Fingerprint spectrum construction method of carbohydrate components in Shenfu injection and application of fingerprint spectrum construction method

Abstract

The invention provides a fingerprint spectrum construction method of carbohydrate components in a ginseng-radix aconiti carmichaeli injection and application of the fingerprint spectrum construction method, and relates to the technical field of injection quality control. According to the construction method of the fingerprint spectrum of the saccharide components in the ginseng-radix aconiti lateralis praeparata injection, an ultra-high performance liquid chromatography-evaporative light scattering detector (UPLC-ELSD) is adopted for drawing the fingerprint spectrum of the saccharide components in the ginseng-radix aconiti lateralis praeparata injection, and compared with existing detection methods such as a high performance liquid chromatography-differential refraction detector (HPLC-RID) and the like, the method has the advantages that the detection sensitivity is higher; meanwhile, the technical advantages that the detection result is more stable and the error is small can be ensured, and a basis is provided for performing quality control on the ginseng-radix aconiti carmichaeli injection by utilizing carbohydrate components.

Description

Technical Field

[0001] The present invention relates to the technical field of injection quality control, and in particular to a method for constructing a fingerprint spectrum of sugar components in a ginseng and aconite injection and an application thereof. Background Art

[0002] Shenfu Injection (SF1) is derived from the traditional classic prescription - Shenfu Decoction, which was first seen in the Jisheng Prescription by Yan Yonghe in the Song Dynasty. It is made of red ginseng and aconite by modern processing technology. Shenfu Injection mainly contains ginsenosides and aconite alkaloids, as well as a small amount of organic acids, nucleosides, amino acids and metal elements. In the prescription, red ginseng greatly replenishes the vital energy, restores the pulse and consolidates the body, and replenishes qi and blood, and is a "drug that greatly replenishes the vital energy"; aconite restores yang and rescues adverse conditions, replenishes fire and assists yang, dispels cold and relieves pain, and is a "first-class drug for restoring yang and rescuing adverse conditions"; the two medicines are matched, which are warming and replenishing, and have multiple functions such as replenishing qi and warming yang, eliminating cold and dampness, invigorating heart yang, restoring yang and rescuing adverse conditions, replenishing qi and consolidating the body, and warming and unblocking the heart meridian. It is mainly used for syncope caused by sudden loss of yang qi (infectious, hemorrhagic, fluid loss shock, etc.), as well as palpitations, wheezing and coughing, stomach pain, diarrhea, arthralgia, etc. caused by yang deficiency (qi deficiency). In recent years, ginseng and aconite injection has been widely used to treat complex diseases such as heart failure, shock, arrhythmia, sepsis, cor pulmonale, tumors, etc., with significant therapeutic effects.

[0003] Up to now, the research on Shenfu Injection has mainly focused on ginsenoside components and aconite alkaloid components, and there is relatively little research on carbohydrate substances. In recent years, Chinese medicine carbohydrates, as a type of biologically active ingredients, have multiple functions such as immune regulation, anti-oxidation, anti-tumor, and hypoglycemic effects, and have become a hot topic of research for various scholars.

[0004] Fingerprint is an effective method to reflect the quality of Chinese medicine preparations as a whole, and has been adopted in the Chinese Pharmacopoeia. Compared with the quantitative detection of individual components, fingerprint can more comprehensively reflect the quality of Chinese medicine, so it has received attention in the quality control of Chinese medicine in recent years. Through methods such as fingerprint similarity calculation, it is possible to quantitatively determine whether the quality of Chinese medicine is consistent between different batches. Shenfu injection contains a variety of sugar components such as fructose, glucose, sucrose and maltose, accounting for more than 60% of the total solid content. These sugar components can usually be used as bioactive substances to maintain or enhance the body's physiological functions. Therefore, it is of great significance and value to use sugar components for the quality control of Shenfu injection.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The first purpose of the present invention is to provide a method for constructing a fingerprint spectrum of carbohydrate components in Shenfu injection. The fingerprint spectrum of carbohydrate components in Shenfu injection constructed by this method has the advantages of high detection sensitivity and can ensure that the detection results are more stable and the error is smaller.

[0007] The second purpose of the present invention is to provide an application of a method for constructing a fingerprint of carbohydrate components in Shenfu injection.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] The present invention provides a method for constructing a fingerprint spectrum of carbohydrate components in a Shenfu injection, comprising the following steps:

[0010] (A) dissolving a sucrose reference substance in a solvent to prepare a reference substance solution;

[0011] Dissolving D-fructose, D-glucose, sucrose and maltose reference substances in a solvent to prepare a mixed reference substance solution;

[0012] The ginseng and aconite injection to be tested is dissolved in a solvent to prepare a test solution.

[0013] (B) Using an ultra-high performance liquid chromatograph and an evaporative light scattering detector, chromatographic detection is performed on the reference solution, the test solution and the mixed reference solution to obtain a fingerprint of the carbohydrate components in the Shenfu injection.

[0014] (C) The common fingerprint characteristic peaks were determined by measuring the fingerprint of the carbohydrate components of more than 10 batches of Shenfu injection, and the control fingerprint of the carbohydrate components in Shenfu injection was established by fitting with the traditional Chinese medicine chromatographic fingerprint similarity evaluation software.

[0015] Furthermore, the sugar components in the sugar component fingerprint of the Shenfu injection include: fructose, glucose, sucrose and maltose.

[0016] Furthermore, the preparation of the reference substance solution in step (A) includes: taking appropriate amounts of D-fructose, D-glucose, sucrose and maltose reference substances, accurately weighing them, adding 50% acetonitrile to dissolve them and preparing mixed reference substance solutions containing 0.2 mg, 0.5 mg, 2 mg and 2 mg per 1 ml, respectively.

[0017] Furthermore, the preparation of the test solution in step (A) includes: accurately measuring 5 ml of Shenfu injection, placing it in a 10 ml volumetric flask, adding 50% acetonitrile to dilute to the scale, and shaking well to obtain the test solution.

[0018] Furthermore, the chromatographic conditions of the ultra-high performance liquid chromatograph in step (B) include:

[0019] Column: ACQUITY BEH Amide (2.1 mm × 150 mm, 1.7 μm) or equivalent chromatographic column, column temperature: 30-40 °C;

[0020] Mobile phase: isocratic elution with acetonitrile containing 0.2% triethylamine and water in a volume ratio of 72-78:20-28, with a flow rate of 0.2-0.3 ml / min.

[0021] Furthermore, the chromatographic conditions of the ultra-high performance liquid chromatograph in step (B) include:

[0022] Column: ACQUITY BEH Amide (2.1 mm × 150 mm, 1.7 μm), column temperature: 35 °C;

[0023] Mobile phase: isocratic elution with acetonitrile containing 0.2% triethylamine and water in a volume ratio of 75:25, flow rate 0.2 ml / min.

[0024] Furthermore, the conditions of the evaporative light scattering detector in step (B) include:

[0025] Drift tube temperature: 50-60°C; carrier gas: nitrogen, pressure 35.0-45.0 psi; gain: 100-500; nebulizer mode: cooling mode.

[0026] Furthermore, the conditions of the evaporative light scattering detector in step (B) include:

[0027] Drift tube temperature: 55°C; carrier gas: nitrogen, pressure 40.0 psi; gain: 100; nebulizer mode: cooling mode.

[0028] Furthermore, the purity of nitrogen in the evaporative light scattering detector is ≥99.9%.

[0029] Furthermore, the method for generating the reference fingerprint in step (C) comprises:

[0030] The detection method of step (B) was used to sample and measure 15 batches of ginseng and aconite injection samples, and then all the spectra were processed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and the chromatograms within 2 to 7 minutes were cut, multi-point correction was performed, and full-spectrum peak matching was performed to generate a control fingerprint of ginseng and aconite injection.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present application provides a method for constructing a fingerprint spectrum of carbohydrate components in Shenfu injection. The method uses an ultra-high performance liquid chromatography-evaporative light scattering detector (UPLC-ELSD) to draw a fingerprint spectrum of the carbohydrate components in Shenfu injection. Compared with existing detection methods such as high performance liquid chromatography-differential refractometer detector (HPLC-RID), the method has higher detection sensitivity and can ensure that the detection results are more stable and the error is smaller, thereby providing a basis for the quality control of Shenfu injection.

[0033] The fingerprint spectrum construction method of the carbohydrate components in the above-mentioned Shenfu injection provided by the present invention can be widely used in the quality control of Shenfu injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 A comparison chart of the chromatograms of the Shenfu injection test solution and the mixed reference solution provided in Example 1 of the present invention;

[0036] Figure 2 Chromatographic comparison chart of Shenfu injection, red ginseng intermediate, aconite intermediate, and mixed reference substance provided in Example 2 of the present invention;

[0037] Figure 3 The reference fingerprint of Shenfu injection provided in Example 2 of the present invention;

[0038] Figure 4 Fingerprints of Shenfu injection of 15 batches of samples provided in Example 2 of the present invention;

[0039] Figure 5 The flow rate provided for Example 3 of the present invention is 0.1 ml / min HPLC investigation spectrum;

[0040] Figure 6 The flow rate provided for Example 3 of the present invention is 0.4 ml / min HPLC investigation spectrum. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. Meanwhile, the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0043] According to one aspect of the present invention, a method for constructing a fingerprint of carbohydrate components in Shenfu injection comprises the following steps:

[0044] (A) dissolving a sucrose reference substance in a solvent to prepare a reference substance solution;

[0045] Dissolving D-fructose, D-glucose, sucrose and maltose reference substances in a solvent to prepare a mixed reference substance solution;

[0046] The ginseng and aconite injection to be tested is dissolved in a solvent to prepare a test solution.

[0047] (B) Using an ultra-high performance liquid chromatograph and an evaporative light scattering detector, chromatographic detection is performed on the reference solution, the test solution and the mixed reference solution to obtain a fingerprint of the carbohydrate components in the Shenfu injection.

[0048] (C) The common fingerprint characteristic peaks were determined by measuring the fingerprint of the carbohydrate components of more than 10 batches of Shenfu injection, and the control fingerprint of the carbohydrate components in Shenfu injection was established by fitting with the traditional Chinese medicine chromatographic fingerprint similarity evaluation software.

[0049] The present application provides a method for constructing a fingerprint spectrum of carbohydrate components in Shenfu injection. The method uses an ultra-high performance liquid chromatography and an evaporative light scattering detector (UPLC-ELSD) to draw a fingerprint spectrum of the carbohydrate components in Shenfu injection. Compared with existing detection methods such as high performance liquid chromatography-differential refractometer detector (HPLC-RID), the method has higher detection sensitivity and can ensure that the detection results are more stable and the error is smaller, thereby providing a basis for the quality control of Shenfu injection.

[0050] The Shenfu injection used for detection and fingerprint map construction in the present invention is the Shenfu injection produced by China Resources Sanjiu (Ya'an) Pharmaceutical Co., Ltd., and the Shenfu injection can be purchased commercially.

[0051] In a preferred embodiment of the present invention, the carbohydrate components in the carbohydrate component fingerprint of the Shenfu injection include: fructose, glucose, sucrose and maltose.

[0052] In a preferred embodiment of the present invention, the preparation of the reference substance solution in step (A) comprises: taking appropriate amounts of D-fructose, D-glucose, sucrose and maltose reference substances, accurately weighing them, adding 50% acetonitrile to dissolve them and preparing mixed reference substance solutions containing 0.2 mg, 0.5 mg, 2 mg and 2 mg per 1 ml, respectively.

[0053] In a preferred embodiment of the present invention, the preparation of the test solution in step (A) comprises: accurately measuring 5 ml of ginseng and aconite injection, placing it in a 10 ml volumetric flask, adding 50% acetonitrile to dilute to the scale, and shaking well to obtain the test solution.

[0054] In a preferred embodiment of the present invention, the chromatographic conditions of the ultra-high performance liquid chromatograph in step (B) include:

[0055] Column: ACQUITY BEH Amide (2.1 mm × 150 mm, 1.7 μm) or equivalent chromatographic column, column temperature: 30-40 °C;

[0056] Mobile phase: isocratic elution with acetonitrile containing 0.2% triethylamine and water in a volume ratio of 72-78:20-28, with a flow rate of 0.2-0.3 ml / min.

[0057] Preferably, the chromatographic conditions of the ultra-high performance liquid chromatograph in step (B) include:

[0058] Column: ACQUITY BEH Amide (2.1 mm × 150 mm, 1.7 μm), column temperature: 35 °C;

[0059] Mobile phase: isocratic elution with acetonitrile containing 0.2% triethylamine and water in a volume ratio of 75:25, flow rate 0.2 ml / min.

[0060] In a preferred embodiment of the present invention, the conditions of the evaporative light scattering detector in step (B) include:

[0061] Drift tube temperature: 50-60°C; carrier gas: nitrogen, pressure 35.0-45.0 psi; gain: 100-500; nebulizer mode: cooling mode.

[0062] Preferably, the conditions of the evaporative light scattering detector in step (B) include:

[0063] Drift tube temperature: 55°C; carrier gas: nitrogen, pressure 40.0 psi; gain: 100; nebulizer mode: cooling mode.

[0064] As a preferred embodiment, the present application studies the UPLC-ELSD method and studies conditions such as chromatographic conditions, chromatographic columns, gas pressure and drift tube temperature, and obtains a method for constructing a fingerprint spectrum of carbohydrate components in Shenfu injection, thereby achieving rapid, efficient and accurate quality control of carbohydrate components in Shenfu injection.

[0065] In the above preferred embodiment, the purity of nitrogen in the evaporative light scattering detector is ≥99.9%.

[0066] In a preferred embodiment of the present invention, the method for generating the reference fingerprint in step (C) comprises:

[0067] The detection method of step (B) was used to sample and measure 15 batches of ginseng and aconite injection samples, and then all the spectra were processed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and the chromatograms within 2 to 7 minutes were cut, multi-point correction was performed, and full-spectrum peak matching was performed to generate a control fingerprint of ginseng and aconite injection.

[0068] The fingerprint spectrum construction method of the carbohydrate components in the above-mentioned Shenfu injection provided by the present invention can be widely used in the quality control of Shenfu injection.

[0069] According to one aspect of the present invention, a quality control method for Shenfu injection comprises:

[0070] (A) According to the fingerprint spectrum construction method of the carbohydrate components in Shenfu injection of the present invention, the fingerprint spectrum of the carbohydrate components in Shenfu injection is obtained;

[0071] (B) Compare the fingerprint of the carbohydrate components in the obtained Shenfu injection with the standard fingerprint. If the similarity is greater than 0.9, it means that the carbohydrate components in the Shenfu injection are of qualified quality.

[0072] The technical solution of the present invention will be further described below in conjunction with embodiments.

[0073] Example 1

[0074] (I) Instruments and test materials:

[0075] Waters ACQUITY UPLC liquid chromatography system equipped with ACQUITY ELSD evaporative light scattering detector and METTLER AE240 electronic balance.

[0076] D-fructose reference substance (China Food and Drug Inspection Institute, 100231-202008, content is 99.9%), D-anhydrous glucose reference substance (China Food and Drug Inspection Institute, 110833-201908, content is 99.9%), sucrose reference substance (China Food and Drug Inspection Institute, 111507-201704, content is 100.0%), maltose reference substance (China Food and Drug Inspection Institute, 100287-201905, content is 94.4%).

[0077] Three batches of Shenfu injection, with batch numbers 19120201006, 200703AK01, and 200701AK02, were provided by China Resources Sanjiu (Ya'an) Pharmaceutical Co., Ltd. Chromatographically pure acetonitrile was purchased from Merck, analytically pure triethylamine was purchased from Beijing Chemical Reagent Company, and water was ultrapure water.

[0078] (II) Detection method:

[0079] 1. UPLC-ELSD chromatographic conditions:

[0080] Column: ACQUITY BEH Amide (2.1 mm×150 mm, 1.7 μm), column temperature: 35° C. Mobile phase: acetonitrile-water (75:25, containing 0.2% triethylamine), flow rate 0.2 ml / min.

[0081] Drift tube temperature: 55°C, nitrogen pressure: 40.0 psi, gain: 100, data rate 2 pps, cooling mode.

[0082] 2. Preparation of reference solution:

[0083] Take appropriate amounts of D-fructose, D-glucose, sucrose and maltose reference substances in step (i), weigh accurately, add 50% acetonitrile to dissolve and prepare mixed reference substance solutions containing 0.2 mg, 0.5 mg, 2 mg and 2 mg respectively per 1 ml.

[0084] 3. Preparation of test solution:

[0085] Accurately measure 5 ml of the ginseng and aconite injection in step (I) of this example, place it in a 10 ml volumetric flask, add 50% acetonitrile to dilute to the mark, shake well, and obtain.

[0086] 4. Determination method:

[0087] Accurately pipette 1μl and 2μl of the reference solution and 1μl of the test solution, inject into the liquid chromatograph, measure, use the logarithm of the injection volume as the abscissa and the logarithm of the peak area as the ordinate, draw the calibration curve and linear equation, and calculate the content.

[0088] (III) Test results and methodological investigation:

[0089] The following test was performed using the above detection method of this embodiment.

[0090] 1. Linear relationship test:

[0091] Take appropriate amount of D-fructose, D-glucose, sucrose and maltose reference substances, weigh accurately, add 50% acetonitrile to dissolve and make mixed reference substance solutions containing 0.5 mg (D-fructose), 1 mg (D-glucose), 5 mg (sucrose) and 5 mg (maltose) per 1 ml, respectively. Add 50% acetonitrile to dilute step by step to obtain a series of reference substance solutions. Draw a standard curve with the logarithm of the injection amount as the abscissa and the logarithm of the peak area as the ordinate. The results show that each sugar has a good linear relationship. The regression equation, linear range and correlation coefficient are shown in Table 1.

[0092] Table 1 Linear regression equations for fructose, glucose, sucrose and maltose:

[0093]

[0094] 2. Precision test:

[0095] The same sample solution was taken and measured 6 times continuously. The RSD of each sugar peak area was less than 3.0%. The results are shown in Table 2.

[0096] Table 2 Fructose, glucose, sucrose, maltose precision test:

[0097]

[0098]

[0099] 3. Stability test:

[0100] The same sample solution was taken and injected at 0h, 2h, 4h, 8h, 12h and 16h for determination. The RSD of each sugar peak area was less than 3.0%, see Table 3.

[0101] Table 3 Stability test of fructose, glucose, sucrose and maltose:

[0102] Element Average peak area RSD(%) fructose 65876 1.4 glucose 214157 1.2 sucrose 2241245 1.8 maltose 604803 1.6

[0103] 4. Repeatability test

[0104] Six test solutions were prepared in parallel from the same batch of samples, and the sugar contents were calculated. The RSDs were all less than 3.0%, see Table 4.

[0105] Table 4 Repeatability test of fructose, glucose, sucrose and maltose:

[0106] Element Average content RSD(%) fructose 0.4 2.4 glucose 1.3 3.2 sucrose 3.9 2.8 maltose 3.6 2.6

[0107] 5. Recovery rate test:

[0108] Take appropriate amount of D-fructose, D-glucose, sucrose and maltose reference substances, weigh accurately, add 50% acetonitrile to dissolve and prepare mixed reference substance solutions containing 1mg, 3mg, 10mg and 10mg per 1ml respectively. Accurately measure 2.5ml of the known content of this product (19120201006) in a 10ml volumetric flask, then accurately add 1ml of the above mixed reference substance solution, mix well, add 50% acetonitrile to dilute to the scale, shake well, and obtain the sample recovery solution. Prepare 6 copies in parallel. Sample injection determination, calculate the recovery rate, the recovery rate of each sugar is between 95.0% and 105.0%, and the RSD is less than 3.0%. See Table 5.

[0109] Table 5 Recovery rates of fructose, glucose, sucrose and maltose:

[0110]

[0111] 6. Sample determination:

[0112] Three batches of Shenfu injection in step (a) were taken and measured according to the detection method of this embodiment. The results are shown in Tables 6 and Figure 1 .

[0113] Figure 1 The chromatogram comparison chart of the annexed injection test solution and the mixed reference solution in this example.

[0114] Figure 1 A is the chromatogram of the test solution of the injection solution in this example; Figure 1 B is the chromatogram of the mixed reference solution of this example. Figure 1 In each spectrum, peak 1 is fructose, peak 2 is glucose, peak 3 is sucrose, and peak 4 is maltose.

[0115] Table 6 Determination results of fructose, glucose, sucrose and maltose contents in Shenfu injection (mg / ml):

[0116]

[0117] Example 2

[0118] (I) Instruments and test materials:

[0119] Waters ACQUITY UPLC liquid chromatography system equipped with ACQUITY ELSD evaporative light scattering detector and METTLER AE240 electronic balance.

[0120] The D-fructose reference substance, D-anhydrous glucose reference substance, sucrose reference substance, and maltose reference substance were the same as those in Example 1.

[0121] There are 15 batches of ginseng and aconite injection, with batch numbers 19120201006, 200703AK01, 200701AK02, 200703AK06, 200703AK07, 201002AU01, 201101AK03, 201103AK05, 201108AK07, 201109AK07, 201110AK05, 201111AK05, 201201AK05, 201202AK01, and 201203AK06.

[0122] Red ginseng intermediates and aconite root intermediates were provided by China Resources Sanjiu (Ya'an) Pharmaceutical Co., Ltd.

[0123] Chromatographically pure acetonitrile was purchased from Merck, analytically pure triethylamine was purchased from Beijing Chemical Reagent Company, and water was ultrapure water.

[0124] (II) Detection method:

[0125] 1. UPLC-ELSD chromatographic conditions: same as in Example 1.

[0126] 2. Preparation of reference solution: Take an appropriate amount of sucrose reference substance from step (i) and add 50% acetonitrile to make a solution containing about 2 mg per 1 mL as the reference solution.

[0127] 3. Preparation of reference solution: Take appropriate amount of D-fructose, D-glucose, sucrose and maltose reference substances in step (i), weigh accurately, add 50% acetonitrile to dissolve and prepare mixed reference solution containing 0.2 mg, 0.5 mg, 2 mg and 2 mg respectively per 1 ml.

[0128] 4. Preparation of test solution: Accurately measure 5 ml of the ginseng and aconite injection solution in step (i) of this example, place it in a 10 ml volumetric flask, add 50% acetonitrile to dilute to the scale, shake well, and obtain the solution.

[0129] 5. Preparation of intermediate solution: Take appropriate amount of red ginseng intermediate and aconite intermediate respectively, prepare single flavor solution according to the preparation method, take 5 ml of each according to the preparation method of the test sample, place them in 10 ml volumetric flasks respectively, add 50% acetonitrile to dilute to the scale, shake well, and you can get red ginseng intermediate solution and aconite intermediate solution.

[0130] 6. Determination method: Accurately pipette 1 μl of reference solution, standard solution and this product respectively, inject into liquid chromatograph and determine.

[0131] (III) Test results and methodological investigation:

[0132] The following test was performed using the above detection method of this embodiment.

[0133] 1. The reference solution, test solution, red ginseng intermediate solution, aconite intermediate solution and mixed reference solution were chromatographically detected by ultra-high performance liquid chromatography-evaporative light scattering detector. The specific results are shown in Figure 2 .

[0134] Figure 2 Chromatographic comparison chart of Shenfu injection, red ginseng intermediate, aconite intermediate and mixed reference substance in this example.

[0135] Figure 2 A is the chromatogram of the test solution of the injection solution in this example; Figure 2 Middle B is the chromatogram of the red ginseng intermediate of this example; Figure 2 Middle C is the chromatogram of the intermediate of the aconite root of this embodiment; Figure 2 D is the chromatogram of the mixed reference solution in this example. Figure 2 In each spectrum, peak 1 is fructose, peak 2 is glucose, peak 3 is sucrose, and peak 4 is maltose.

[0136] 2. Precision experiment:

[0137] The same test solution was sampled continuously for 6 times. Taking the glucose chromatographic peak as a reference, the average relative retention times of the other three main chromatographic peaks were measured to be 0.47, 0.75, and 0.90, with RSDs of 0.9%, 0.7%, and 0.6%, respectively. The average relative peak areas were 0.55, 3.48, and 3.77, with RSDs of 1.1%, 1.1%, and 1.3%, respectively. The results showed that the method had good precision.

[0138] 3. Stability test:

[0139] The same test solution was taken and its fingerprint was determined at 0, 2, 4, 6 and 8 h, respectively. Taking the glucose chromatographic peak as a reference, the average relative retention times of the other three main chromatographic peaks were 0.47, 0.75 and 0.90, and the RSDs were 0.7%, 0.5% and 1.0%, respectively. The average relative peak areas were 0.55, 3.48 and 3.77, and the RSDs were 2.3%, 2.5% and 2.1%, respectively. The results showed that the test solution was stable at room temperature for 8 hours.

[0140] 4. Repeatability experiment:

[0141] From the same batch of samples, 6 test solutions were prepared in parallel, and their fingerprints were determined respectively. Taking the glucose chromatographic peak as a reference, the average relative retention times of the other three main chromatographic peaks were measured to be 0.47, 0.75, and 0.90, and the RSDs were 0.6%, 0.6%, and 0.4%, respectively. The average relative peak areas were 0.55, 3.48, and 3.77, and the RSDs were 1.4%, 2.1%, and 1.9%, respectively. The results showed that this method had good repeatability.

[0142] 5. Generation of control fingerprint:

[0143] Take 15 batches of samples given in step (a), sample injection and determination according to the detection method of this application, use the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" to process all spectra, cut to obtain chromatograms within 2-7 minutes, multi-point correction, full spectrum peak matching, generate a control fingerprint of Shenfu injection, see details Figure 3 .

[0144] Figure 3 This is the reference fingerprint of the Shenfu injection provided in this example. Figure 3 In the chromatographic peak 1, fructose, peak 2, glucose, peak 3, sucrose, and peak 4, maltose.

[0145] 6. Sample similarity determination:

[0146] Figure 4 Fingerprints of ginseng and aconite injection for 15 batches of samples provided in this example. Figure 4 In the chromatographic peak 1, fructose, peak 2, glucose, peak 3, sucrose, and peak 4, maltose.

[0147] Compare the above-mentioned reference fingerprint of Shenfu injection with the reference fingerprint of Shenfu injection in step (ii) 5 ( Figure 3 ) were used for similarity determination. The specific results are shown in Table 7.

[0148] Table 7 Similarity determination results of fingerprints of Shenfu injection:

[0149]

[0150]

[0151] Example 3

[0152] In this example, except for the selected conditions and the specifically listed conditions, other solution preparation conditions are the same as those in Example 2.

[0153] (I) Investigation of chromatographic conditions:

[0154] 1. Investigation of gas flow rate of nebulizer:

[0155] Preparation of test solution: Accurately measure 5 ml of Shenfu injection (19120201006), place in a 10 ml volumetric flask, add 50% acetonitrile to dilute to the scale, shake well, and obtain.

[0156] The above-mentioned test solution was measured by liquid chromatography under the same liquid phase conditions (based on the UPLC-ELSD chromatographic conditions of Example 1, only the nebulizer gas flow rate was adjusted), except that the nebulizer gas flow rate pressure was 20, 30, 35, 40, 45, and 50 psi, respectively, to examine the effect of the nebulizer gas flow rate on the chromatographic peak.

[0157] The results show that the smaller the flow rate, the sharper the response, but the baseline pressure response increases, and the optimal nebulizer flow pressure is 40.0 psi.

[0158] 2. Investigation of the temperature of the evaporative light detector:

[0159] Preparation of test solution: Accurately measure 5 ml of Shenfu injection (19120201006), place in a 10 ml volumetric flask, add 50% acetonitrile to dilute to the scale, shake well, and obtain.

[0160] Except for the drift tube temperatures of 45°C, 50°C, 55°C, 60°C, and 65°C, the above-mentioned test solution was measured by liquid chromatograph under the same liquid phase conditions (based on the UPLC-ELSD chromatographic conditions of Example 1, only the nebulizer gas flow rate was adjusted) to examine the effect of the evaporative light detector temperature on the chromatographic peak.

[0161] The results showed that the peak shape and separation effect at 45℃ and 65℃ did not meet the requirements. The separation and response values ​​of the peaks of each component were not significantly affected when the drift tube temperature was 50℃, 55℃ and 60℃, and there was only a slight difference in retention time. In view of the rapid heating of the instrument and low energy consumption at a lower temperature, 50℃ was used as the optimal temperature.

[0162] 3. Investigation of mobile phase of ultra-high performance liquid chromatography:

[0163] Preparation of test solution: Accurately measure 5 ml of Shenfu injection (19120201006), place in a 10 ml volumetric flask, add 50% acetonitrile to dilute to the scale, shake well, and obtain.

[0164] Except for the different selection of mobile phase, the above-mentioned test solution was measured by liquid chromatography under the same liquid phase conditions (based on the UPLC-ELSD chromatographic conditions of Example 1, only the nebulizer gas flow rate was adjusted) to examine the influence of the mobile phase of the ultra high performance liquid chromatography on the chromatographic peak.

[0165] The specific mobile phase selection is:

[0166] Group 1, mobile phase: acetonitrile-water (75:25). The difference between the mobile phases of Group 1 and Example 1 is that acetonitrile does not contain triethylamine.

[0167] Group 2, mobile phase: methanol-water (75:25). The difference between the mobile phases of Group 1 and Example 1 is that acetonitrile does not contain triethylamine.

[0168] The results showed that when the mobile phase was acetonitrile-water (75:25) without the addition of triethylamine, the peak shape and separation could not meet the requirements, and fructose and glucose could not be separated; when the mobile phase was methanol-water (75:25) without the addition of triethylamine, the peak shape had severe tailing and the peak elution time was longer.

[0169] 4. Investigation of column temperature of ultra-high performance liquid chromatography:

[0170] Preparation of test solution: Accurately measure 5 ml of ginseng and aconite injection in 19120201006-1, place in a 10 ml volumetric flask, add 50% acetonitrile to dilute to the scale, shake well, and obtain.

[0171] Except that the column temperature of the ultra high performance liquid chromatograph was 20, 30, 35, 40, and 50° C., respectively, under the same liquid phase conditions (based on the UPLC-ELSD chromatographic conditions of Example 1, only the column temperature of the ultra high performance liquid chromatograph was adjusted to measure the above-mentioned test solution by liquid chromatograph to investigate the effect of the column temperature of the ultra high performance liquid chromatograph on the chromatographic peak.

[0172] The results showed that the lower the temperature, the longer the peak time, and the higher the temperature, the shorter the peak time. The peak tailing was serious at 20°C, and the peaks could not be separated at 50°C. There was no significant difference in the separation and response values ​​of the component peaks when the column temperature was 30°C, 35°C, and 40°C, among which 35°C was the optimal condition.

[0173] 5. Investigation of flow rate of ultra-high performance liquid chromatography:

[0174] Preparation of test solution: Accurately measure 5 ml of ginseng and aconite injection in 19120201006-1, place in a 10 ml volumetric flask, add 50% acetonitrile to dilute to the scale, shake well, and obtain.

[0175] Except that the flow rates of the ultra high performance liquid chromatograph were 0.1, 0.2, 0.3, and 0.4 ml / min, respectively, under the same liquid phase conditions (based on the UPLC-ELSD chromatographic conditions of Example 1, only the flow rate of the ultra high performance liquid chromatograph was adjusted to measure the above-mentioned test solution by liquid chromatograph to investigate the effect of the flow rate of the ultra high performance liquid chromatograph on the chromatographic peak.

[0176] Figure 5 The flow rate provided for Example 3 of the present invention is 0.1 ml / min HPLC investigation spectrum;

[0177] Figure 6 The flow rate provided for Example 3 of the present invention is 0.4 ml / min HPLC investigation spectrum.

[0178] The results show that the lower the flow rate, the longer the retention time, the tailing of the peak, and the failure to meet the requirements (see Figure 5 The flow rate is 0.1 ml / min, see Figure 6 The flow rate is 0.4ml / min). When the flow rate is 0.2 and 0.3ml / min, the separation and response values ​​of the peaks of each component are not significantly affected, and there is only a slight difference in the retention time. Therefore, the flow rate is selected to be 0.2-0.3ml / min.

[0179] (II) Sensitivity investigation:

[0180] The UPLC-ELSD method and HPLC-RID method used in the present invention were used to investigate the detection sensitivity. The specific results are shown in Table 8:

[0181] Table 8:

[0182]

[0183]

[0184] As can be seen from the above table, the UPLC-ELSD method used in the present invention has higher detection sensitivity than the HPLC-RID method, ensuring that the detection results are more stable and the error is smaller.

[0185] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a fingerprint of carbohydrate components in Shenfu injection, characterized in that: The following steps are involved: (A) dissolving a sucrose reference substance in a solvent to prepare a reference substance solution; Dissolving D-fructose, D-glucose, sucrose and maltose reference substances in a solvent to prepare a mixed reference substance solution; Dissolve the Shenfu injection to be tested in a solvent to prepare a test solution; (B) using ultra-high performance liquid chromatography-evaporative light scattering detector to perform chromatographic detection on the reference solution, the test solution and the mixed reference solution to obtain a fingerprint of the carbohydrate components in the Shenfu injection; (C) The common fingerprint characteristic peaks were determined by measuring the fingerprint of the carbohydrate components of more than 10 batches of Shenfu injection, and the control fingerprint of the carbohydrate components in Shenfu injection was established by fitting with the traditional Chinese medicine chromatographic fingerprint similarity evaluation software.

2. The method for constructing the fingerprint of the carbohydrate components in Shenfu injection according to claim 1, characterized in that: The carbohydrate components in the carbohydrate component fingerprint of the Shenfu injection include: Fructose, glucose, sucrose and maltose.

3. The method for constructing the fingerprint of the carbohydrate components in Shenfu injection according to claim 1, characterized in that: The preparation of the reference solution in step (A) comprises: Take appropriate amounts of D-fructose, D-glucose, sucrose and maltose reference substances, weigh accurately, add 50% acetonitrile to dissolve and prepare mixed reference substance solutions containing 0.2 mg, 0.5 mg, 2 mg, and 2 mg per ml respectively.

4. The method for constructing the fingerprint of the carbohydrate components in Shenfu injection according to claim 1, characterized in that: The preparation of the test solution in step (A) comprises: Accurately measure 5 ml of Shenfu injection, place it in a 10 ml volumetric flask, add 50% acetonitrile to dilute to the scale, shake well, and obtain the test solution.

5. The method for constructing the fingerprint of carbohydrate components in Shenfu injection according to claim 1, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatograph in step (B) include: Column: ACQUITY BEH Amide (2.1 mm × 150 mm, 1.7 μm) or equivalent column, column temperature: 30-40 °C; Mobile phase: isocratic elution with acetonitrile containing 0.2% triethylamine and water in a volume ratio of 72-78:20-28, with a flow rate of 0.2-0.3 ml / min.

6. The method for constructing the fingerprint of the carbohydrate components in Shenfu injection according to claim 5, characterized in that: The chromatographic conditions of the ultra-high performance liquid chromatograph in step (B) include: Column: ACQUITY BEH Amide (2.1 mm × 150 mm, 1.7 μm), column temperature: 35 °C; Mobile phase: isocratic elution with acetonitrile containing 0.2% triethylamine and water in a volume ratio of 75:25, flow rate 0.2 ml / min.

7. The method for constructing the fingerprint of carbohydrate components in Shenfu injection according to claim 1, characterized in that: The conditions of the evaporative light scattering detector in step (B) include: Drift tube temperature: 50-60°C; carrier gas: nitrogen, gas pressure 35.0-45.0 psi; gain: 100-500; nebulizer mode: cooling mode.

8. The method for constructing the fingerprint of the carbohydrate components in Shenfu injection according to claim 7, characterized in that: The conditions of the evaporative light scattering detector in step (B) include: Drift tube temperature: 55°C; carrier gas: nitrogen, gas pressure 40.0 psi; gain: 100; nebulizer mode: cooling mode; And / or, the purity of nitrogen in the evaporative light scattering detector is ≥99.9%.

9. The method for constructing the fingerprint of the carbohydrate components in Shenfu injection according to claim 1, characterized in that: The method for generating the reference fingerprint in step (C) comprises: The detection method of step (B) was used to sample and measure 15 batches of ginseng and aconite injection samples, and then all the spectra were processed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System", and the chromatograms within 2 to 7 minutes were cut, multi-point correction was performed, and full-spectrum peak matching was performed to generate a control fingerprint of ginseng and aconite injection.

10. Application of the fingerprint spectrum construction method of the carbohydrate components in Shenfu injection according to any one of claims 1 to 9 in the quality control of Shenfu injection.

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