸和水的体积比为1:15:1:1:2；所述的甲苯、乙酸乙酯、甲酸和水的体积比为3:15:2:1。
  4. 根据权利要求3所述的夏桑菊颗粒定性和定量检测方法，其特征在于，所述的展开剂为体积比为3:15:2:1的甲苯、乙酸乙酯、甲酸和水的混合液。
  5. 根据权利要求1所述的夏桑菊颗粒定性和定量检测方法，其特征在于，所述的超高效液相色谱含量测定的具体步骤如下：
    (a)对照品溶液的制备；
    (b)供试品溶液的制备；
    (c)色谱条件：采用C18反相色谱柱；以乙腈(A)和1％乙酸水溶液(B)作为流动相，按如下洗脱程序进行梯度洗脱：0-4min，8％-10％A；4-6min，10％-19.5％A；6-13min，19.5％A；13-18min，19.5％-35％A；18-20min，35％-90％A；流速为0.4mL/min，柱温为30℃，检测波长为325nm，进样量为2μL；
    (d)相对校正因子和相对保留时间的计算：以绿原酸内参物，计算新绿原酸、隐绿原酸、 咖啡酸的相对保留时间和相对校正因子；以迷迭香酸为内参物，计算异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷的相对保留时间和相对校正因子；
    (e)测定：测定夏桑菊颗粒中绿原酸和迷迭香酸的含量，依据相对校正因子计算新绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷7种成分的含量。
  6. 根据权利要求5所述的夏桑菊颗粒定性和定量检测方法，其特征在于，步骤(c)中所述的C18反相色谱柱为Waters Acquity UPLC HSS T3(100mm×2.1mm,1.8μm)、Waters Acquity UPLC CORTECS T3(100mm×2.1mm,1.6μm)、Waters Acquity UPLC BEH C18(100mm×2.1mm,1.7μm)或Thermo scientific Hypersil GOLD(100mm×2.1mm,1.9μm)中的一种；优选为Waters Acquity UPLC HSS T3(100mm×2.1mm，1.8μm)。
  7. 根据权利要求5所述的夏桑菊颗粒定性和定量检测方法，其特征在于，步骤(d)中所述的绿原酸对于新绿原酸、隐绿原酸、咖啡酸的相对保留时间分别为0.561-0.635、1.120-1.343、1.301-1.355；所述的迷迭香酸对于异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷的相对保留时间分别为0.779-0.866、0.815-0.888、0.855-0.906、1.522-1.699。
  8. 根据权利要求7所述的夏桑菊颗粒定性和定量检测方法，其特征在于，步骤(d)中所述的绿原酸对于新绿原酸、隐绿原酸、咖啡酸的相对保留时间分别为0.561、1.153、1.309；所述的迷迭香酸对于异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷的相对保留时间分别为0.787、0.815、0.855、1.547。
  9. 根据权利要求5所述的夏桑菊颗粒定性和定量检测方法，其特征在于，步骤(d)中所述绿原酸对于新绿原酸、隐绿原酸、咖啡酸的相对校正因子分别为0.788-0.884、0.894-0.927、0.462-0.517；所述的迷迭香酸对于异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷的相对校正因子分别为1.269-1.403、1.276-1.444、0.659-0.747、1.075-1.272。
  10. 根据权利要求9所述的夏桑菊颗粒定性和定量检测方法，其特征在于，步骤(d)中所述绿原酸对于新绿原酸、隐绿原酸、咖啡酸的相对校正因子分别为0.804、0.904、0.499；所述的迷迭香酸对于异迷迭香酸苷、异绿原酸B、异绿原酸A、蒙花苷的相对校正因子分别为1.352、1.321、0.671、1.152。
  11. 根据权利要求2所述的夏桑菊颗粒定性和定量检测方法，其特征在于，步骤(1)中所述的供试品溶液的制备方法为：取夏桑菊颗粒10g，研细，加无水乙醇30mL，超声处理30分钟，滤过，滤液旋干，残渣溶解于2mL无水乙醇，作为供试品溶液。
  12. 根据权利要求2所述的夏桑菊颗粒定性和定量检测方法，其特征在于，步骤(2)中所述的对照药材溶液的制备方法为：取夏枯草对照药材0.5g、桑叶对照药材1.0g、野菊花对 照药材1.0g，分别加15mL无水乙醇超声处理30分钟，滤过，滤液旋干，残渣溶解于2mL无水乙醇，作为对照药材溶液。
  13. 根据权利要求2所述的夏桑菊颗粒定性和定量检测方法，其特征在于，步骤(3)中所述的对照品溶液的制备方法为：取迷迭香酸、东莨菪苷、蒙花苷对照品，加甲醇制成每1mL含以上对照品各0.3-0.5mg的混合溶液，作为对照品溶液。
  14. 根据权利要求5所述的夏桑菊颗粒定性和定量检测方法，其特征在于，步骤(a)中所述的对照品溶液的制备方法为：分别取新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷适量，精密称定，加甲醇溶解，得到对照品溶液；所述的对照品溶液中新绿原酸、绿原酸、隐绿原酸、咖啡酸、异迷迭香酸苷、异绿原酸B、异绿原酸A、迷迭香酸、蒙花苷的质量浓度分别为0.192mg/mL、0.192mg/mL、0.185mg/mL、0.131mg/mL、0.160mg/mL、0.162mg/mL、0.135mg/mL、0.262mg/mL和0.198mg/mL。
  15. 根据权利要求5所述的夏桑菊颗粒定性和定量检测方法，其特征在于，步骤(b)中所述的供试品溶液的制备方法为：取夏桑菊颗粒研细粉末1.25g，加50％乙醇溶液25mL，超声处理30分钟，冷却至室温，补足失重，摇匀，3500rpm离心10分钟，取上清液，用0.22μm滤膜过滤，取续滤液作为供试品溶液。
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