High performance liquid phase content determination method of rhizoma nardostachyos medicinal material or preparation

Abstract

The invention provides a high performance liquid phase content determination method for a nardostachyos root and rhizome medicinal material or a nardostachyos root and rhizome preparation. The invention further provides a method for judging qualified products of rhizoma nardostachyos medicinal materials or preparations. According to the method for determining the content of the rhizoma nardostachyos, the total amount of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid is taken as an index, so that the internal quality of the rhizoma nardostachyos and related preparations thereof can be controlled on the whole and macroscopically, the curative effect of the medicine is ensured, and more regular quality control on the medicinal materials and the related preparations thereof is realized. The method is good in stability, high in precision, good in reproducibility, convenient and easy to master.

Description

TECHNICAL FIELD

[0001] The application relates to a high-performance liquid chromatography content determination method for Nardostachys jatamansi DC. BACKGROUND

[0002] Nardostachys jatamansi DC. is recorded in the Chinese Pharmacopoeia 2020 edition as the dried root and rhizome of Nardostachys jatamansi DC. of the family Adoxaceae. Nardostachys jatamansi DC. smells fragrant, tastes bitter and sweet, and is warm in nature. It belongs to the spleen and stomach channels. It has the effects of regulating qi and relieving pain, relieving stagnation and activating the spleen, and externally relieving dampness and swelling. It is used for abdominal distension, loss of appetite, and vomiting, and externally for toothache and foot swelling. Terpenes, flavonoids, and phenolic acids are the main chemical components in Nardostachys jatamansi DC. [2] , such as nardosinone, chlorogenic acid, and linarin (Liu Yinghui, et al., Simultaneous determination of chlorogenic acid and linarin in Nardostachys jatamansi by RP-HPLC, New Drugs and Clinical Pharmacology, May 2012, Vol. 23, No. 3). Nardostachys jatamansi DC. also contains a large amount of volatile oil. Nardosinone is one of the main active ingredients and has various pharmacological activities. Therefore, the content determination is mainly to analyze the contents of active ingredients such as nardosinone and chlorogenic acid by high-performance liquid chromatography, and to analyze the volatile oil components in Nardostachys jatamansi DC. by gas chromatography. Li Yanbang, Liu Guolin, Qiao Jing, et al. Simultaneous determination of chlorogenic acid and nardosinone in Nardostachys jatamansi from different habitats by HPLC [J]. Chinese Medicine Information, 2015, 32 (6): 27-30. A method for simultaneously determining the contents of chlorogenic acid and nardosinone in Nardostachys jatamansi by high-performance liquid chromatography was established. The results show that the mass fraction of chlorogenic acid in 25 Nardostachys jatamansi from different habitats is 0.026% to 0.355%, and the mass fraction of nardosinone is 0.109% to 2.013%.

[0003] At present, the content determination method of Nardostachys jatamansi DC. is mainly referred to in the Chinese Pharmacopoeia 2020 edition. There are few literatures on the qualitative and quantitative research of nardosinone in Nardostachys jatamansi DC. formula granules and related preparations. We found through research that after Nardostachys jatamansi DC. is decocted for medicine, nardosinone is almost not detected in the decoction, and it is also difficult to detect nardosinone in the volatile oil. Therefore, it is difficult to use the content limit of nardosinone as a quality control index to evaluate the quality of Nardostachys jatamansi DC., medicinal slices, standard decoction, extract, and formula granules and related preparations. Some literatures take linarin as the content determination index. Liu Chengping, Zeng Yuan'er, and Gao Feng. Thermal stability of linarin in Chrysanthemum indicum L. [J]. Today's Pharmacy, 2011, 21 (02): 88-90+93. It is reported that the content of linarin will be greatly reduced due to heat, and the drying temperature of extract and preparation in the actual production process is usually above 100 DEG C, and the time is generally more than 12 h. Therefore, the content of linarin will be greatly reduced, which may lead to the phenomenon that the content of linarin in many Nardostachys jatamansi DC. preparations is small or even undetectable.

[0004] Most existing methods for detecting the content of spikenard are single-indicator or dual-indicator methods. Modern research shows that phenolic acid components such as chlorogenic acid have antibacterial, antiviral, white blood cell-increasing, hepatoprotective, choleretic, antitumor, blood pressure-lowering, blood lipid-lowering, free radical scavenging, and central nervous system-stimulating effects.

[0005] Application No. 202310858330.3, Invention Title: A Method for Constructing Characteristic Chromatography and Quality Detection of Nardostachys jatamansi and its Preparations. This invention provides a method for constructing characteristic chromatography and quality detection of Nardostachys jatamansi and its preparations. In the method for constructing the characteristic chromatography, the chromatographic conditions include: using acetonitrile and an aqueous solution containing formic acid as the mobile phase, performing gradient elution. The gradient elution program includes: 0→5min→40min→50min, and the volume percentage of acetonitrile in the mobile phase is: 10%→10%→35%→50%. Under this specific elution program, nine common characteristic peaks are obtained. The characteristic peaks detected in this literature include: chlorogenic acid, cryptochlorogenic acid, deoxynapterinol A, nardostachys neoketone diol, and nardostachys ketone A.

[0006] Since chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid are all present in Nardostachys jatamansi, and their contents vary by 1 to 20 times (0.026%–0.54%) in different producing areas (Wu Jie, et al., Quality Evaluation Study of Nardostachys jatamansi from Different Producing Areas, Sichuan Forestry Science and Technology, August 2017, Vol. 38, No. 4), the literature also mentions that there are certain differences in volatile oil, macroelements, chlorogenic acid, and nardostachys ketone components in Nardostachys jatamansi from different producing areas. This may be related to factors such as climate, growth environment, and soil in different producing areas. Although the current Chinese Pharmacopoeia does not list these three as legal indicators, some studies have used "chlorogenic acid + nardostachysone" as common peaks in the HPLC fingerprint of nardostachysone, which is used to determine authenticity and quality (Wang Ziyang et al., Quality Evaluation of Nardostachysone from Different Origins by Combining HPLC Fingerprint and Chemical Pattern Recognition and Multi-Component Quantification, Chinese Traditional and Herbal Drugs, February 2024, Vol. 55, No. 3, identified chlorogenic acid, syringic acid, nardostachysone, and aristolochic acid as four potential marker components for evaluating the quality differences of nardostachysone. Because chlorogenic acids have antioxidant, antibacterial, and anti-inflammatory activities, they are considered one of the material bases for the "regulating qi and relieving pain, relieving depression and invigorating the spleen" effects of nardostachysone. The ratio and content of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid vary significantly with the harvesting period and drying method. Imbalance in the ratio will change the antibacterial spectrum and free radical scavenging rate of the extract, thus affecting the clinical efficacy. Currently, there is no method to simultaneously detect these three indicators to control the quality of nardostachysone and its preparations. Summary of the Invention

[0007] This invention provides a high-performance liquid chromatography method for the determination of the content of Nardostachys jatamansi medicinal material or preparation, which simultaneously detects chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid under the same conditions.

[0008] The application provides a high performance liquid chromatography content determination method of Gansong medicinal materials or preparations, which comprises the following steps:

[0009] a. Preparation of test sample solution:

[0010] The Gansong medicinal materials or preparations are dissolved by solvent, extracted, and Gansong medicinal materials or preparations are obtained;

[0011] b. The Gansong medicinal materials or preparations are determined by high performance liquid chromatography, and the characteristic map of Gansong medicinal materials or preparations is obtained;

[0012] The high performance liquid chromatography conditions are as follows: the chromatographic column is a C18 column; acetonitrile is used as mobile phase A, and 0.1% phosphoric acid solution is used as mobile phase B for gradient elution, and the gradient elution conditions are as follows:

[0013]

[0014] The preparation method of the test sample solution is as follows: the Gansong medicinal materials or preparations are extracted by water or methanol aqueous solution, filtered, and the filtered solution is obtained.

[0015] The extraction method is reflux extraction or ultrasonic extraction.

[0016] The ultrasonic extraction conditions are as follows: the power is 600 W, the frequency is 40 KHz, and the ultrasonic time is 30 min; the concentration of the methanol aqueous solution is 70% methanol.

[0017] The application also comprises preparation of a control solution, and the control is chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid.

[0018] The chlorogenic acid control, neochlorogenic acid control and cryptochlorogenic acid control are prepared into methanol aqueous solution as the control solution.

[0019] The chromatographic conditions are as follows: the C18 column has a column length of 150 mm, an inner diameter of 2.1 mm and a particle size of 1.8 μm; the flow rate is 0.1-0.3 ml / min; the column temperature is 25-35 DEG C; the detection wavelength is 254 nm; and the theoretical plate number calculated according to the chlorogenic acid peak should be not less than 5000.

[0020] Preferably, the flow rate is 0.2 ml / min, and the column temperature is 30 DEG C.

[0021] The Gansong medicinal materials or preparations comprise medicinal materials, decoction pieces, standard decoction, medicinal material extracts and formula granules.

[0022] The total content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the Gan Song medicinal material is not less than 0.54%; the total content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the Gan Song decoction piece is not less than 0.52%; the total content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the Gan Song standard decoction is not less than 16.3 mg / g; the total content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the Gan Song extract is not less than 16.7 mg / g; and the total content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the Gan Song formula granule is not less than 13.9 mg / g.

[0023] The application also provides a method for judging Gan Song medicinal material or preparation qualified products, which comprises the following steps:

[0024] a, taking a sample to be tested;

[0025] b, determining the total content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid according to the high performance liquid content determination method of the Gan Song medicinal material or preparation;

[0026] c, if the total content is higher than the lower limit value, it is a qualified product.

[0027] The simultaneous determination of the total content of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid is necessary, and there are difficulties in detection, specifically:

[0028] 1, too similar chemical structure

[0029] Neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid are isomers, only different in the connection position of the caffeoyl group, and the separation degree of the conventional C18 column is often <1.2, and they are easily co-eluted.

[0030] 2, large content span and complex matrix

[0031] · Gan Song contains a large amount of sesquiterpenes (Gansu new ketone, etc.) and volatile oil, which will produce ultraviolet absorption interference near 325 nm; · The total content of chlorogenic acid is only about 0.05%, and the content of neochlorogenic acid and cryptochlorogenic acid is often less than 0.01%, so high sensitivity and wide linear range need to be considered.

[0032] 3, easy transformation of sample pretreatment

[0033] Chlorogenic acid can undergo ester bond migration under high temperature, strong light or alkaline conditions When the ultrasonic extraction time is too long or the pH of the methanol-water extraction liquid is >6, “false positive” or proportion drift will occur.

[0034] 4, expensive and poor stability of standard

[0035] The market price of neochlorogenic acid and cryptochlorogenic acid reference substance is about 1500-2000 yuan per 10 mg, and it needs to be stored in the dark at -20℃; without internal standard correction, the reproducibility is difficult to meet the quantitative requirements at trace level (<0.005%).

[0036] 5. The current pharmacopoeia method is insufficient. The 2020 edition of the Chinese Pharmacopoeia only includes the determination of "chlorogenic acid" as a single index. The chromatographic gradient is simple and cannot separate the three isomers. If the conditions under honeysuckle or chrysanthemum are directly applied, cryptochlorogenic acid will be misidentified as chlorogenic acid, resulting in an overestimation of 15-30%.

[0037] This invention discloses the construction and application of a high-performance liquid chromatography (HPLC) method for determining the content of Nardostachys jatamansi. This method establishes a content determination method with the total amount of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid as indicators, which fully demonstrates the chemical composition characteristics of Nardostachys jatamansi and comprehensively reflects the quality information of Nardostachys jatamansi, thereby enabling comprehensive and effective control of the quality of Nardostachys jatamansi medicinal materials, decoction pieces, and related preparations.

[0038] The beneficial effects of this invention are:

[0039] (1) Establish a method for determining the content of Nardostachys jatamansi, using the total amount of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid as indicators. This can control the intrinsic quality of Nardostachys jatamansi and its related preparations as a whole and macroscopically, ensuring the efficacy of the drug and enabling more standardized quality control of the medicinal materials and their related preparations.

[0040] (2) The method of the present invention has good stability, high precision, good reproducibility, and is convenient and easy to master. Attached Figure Description

[0041] Figure 1 Mobile phase selection diagram;

[0042] Figure 2 Ultraviolet absorption spectrum of neochlorogenic acid-reference standard;

[0043] Figure 3 Neochlorogenic acid - UV absorption spectrum of the test sample;

[0044] Figure 4 Chlorogenic acid-reference standard UV absorption spectrum;

[0045] Figure 5 Chlorogenic acid - UV absorption spectrum of the test sample;

[0046] Figure 6 Cryptochlorogenic acid-reference standard UV absorption spectrum;

[0047] Figure 7 Cryptochlorogenic acid - UV absorption spectrum of the test sample;

[0048] Figure 8 Flow velocity study results;

[0049] Figure 9 Column temperature investigation results;

[0050] Figure 10 Investigation into the specificity of Nardostachys jatamansi;

[0051] Figure 11 Neochlorogenic acid standard curve chart;

[0052] Figure 12 Chlorogenic acid standard curve chart;

[0053] Figure 13 Cryptochlorogenic acid standard curve chart;

[0054] Figure 14 Chromatogram of different instruments;

[0055] Figure 15 Different gradient chromatographic condition screening test. DETAILED DESCRIPTION

[0056] Example 1 High performance liquid content determination method of gansong medicinal material or preparation

[0057] 1. Experimental instruments and materials

[0058] Liquid chromatograph: Shimadzu LC-40AD type ultra-high performance liquid chromatograph, Waters H-Class Plus type ultra-high performance liquid chromatograph;

[0059] Electronic balance: ME204E / 02, MS205DU, XP26 (Mettler-Toledo Instruments Co., Ltd.);

[0060] Ultrapure water machine: cell type 1810A (Shanghai Moore Scientific Instruments Co., Ltd.);

[0061] Ultrasonic cleaner: KQ-600DB type (600W, 40KHz; Kunshan Ultrasonic Instrument Co., Ltd.);

[0062] Chlorogenic acid (China Food and Drug Inspection Research Institute, batch number: 110753-202119, purity: 96.3%);

[0063] Neochlorogenic acid (Chengdu Desit Bio-technology Co., Ltd., batch number: DSTDX001504, purity: 98%);

[0064] Cryptochlorogenic acid (Chengdu Desit Bio-technology Co., Ltd., batch number: DST220104-035, purity: 98%);

[0065] Acetonitrile and phosphoric acid are chromatographically pure; water is ultrapure water; and other reagents are analytically pure;

[0066] Radix et Rhizoma Phlomidis batch number: 23001, 23002, 23003, 23004, 23005, 23006, 23007, 23008, 23009, 23010, 23011, 23012, 23013, 23014, 23015, 23016;

[0067] Radix et Rhizoma Phlomidis slice batch number: YP01, YP02, YP03, YP04, YP05, YP06, YP07, YP08, YP09, YP10, YP11, YP12, YP13, YP14, YP15, YP16;

[0068] Radix et Rhizoma Phlomidis standard decoction batch number: BT01, BT02, BT03, BT04, BT05, BT06, BT07, BT08, BT09, BT10, BT11, BT12, BT13, BT14, BT15, BT16;

[0069] Radix et Rhizoma Phlomidis intermediate batch number: TQW01, TQW02, TQW03;

[0070] Radix et Rhizoma Phlomidis formula granules batch number: GSP01, PFKL01, PFKL02, PFKL03.

[0071] 2. Experimental conditions

[0072] 2.1 Chromatographic conditions and system suitability test

[0073] The chromatographic conditions and system suitability test used octadecylsilane-bonded silica gel as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the provisions in the table below, flow rate 0.2 ml per minute; column temperature 30°C; detection wavelength 254 nm. The theoretical plate number should not be less than 5000 calculated on the chlorogenic acid peak.

[0074]

[0075] 2.2 Preparation of control solution

[0076] The control solution was prepared by accurately weighing appropriate amounts of chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid controls, and adding 70% methanol to make a mixture solution containing 50 μg per 1 ml as the control solution.

[0077] 2.3 Preparation of test solution

[0078] Take about 1.0 g of the powder of the medicinal material (pass through a No. 2 sieve), accurately weigh, place in a conical flask with a stopper, accurately add 50 ml of water, heat and reflux for 30 minutes, filter, accurately take 25 ml of the filtrate, evaporate to dryness, accurately add 25 ml of 70% methanol to the residue, seal tightly, weigh, ultrasonic treatment (600 W, 40 KHz) for 30 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is the test solution.

[0079] Take about 1.0 g of the powder of the medicinal material (pass through a No. 2 sieve), accurately weigh, place in a conical flask with a stopper, accurately add 50 ml of water, heat and reflux for 30 minutes, filter, accurately take 25 ml of the filtrate, evaporate to dryness, accurately add 25 ml of 70% methanol to the residue, seal tightly, weigh, ultrasonic treatment (600 W, 40 KHz) for 30 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is the test solution.

[0080] Take about 0.1 g of the standard decoction, accurately weigh, place in a conical flask with a stopper, accurately add 25 ml of 70% methanol, seal tightly, weigh, ultrasonic treatment (600 W, 40 KHz) for 30 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is the test solution.

[0081] Take about 0.1 g of the standard decoction, accurately weigh, place in a conical flask with a stopper, accurately add 25 ml of 70% methanol, seal tightly, weigh, ultrasonic treatment (600 W, 40 KHz) for 30 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is the test solution.

[0082] Take about 0.1 g of the standard decoction, accurately weigh, place in a conical flask with a stopper, accurately add 25 ml of 70% methanol, seal tightly, weigh, ultrasonic treatment (600 W, 40 KHz) for 30 minutes, cool, re-weigh, make up the weight loss with 70% methanol, shake well, filter, and take the filtrate, which is the test solution.

[0083] 2.4 Determination method

[0084] Accurately take 1 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine, which is the test solution.

[0085] 3. Content determination method

[0086] 3.1 Chromatographic conditions and system suitability test

[0087] 3.1.1 Selection of mobile phase

[0088] On the basis of the above experimental conditions, acetonitrile-0.1% phosphoric acid was selected for investigation, see Figure 1 , Table 1.

[0089] Table 1 Analysis results of mobile phase

[0090]

[0091] The results showed that acetonitrile-0.1% phosphoric acid as the mobile phase, the peak shape and separation effect of the target peak was good, so acetonitrile-0.1% phosphoric acid solution was selected as the mobile phase for subsequent investigation.

[0092] 3.1.2 Wavelength selection

[0093] On the basis of the above experimental conditions, the diode array detector was used to scan the whole wavelength of chlorogenic acid reference solution, greenish chlorogenic acid reference solution and test sample solution. See Figures 2-7 .

[0094] By comparing the maximum absorption wavelength of new greenish chlorogenic acid, greenish chlorogenic acid and greenish chlorogenic acid, referring to the chromatogram, the index peak has greater absorption at the detection wavelength of 245nm, and the chromatogram baseline is more stable, and combined with the standard of Gansong in Chinese Pharmacopoeia 2020 edition, the detection wavelength is determined as 245nm.

[0095] 3.1.3 Flow rate investigation

[0096] According to the above experimental conditions, the flow rate of 0.1ml / min, 0.2ml / min, 0.3ml / min was investigated. See Figure 8 , Table 2.

[0097] Table 2 Analysis results of different flow rates

[0098]

[0099] The results showed that when the flow rate was 0.1ml / min, 0.2ml / min, 0.3ml / min, the chromatogram peak shape and separation effect met the requirements, and the flow rate was determined as 0.2ml / min.

[0100] 3.1.4 Column temperature investigation

[0101] On the basis of the above experimental conditions, the column temperature of 25℃, 30℃, 35℃ was investigated. See Figure 9 , Table 3.

[0102] Table 3 Analysis results of different column temperatures

[0103]

[0104] The results show that when the column temperature is 25℃, 30℃ and 35℃, the peak shape and separation effect of the chromatogram meet the requirements, and 30℃ is selected as the detection column temperature.

[0105] The chromatographic conditions and system suitability test of Gan Song dispensing granules characteristic map are as follows: octadecylsilane bonded silica as the filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the following table, flow rate 0.2 ml / min; column temperature 30℃; detection wavelength 254 nm. The theoretical plate number should not be less than 5000 calculated by the chlorogenic acid peak.

[0106]

[0107] 3.2 Preparation of test solution

[0108] 3.2.1 Extraction method investigation

[0109] Take GSP01, grind finely, take about 0.1 g, accurately weigh, put into a conical flask with a plug, accurately add 70% methanol 25 ml, tightly plug, weigh, ultrasonic (600 W, 40 KHz) treatment for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and get it. Accurately take 1 μl of each test solution, inject into the liquid chromatograph, calculate the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid under different extraction methods, and the results are shown in Table 4.

[0110] Table 4 Extraction method investigation

[0111]

[0112] The results show that the extraction method has little effect on the content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid, so the ultrasonic extraction method is selected because it is simple to operate.

[0113] 3.2.2 Extraction solvent investigation

[0114] Take GSP01, grind finely, take about 0.1 g, accurately weigh, put into a conical flask with a plug, accurately add methanol, 70% methanol, 30% methanol, ethanol, 70% ethanol, 30% ethanol, water 25 ml respectively, tightly plug, weigh, ultrasonic (power 600 W, frequency 40 kHz) treatment for 30 minutes, cool, weigh again, make up the weight loss with the corresponding solvent respectively, shake well, filter, take the filtrate, and get it. Accurately take 1 μl of each test solution, inject into the liquid chromatograph, calculate the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid under different extraction solvents, and the results are shown in Table 5.

[0115] Table 5 Extraction solvent investigation results

[0116]

[0117] From the above, the extraction solvent is 70% methanol, the extraction efficiency is the highest, so the extraction solvent is 70% methanol.

[0118] 3.2.3 Investigation of the amount of extraction solvent added

[0119] Take GSP01, grind finely, take about 0.1g, accurately weigh, put in a conical flask with a plug, respectively accurately add 70% methanol 15ml, 25ml, 50ml, tightly plug, weigh, ultrasonic treatment (power 600W, frequency 40KHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and get it. Respectively, accurately take 1μl of test solution, inject into liquid chromatograph, calculate the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid under different extraction solvents, and the results are shown in Table 6.

[0120] Table 6 Investigation of the amount of extraction solvent added

[0121]

[0122] The results show that when the amount of extraction solvent added is 15ml, 25ml, 50ml, the content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid has little difference, so the amount of solvent added is temporarily set as 25ml.

[0123] 3.2.4 Extraction time investigation

[0124] Take GSP01, grind finely, take about 0.1g, accurately weigh, put in a conical flask with a plug, accurately add 70% methanol 25ml, tightly plug, weigh, ultrasonic treatment (power 600W, frequency 40KHz) for 15, 30, 45 minutes respectively, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and get it. Respectively, accurately take 1μl of test solution, inject into liquid chromatograph, calculate the total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid under different extraction solvents, and the results are shown in Table 7.

[0125] Table 7 Investigation of the amount of extraction solvent added

[0126]

[0127] The results show that the extraction time is 30min, which can be completely extracted, so the extraction time is temporarily set as 30min.

[0128] In summary, the preparation method of the Gan Song content determination test sample is as follows: take the product, grind finely, take about 0.1 g, accurately weigh, place in a conical flask with a plug, accurately add 25 ml of 70% methanol, tightly plug, weigh, ultrasonic treatment (power 600 W, frequency 40 KHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and obtain.

[0129] 3.2.5 Gan Song content determination method

[0130] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel as filler (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, gradient elution according to the following table, flow rate 0.2 ml per minute; column temperature 30°C; detection wavelength 254 nm. The theoretical plate number should not be less than 5000 calculated by the chlorogenic acid peak.

[0131]

[0132] Preparation of reference solution: take new chlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, accurately weigh, add 70% methanol to make a mixed solution containing 50 μg per 1 ml, and obtain.

[0133] Preparation of test sample solution: take Gan Song formula granules, grind finely, take about 0.1 g, accurately weigh, place in a conical flask with a plug, accurately add 25 ml of 70% methanol, tightly plug, weigh, ultrasonic treatment (power 600 W, frequency 40 KHz) for 30 minutes, cool, weigh again, make up the weight loss with 70% methanol, shake well, filter, take the filtrate, and obtain.

[0134] Determination: accurately pipette 1 μl of reference solution and test sample solution respectively, inject into the liquid chromatograph, and determine.

[0135] 3.3 Methodology investigation

[0136] 3.3.1 Specificity investigation

[0137] Preparation of reference solution: take new chlorogenic acid reference substance, chlorogenic acid reference substance, cryptochlorogenic acid reference substance, accurately weigh, add 70% methanol to make a solution containing 50 μg per 1 ml, as the reference solution.

[0138] Preparation of test solution: GSP01 was finely ground, about 0.1 g was accurately weighed into a conical flask with a stopper, 25 ml of 70% methanol was added accurately, the flask was tightly stoppered, the weight was determined again, and the mixture was ultrasonically treated (power 600 W, frequency 40 KHz) for 30 min. The flask was allowed to cool, the weight was determined again, the lost weight was made up with 70% methanol, the mixture was shaken and filtered. The filtrate was collected to give the test solution.

[0139] Preparation of negative control solution: The negative control solution was prepared according to the above test solution preparation method, except that Gansong was not added. The results are shown in Table 1. Figure 10 .

[0140] As can be seen from the figure, the negative solution has no interference with the determination of the test peak, indicating that the method has good specificity.

[0141] 3.3.2 Precision examination

[0142] The mixed solution of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid was continuously injected 6 times, the peak area was recorded, and the RSD value was calculated. The results are shown in Table 8.

[0143] Table 8 Precision examination results

[0144]

[0145] As can be seen from the above, the peak area RSD value of neochlorogenic acid in the precision examination was 0.27%, the peak area RSD value of chlorogenic acid was 0.27%, and the peak area RSD value of cryptochlorogenic acid was 0.22%, indicating that the precision of the instrument was good.

[0146] 3.3.3 Linear relationship

[0147] Take the new chlorogenic acid control (purity 98.0%) in the right amount, placed in 20 ml volumetric flask, with 70% methanol solution to make every 1 ml containing new chlorogenic acid 149.939100 μg solution, dilution to concentration of 29.987820 μg / ml, 14.993910 μg / ml, 7.496955 μg / ml, 2.998982 μg / ml, 1.499391 μg / ml. Another green take chlorogenic acid control (purity 96.3%) in the right amount, placed in 20 ml volumetric flask, with 70% methanol solution to make every 1 ml containing green 149.98900 μg solution, dilution to concentration of 29.99780 μg / ml, 14.99890 μg / ml, 7.499890 μg / ml, 2.99978 μg / ml, 1.49989 μg / ml. Take cryptochlorogenic acid control (purity 98.0%) in the right amount, placed in 20 ml volumetric flask, with 70% methanol solution to make every 1 ml containing cryptochlorogenic acid 145.9500 μg solution, dilution to concentration of 29.1900 μg / ml, 14.5950 μg / ml, 7.2975 μg / ml, 2.9190 μg / ml, 1.4595 μg / ml. Respectively, 1 μl precision injection into liquid chromatograph, determination, get peak area, with concentration (X, μg / mL) as the abscissa, peak area (Y) as the ordinate draw response curve, the results are shown in table 9, 10, 11, Figure 11 、 12 , 13.

[0148] Table 9 new chlorogenic acid standard curve analysis results

[0149]

[0150] Table 10 green acid standard curve analysis results

[0151]

[0152] Table 11 cryptochlorogenic acid standard curve analysis results

[0153]

[0154] The results show that: the new chlorogenic acid injection concentration in 1.499391-149.93100 μg / ml, linear relationship y=4945.9x-3773.4, R 2 =0.9998, indicating that the new chlorogenic acid injection concentration in 1.499391-149.93100 μg / ml, good linear relationship. Green acid injection concentration in 1.49989-149.98900 μg / ml, linear relationship y=5997.4x-3473.5, R 2= 0.9999, the sample concentration of chlorogenic acid was in the range of 1.49989-149.98900 μg / ml, which showed a good linear relationship. The sample concentration of cryptochlorogenic acid was in the range of 1.4595-145.9500 μg / ml, which showed a good linear relationship. 2 = 0.9998, the sample concentration of cryptochlorogenic acid was in the range of 1.4595-145.9500 μg / ml, which showed a good linear relationship.

[0155] 3.3.4 repeatability

[0156] GSP01 was taken, finely ground, and about 0.1 g was accurately weighed into six portions. The same operator prepared the sample solution according to the proposed experimental method, and 1 μl of the sample solution was accurately taken into the liquid chromatograph. The total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid in the six samples was calculated, and the results are shown in Table 12.

[0157] Table 12 Repeatability test results

[0158]

[0159]

[0160] The results showed that the RSD value of neochlorogenic acid in the six repeated samples was 0.39%, the RSD value of chlorogenic acid was 0.41%, and the RSD value of cryptochlorogenic acid was 0.69%. The method had good repeatability.

[0161] 3.3.5 intermediate precision

[0162] Two sample solutions were prepared by personnel A1 at T1 time according to the proposed experimental method, and were measured on C1 (Waters H-Class Plus) machine. Two sample solutions were prepared by personnel A2 at T2 time according to the proposed experimental method, and were measured on C2 (Shimadzu L-40AB X3) machine. The total amount of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid was calculated, respectively. The results are shown in Table 13. Figure 14 .

[0163] Table 13 Intermediate precision test results

[0164]

[0165] From the above, the neochlorogenic acid in Agilent 1290 ultra-high performance liquid chromatograph was not separated, so the method had poor durability for Agilent instrument, and the content calculation was not performed. Different personnel detected sample GSP01 on Waters and Shimadzu instruments at different times, and the RSD value of the sample content determination results was 1.34%. The method had good intermediate precision.

[0166] 3.3.6 Spiked recovery

[0167] About 0.05 g of test sample (batch number: GSP01, the content of neochlorogenic acid was 5.3 mg / g, the content of chlorogenic acid was 5.7 mg / g, and the content of cryptochlorogenic acid was 5.3 mg / g) was precisely weighed, 6 samples in total, and a certain amount of neochlorogenic acid reference substance, chlorogenic acid reference substance, and cryptochlorogenic acid reference substance was precisely added, respectively. The preparation of test sample solution was carried out according to the proposed method, and the recovery rate was calculated. The results are shown in Table 14. The calculation formula is as follows:

[0168]

[0169] Table 14 Results of spiked recovery experiment

[0170]

[0171] From the above, the average recovery rate of neochlorogenic acid was 99.1%, the average recovery rate of chlorogenic acid was 100.0%, and the average recovery rate of cryptochlorogenic acid was 98.3%. The accuracy of the method is good.

[0172] 3.3.7 Durability investigation

[0173] 3.3.7.1 Investigation of column durability

[0174] Three different types of C18 chromatographic columns (chromatographic column 1: ACQUITY HSS T3, 2.1*150mm, 1.8μm; chromatographic column 2: ACQUITY BEH C18, 2.1*150mm, 1.7μm; chromatographic column 3: ZORBAX Eclipse Plus C18, 2.1*150mm, 1.8μm) were used to detect GSP01. 1 μl of test sample solution was precisely taken and injected into the liquid chromatograph, and the total amount of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the sample was calculated. The results are shown in Table 15.

[0175] Table 15 Results of investigation of column durability

[0176]

[0177] From the above, the RSD value of the sample content determination results was 2.96% when different types of chromatographic columns were used for detection, indicating that the column durability of the method was good.

[0178] 3.3.8 Stability experiment

[0179] According to the above experimental conditions, a test solution was prepared, and the peak area of uridine was measured at 0 h, 6 h, 12 h, 20 h, and 26 h, respectively. The results are shown in Table 16.

[0180] Table 16 Stability test results

[0181]

[0182] As can be seen from the above, the RSD value of the peak area of neochlorogenic acid in the sample solution within 26 h is 0.78%, the RSD value of the peak area of chlorogenic acid is 0.92%, and the RSD value of the peak area of cryptochlorogenic acid is 0.79%, indicating that the test solution is stable within 26 hours.

[0183] 3.4 Sample content determination verification

[0184] The proposed method was used to test 16 batches of Gansong medicinal materials, 16 batches of Gansong decoction pieces, 16 batches of Gansong standard decoction, 3 batches of Gansong extract, and 3 batches of Gansong formula granules. The results are shown in Tables 16-20.

[0185] Table 16 Content determination results of 16 batches of Gansong medicinal materials

[0186]

[0187]

[0188] Table 17 Content determination results of 16 batches of Gansong decoction pieces

[0189]

[0190]

[0191] Table 18 Content determination results of 16 batches of Gansong standard decoction

[0192]

[0193] Table 19 Content determination results of 3 batches of Gansong extract

[0194]

[0195] Table 20 Content determination results of 3 batches of Gansong formula granules

[0196]

[0197]

[0198] As can be seen from the above, the content determination method of Gansong can effectively detect Gansong medicinal materials, decoction pieces, standard decoction, extract, and formula granules samples, and the method is stable and feasible.

[0199] Example 2: Screening test of chromatographic conditions of the present invention

[0200] Chromatographic conditions and system suitability test: Octadecylsilane-bonded silica gel was used as the packing material (column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid solution was used as mobile phase B. Gradient elution was performed according to the specifications in the table below, with a flow rate of 0.2 mL / min; column temperature was 30℃; detection wavelength was 254 nm. The theoretical plate number, calculated based on the chlorogenic acid peak, should not be less than 5000.

[0201] Gradient 1:

[0202]

[0203] Gradient 2:

[0204]

[0205] Gradient 3:

[0206]

[0207] Depend on Figure 15 It can be seen that gradient 3 (i.e., the content determination method of the present invention) is superior.

[0208] Modern research indicates that phenolic acids, such as chlorogenic acid, possess antibacterial, antiviral, white blood cell-boosting, hepatoprotective, choleretic, antitumor, blood pressure-lowering, blood lipid-lowering, free radical scavenging, and central nervous system-stimulating effects. Furthermore, most existing methods for determining the content of Nardostachys jatamansi are single-indicator or dual-indicator methods; therefore, it is necessary for this invention to simultaneously determine the total amount of chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid.

Claims

1. A high-performance liquid chromatography method for determining the content of Nardostachys jatamansi medicinal material or preparation, characterized in that: It includes the following steps: a. Preparation of the test solution: The raw material or preparation of Nardostachys jatamansi is dissolved and extracted using a solvent to obtain the test solution; b. The test solution is determined by high performance liquid chromatography to obtain the characteristic chromatogram of Nardostachys jatamansi or its preparation; The high-performance liquid chromatography (HPLC) conditions are as follows: a C18 column; acetonitrile as mobile phase A, 0.1% phosphoric acid solution as mobile phase B, and gradient elution; the gradient elution conditions are as follows:

2. The high-performance liquid chromatography method for determining the content of Nardostachys jatamansi medicinal material or preparation according to claim 1, characterized in that: The preparation method of the test solution is as follows: take Nardostachys jatamansi medicinal material or preparation, extract with water or methanol aqueous solution, filter, and take the filtrate to obtain the test solution.

3. The high-performance liquid chromatography method for determining the content of Nardostachys jatamansi medicinal material or preparation according to claim 2, characterized in that: The extraction method described is reflux extraction or ultrasonic extraction.

4. The high-performance liquid chromatography method for determining the content of Nardostachys jatamansi medicinal material or preparation according to claim 3, characterized in that: The ultrasonic extraction conditions are: power 600W, frequency 40KHz; ultrasonic time 30min; and the concentration of the methanol aqueous solution is 70% methanol.

5. The high-performance liquid chromatography method for determining the content of Nardostachys jatamansi medicinal material or preparation according to claim 1, characterized in that: It also includes the preparation of reference solutions, wherein the reference standards are: chlorogenic acid, neochlorogenic acid, and cryptochlorogenic acid; the preparation method is as follows: Prepare a reference solution by adding methanol-water solution to chlorogenic acid reference standard, neochlorogenic acid reference standard and cryptochlorogenic acid reference standard.

6. The high-performance liquid chromatography method for determining the content of Nardostachys jatamansi medicinal material or preparation according to claim 1, characterized in that: The chromatographic conditions are as follows: C18 column: column length 150 mm, inner diameter 2.1 mm, particle size 1.8 μm; flow rate 0.1-0.3 ml / min; column temperature 25-35℃; detection wavelength 254 nm; theoretical plate number calculated based on chlorogenic acid peak should not be less than 5000.

7. The high-performance liquid chromatography method for determining the content of Nardostachys jatamansi medicinal material or preparation according to claim 5, characterized in that: The flow rate was 0.2 ml per minute; the column temperature was 30°C.

8. The high-performance liquid chromatography method for determining the content of Nardostachys jatamansi medicinal material or preparation according to any one of claims 1-7, characterized in that: The aforementioned Nardostachys jatamansi medicinal materials or preparations include medicinal materials, processed slices, standard decoctions, medicinal material extracts, and formulated granules.

9. The high-performance liquid chromatography method for determining the content of Nardostachys jatamansi medicinal material or preparation according to claim 8, characterized in that: The total content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the *Nardostachys jatamansi* medicinal material is not less than 0.54%; the total content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the *Nardostachys jatamansi* decoction is not less than 0.52%; the total content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the *Nardostachys jatamansi* standard decoction is not less than 16.3 mg / g; the total content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the *Nardostachys jatamansi* extract is not less than 16.7 mg / g; and the total content of neochlorogenic acid, chlorogenic acid, and cryptochlorogenic acid in the *Nardostachys jatamansi* formula granules is not less than 13.9 mg / g.

10. A method for determining the quality of *Nardostachys chinensis* medicinal materials or preparations, characterized in that: It includes the following steps: a. Take the sample to be tested; b. Determine the total content of neochlorogenic acid, chlorogenic acid and cryptochlorogenic acid by the high performance liquid chromatography method according to any one of claims 1-8; c. If the total content is higher than the lower limit value described in claim 9, then it is a qualified product.

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