A method for determining the contents of three components in Qianggu capsules by one test and multiple evaluations

Abstract

The present invention relates to a drug analysis and detection method, and specifically to a method for determining the contents of three components in a bone-strengthening capsule by using a one-test-multiple-evaluation method. The determination method of the present invention comprises the following steps: 1) using naringin as an internal standard for one-test-multiple-evaluation, and establishing a relative correction factor between naringin and caffeic acid and eriocin; 2) calculating the contents of caffeic acid and eriocin in the bone-strengthening capsule by the relative correction factor, and determining them by liquid chromatography. The results obtained by this method have no significant difference from those by the external standard method, have high repeatability, stability and credibility, and can provide a reference basis for the quality evaluation of the bone-strengthening capsule.

Description

Technical Field

[0001] The invention relates to a drug analysis and detection method, and in particular to a method for determining the contents of three components in a bone strengthening capsule by adopting a one-test-multiple-evaluation method. Background Art

[0002] The main ingredient of the bone-strengthening capsule is the total flavonoids of Drynaria fortunei, which is included in the "National Drug Standards of the State Food and Drug Administration". It has the effects of tonifying the kidney, strengthening bones, and relieving pain. It is clinically used for bone atrophy caused by kidney yang deficiency, with symptoms of brittle bones, pain in the waist, back or limbs, chills, cold limbs or cramps, lower limb weakness, and frequent urination at night; primary osteoporosis, bone loss with the above symptoms. The total flavonoids of Drynaria fortunei are mainly composed of caffeic acid, sinogeranin, and naringin. Studies have found that naringin has the effect of promoting bone formation. When naringin is applied to osteoblasts, it is found that naringin can better promote the activity of osteoblasts; studies have shown that naringin directly affects bone metabolism through the osteoclast signaling pathway, and has the effect of promoting bone damage repair and slowing down osteoporosis. Both sinogeranin and naringin in Drynaria fortunei are equivalent in promoting the proliferation and differentiation of osteoblasts.

[0003] Zhu Changcheng et al. used the HPLC method to determine the content of naringin in different varieties of Drynariae. The test results are as follows: Naringin has a good linear relationship in the range of 157.5-472.5ng, and the RSD of precision, stability and repeatability are all less than 2%; the microwave method is more efficient in extracting naringin from medicinal materials, and the efficiency of extraction with an extractor is low, but in comparison, the efficiency of using pure methanol as a solvent is higher. The new extraction method used in this experiment, namely the flash extractor extraction method, is simple, stable and has good repeatability. However, its extraction efficiency is not high.

[0004] The Chinese patent application "A method for determining the content of naringin in bone drynaria" discloses a method for determining the content of naringin in bone drynaria, comprising the following steps: (1) crushing the bone drynaria sample, passing it through a No. 3 sieve, and taking 0.25g of the sieved sample powder; mixing the sample powder with 1g of quartz sand, placing it in an ASE extraction cell with a fiber filter membrane, adding quartz sand to be parallel to the cell mouth, and extracting with methanol; collecting the extract and using methanol to make the volume to 50ml; (2) using HPLC to determine the content of naringin in the methanol extract. This patent makes the method for determining the content of naringin in bone drynaria simpler and more accurate. However, it can only determine the content of one component in the bone drynaria sample and has a high cost.

[0005] The one-test-multiple-evaluation method is a multi-index synchronous quality control method that measures the content of a representative ingredient in traditional Chinese medicine and infers the content of other multiple ingredients to be measured in Drynaria Rhizoma based on the proofreading correction factor. The one-test-multiple-evaluation method can measure the content of multiple ingredients at the same time, has been widely used in the quality control of Chinese patent medicines, and solves the problem of large dosage or high price of reference materials in the detection process. At present, the determination method of total flavonoids in Drynaria Rhizoma is relatively complicated, and can only determine the content of one ingredient in Drynaria Rhizoma, with high determination cost and poor extraction effect. Therefore, it is very necessary to provide a method that can determine multiple contents of total flavonoids in Drynaria Rhizoma, and has good extraction effect, strong stability and can reduce costs. Summary of the invention

[0006] The purpose of the present invention is to provide a method for determining the contents of caffeic acid, neoeriocin and naringin in a bone-strengthening capsule, which has the characteristics of good repeatability, high stability and strong credibility, greatly saves resources and reduces detection costs.

[0007] To achieve the above-mentioned purpose of the invention, the technical scheme of the present invention is: a method for determining the contents of three components in a bone-strengthening capsule by a one-measurement-multiple-evaluation method, characterized in that naringin is used as an internal standard for one-measurement-multiple-evaluation, a relative correction factor between naringin and caffeic acid and eriocin is established, the contents of caffeic acid and eriocin in the bone-strengthening capsule are calculated by the relative correction factor, and the determination is carried out by liquid chromatography, comprising the following steps:

[0008] (1) Preparation of reference substance solution: Caffeic acid, neoeriocin and naringin are dissolved in a solvent to prepare a reference substance stock solution, fixed to volume, and mixed to obtain a reference substance solution;

[0009] (2) Preparation of test solution:

[0010] The Qianggu capsule sample and the solvent are mixed, ultrasonically treated to dissolve, cooled, fixed to volume, mixed, filtered, and the filtrate is taken to obtain the test solution;

[0011] (3) Chromatographic conditions:

[0012] Mobile phase A: methanol; Mobile phase B: phosphoric acid aqueous solution;

[0013] Elution conditions:

[0014] From 0min to 50min, the volume fraction of mobile phase A increased from 10-30% to 50-70%;

[0015] 50min-60min, the volume fraction of mobile phase A is 70-90%;

[0016] 60min-70min, the volume fraction of mobile phase A is 10-30%;

[0017] Detection wavelength: 250nm-260nm; flow rate: 0.8-1.2mL / min; column temperature: 25-40℃;

[0018] Chromatographic column: C18 column;

[0019] (4) Determination: The sample solution was injected into a high performance liquid chromatograph and the contents of caffeic acid and erythroposide were calculated by the internal standard method using the relative correction factors with naringin.

[0020] According to some embodiments of the present invention, the solvent in the above steps (1) and (2) is methanol.

[0021] According to some embodiments of the present invention, in the above step (1), the concentration of the reference solution is 36.64-306.3 μg / mL.

[0022] According to some embodiments of the present invention, the ultrasonic treatment time is 20-40 minutes, the power is 300-800W, and the frequency is 30-50KHz;

[0023] Preferably, the ultrasonic treatment time is 30 min, the power is 500 W, and the frequency is 40 KHz.

[0024] According to some embodiments of the present invention, the detection wavelength is 256 nm, the flow rate is 1 mL / min, and the column temperature is 30°C.

[0025] According to some embodiments of the present invention, the conditions of the gradient elution are:

[0026] From 0 min to 50 min, the volume fraction of mobile phase A increased from 20% to 60%;

[0027] 50min-60min, the volume fraction of mobile phase A is 80%;

[0028] 60min-70min, the volume fraction of mobile phase A is 20%.

[0029] According to some embodiments of the present invention, the mobile phase B is a phosphoric acid aqueous solution with a mass fraction of 0.01-1%;

[0030] Preferably, the mobile phase B is a phosphoric acid aqueous solution with a mass fraction of 0.1%.

[0031] According to some embodiments of the present invention, the C18 column is selected from at least one of an Eclipse Plus C18 column, an Aglient ZORBAX SB-C18 column, an Aglient TC-C18 (2) column, and a 4.6 mm×250 mm, 5 μm chromatographic column;

[0032] Preferably, the C18 column is an Aglient ZORBAX SB-C18 column.

[0033] According to some embodiments of the present invention, the calculation formula of the relative correction factor in step (4) is (f) = (Ai×Cr) / (Ar×Ci);

[0034] Where Ai is the peak area of ​​the internal standard, Ar is the peak area of ​​the component to be measured, Ci is the concentration of the internal standard, and Cr is the concentration of the component to be measured.

[0035] The present invention also provides an application of a method for determining the contents of three components in a bone-strengthening capsule by a one-test-multiple-evaluation method in detecting the contents of caffeic acid, neoeriocin and naringin in the bone-strengthening capsule.

[0036] The quality evaluation method of the bone-strengthening capsule provided by the present invention, a one-test-multiple-evaluation method, solves the problems of high cost, large amount of reference substance and high price of the current quality evaluation method of the bone-strengthening capsule. The detection method has high repeatability, strong stability and extremely high credibility, and can provide a reference basis for the quality evaluation of the bone-strengthening capsule.

[0037] The beneficial effects of the present invention are:

[0038] The present invention provides a one-test-multiple-evaluation method for simultaneously determining the contents of caffeic acid, neoeriocin and naringin in a strong bone capsule. Through methodological verification, relative correction factor confirmation, repeatability investigation, chromatographic peak positioning and accuracy evaluation, the one-test-multiple-evaluation method has no significant difference in content determination results with the external standard method. Moreover, the present invention has high repeatability, stability and credibility, and can provide a reference basis for the quality evaluation of strong bone capsules. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The HPLC chromatogram of the mixed reference solution in Example 1 of the present invention is shown in Figure 1, wherein Peak 1 is caffeic acid; Peak 2 is neoeriocin; and Peak 3 is naringin.

[0040] Figure 2 The HPLC chromatogram of the total flavonoids test solution of Drynaria fortunei in Example 1 of the present invention is shown in Figure 1, wherein Peak 1 is caffeic acid; Peak 2 is neoeriocin; and Peak 3 is naringin.

[0041] Figure 3 The HPLC chromatogram of the caffeic acid test solution in Example 1 of the present invention is shown in FIG.

[0042] Figure 4 The HPLC chromatogram of the new eriocin test solution in Example 1 of the present invention.

[0043] Figure 5 This is a high performance liquid chromatogram of the naringin test solution in Example 1 of the present invention.

[0044] Figure 6 This is the HPLC chromatogram of the Drynaria Rhizoma total flavonoids test solution when 50% methanol is used as the extraction solvent in Example 2 of the present invention, wherein Peak 1 is caffeic acid; Peak 2 is eriocin; and Peak 3 is naringin.

[0045] Figure 7 This is a high performance liquid chromatogram of the Drynaria Rhizoma total flavonoids test solution when anhydrous methanol is used as the extraction solvent in Example 2 of the present invention, wherein Peak 1 is caffeic acid; Peak 2 is neoerinoside; and Peak 3 is naringin.

[0046] Figure 8 This is the HPLC chromatogram of the Drynaria Rhizoma total flavonoids test solution when 50% ethanol is used as the extraction solvent in Example 2 of the present invention, wherein Peak 1 is caffeic acid; Peak 2 is neoerinoside; and Peak 3 is naringin.

[0047] Fig. 9 This is a high performance liquid chromatogram of the Drynaria Rhizoma total flavonoids test solution when anhydrous ethanol is used as the extraction solvent in Example 2 of the present invention, wherein Peak 1 is caffeic acid; Peak 2 is neoerinoside; and Peak 3 is naringin.

[0048] Fig.10 This is the HPLC chromatogram of the Drynaria Rhizoma total flavonoids test solution when water is used as the extraction solvent in Example 2 of the present invention, wherein Peak 1 is caffeic acid; Peak 2 is neoerinoside; and Peak 3 is naringin.

[0049] Fig.11 This is the HPLC chromatogram of the total flavonoids test solution of Drynaria fortunei in Example 2 of the present invention (Method 1), wherein Peak 1 is caffeic acid; Peak 2 is neoeriocin; and Peak 3 is naringin.

[0050] Fig.12 This is the HPLC chromatogram of the total flavonoids test solution of Drynaria fortunei in Example 2 of the present invention (Method 2), wherein Peak 1 is caffeic acid; Peak 2 is neoeriocin; and Peak 3 is naringin.

[0051] Fig.13 This is the HPLC chromatogram of the total flavonoids test solution of Drynaria fortunei in Example 2 of the present invention (Method 3), wherein Peak 1 is caffeic acid; Peak 2 is neoeriocin; and Peak 3 is naringin.

[0052] Fig.14 This is the HPLC chromatogram of the total flavonoids test solution of Drynaria fortunei in Example 2 of the present invention (Method 4), wherein Peak 1 is caffeic acid; Peak 2 is neoeriocin; and Peak 3 is naringin.

[0053] Fig.15 This is the HPLC chromatogram of the total flavonoids test solution of Drynaria fortunei in Example 2 of the present invention (Method 5), wherein Peak 1 is caffeic acid; Peak 2 is neoeriocin; and Peak 3 is naringin.

[0054] Fig.16 This is the HPLC chromatogram of the total flavonoids test solution of Drynaria fortunei in Example 2 of the present invention (Method 6), wherein Peak 1 is caffeic acid; Peak 2 is neoeriocin; and Peak 3 is naringin.

[0055] Fig.17 It is a high performance liquid chromatogram of the drynaria total flavonoids test solution in comparative example 1 of the present invention, wherein peak 2 is new eriocin. DETAILED DESCRIPTION

[0056] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific examples below, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the materials, reagents, etc. used can all be obtained from commercial sources.

[0057] The reagents and instruments used in the following examples are as follows:

[0058] 1. Reagents

[0059] Total flavonoids from Drynaria fortunei (batch numbers 191125-1, 210126-3, 210304-3, 210206-1, 210205-3, 210223-3, 191113-3, 210301-3, 210428-3) were obtained from Beijing Qihuang Pharmaceutical Co., Ltd. Caffeic acid (110833-202008), eosin (111507-202109), and naringin (110722-201815) were purchased from the China Food and Drug Administration. Methanol was chromatographically pure, water was ultrapure water, and other reagents were analytically pure.

[0060] 2. Instruments

[0061] LC-2030C 3D Plus high performance liquid chromatograph (Shimadzu, Japan); LC-20AD high performance liquid chromatograph (Shimadzu, Japan); BP211D electronic balance (Sartorius, Germany); KQ-250DA CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.).

[0062] Example 1

[0063] This embodiment 1 is a method for determining the contents of three components in a bone strengthening capsule, comprising the following steps:

[0064] S1. Solution preparation:

[0065] Preparation of reference stock solution:

[0066] Accurately weigh 9.16 mg and 9.05 mg of caffeic acid and new north American erythroside reference substances, respectively, and place them in a 25 ml volumetric flask; accurately weigh 10.21 mg of naringin reference substance, place it in a 10 ml volumetric flask, add methanol to dilute to the scale, shake well, and obtain reference substance stock solutions with concentrations of 366.4, 362, and 1021 μg / mL, respectively.

[0067] Preparation of mixed reference solution:

[0068] Accurately pipette 1 mL, 5 mL and 3 mL of the reference stock solutions of caffeic acid, neoeribotrya glycoside and naringin respectively, place them in the same 10 mL volumetric flask, add methanol to dilute to the scale, shake well, and use them as the mixed reference solution.

[0069] Preparation of test solution:

[0070] Take about 0.1g of total flavonoids from Drynaria fortunei and weigh it accurately, put it in a 100mL volumetric flask, add an appropriate amount of methanol, and ultrasonically treat it (500W, 40kHz) for 30 minutes. Let it cool, add methanol to dilute to the scale, shake well, filter, and take the filtrate to obtain the product.

[0071] S2: Liquid chromatography detection:

[0072] The test solution and the mixed reference solution prepared in step S1 are tested by liquid chromatography.

[0073] The chromatographic conditions were as follows:

[0074] The chromatograph is one of: LC-2030C 3D Plus high performance liquid chromatograph and LC-20AD high performance liquid chromatograph.

[0075] The chromatographic column is one of: Eclipse Plus C18 column, ZORBAX SB-C18 column, and TC-C18 column.

[0076] Mobile phase A was: methanol;

[0077] Mobile phase B is: 0.1% phosphoric acid aqueous solution;

[0078] The gradient elution program was:

[0079] From 0 min to 50 min, the volume fraction of mobile phase A increased from 20% to 60%;

[0080] 50min-60min, the volume fraction of mobile phase A is 80%;

[0081] 60min-70min, mobile phase A is 20%;

[0082] Detection wavelength: 265nm;

[0083] Column temperature: 30°C;

[0084] The injection volume was: 10 μL;

[0085] The volume flow rate is: 1.0mL / min.

[0086] The HPLC chromatogram of the mixed reference solution in this example is shown in Figure 1 ,from Figure 1 It is learned that the detection method of this embodiment is used to achieve effective separation of the above substances.

[0087] The HPLC chromatogram of the test solution in this example is shown in Figure 2 , the HPLC of caffeic acid test solution is shown in Figure 3 , the HPLC chromatogram of the new eriocin is shown in Figure 4 , the HPLC chromatogram of naringin is shown in Figure 5 ,from Figure 2-Figure 5 It can be seen that the liquid chromatography detection method of this embodiment can separate the components of the substance to be tested in the test solution.

[0088] Example 2

[0089] This example is to investigate the determination method in Example 1:

[0090] Drawing of standard curve:

[0091] 2.5, 2.0, 1.5, 1.0, and 0.5 mL of the mixed reference solution prepared in Example 1 were accurately drawn, fixed to volume with methanol, and mixed. According to the chromatographic conditions in Example 1, the chromatographic peak area of ​​each reference substance was recorded. The corresponding peak area (Y) was regressed with the injection amount (X) of each reference substance to obtain the regression equation of the three components. The results show that the linear relationship of each component is good within its respective range, as shown in Table 1.

[0092] Table 1

[0093]

[0094] Precision experiment:

[0095] The mixed reference solution prepared in Example 1 was injected into a high performance liquid chromatograph for detection, and the sample was injected 6 times continuously to record the peak area RSD of caffeic acid, neoeriocin and naringin. The results showed that the peak area RSD (n = 6) of each component was 0.39%, 0.23% and 0.37%, respectively, indicating that the instrument had good precision.

[0096] Repeatability experiment:

[0097] Take an appropriate amount of total flavonoids from the same batch of Drynaria fortunei (batch number 210223-3), prepare 6 test solutions according to the method in Example 1, and measure the samples under the chromatographic conditions of Example 1. The average contents of caffeic acid, eriocin and naringin are 0.0228 mg / mL, 0.1513 mg / mL and 0.2324 mg / mL, and the RSDs of the corresponding peak areas are 0.64%, 0.70% and 0.71%, respectively. The results show that the method has good repeatability.

[0098] Stability test:

[0099] Take the same batch of Drynaria total flavonoids (batch number 210223-3) test solution, and inject and measure the peak area under the chromatographic conditions of Example 1 at 0h, 2h, 4h, 8h, 12h, and 24h after preparation, and obtain the peak area RSD of the three active ingredients of the test sample. It is calculated that the peak area RSD of caffeic acid, eosin, and naringin are 0.85%, 0.96%, and 0.76%, respectively, indicating that the test solution is stable within 24h.

[0100] Sample recovery experiment:

[0101] Accurately weigh about 0.1 g of a sample of total flavonoids from the same batch of Drynaria fortunei (batch number 210223-3) with a known content, a total of 6 portions, and place them in a 100 mL volumetric flask, accurately add 1 mL of caffeic acid (0.3664 mg / mL), 1 mL of elaiocarpon (0.905 mg / mL), and 1 mL of naringin (1.876 mg / mL) reference solution, prepare the test solution according to the method in Example 1, and inject and determine the sample under the chromatographic conditions of Example 1, calculate the sample recovery, and the average sample recoveries of caffeic acid, 100.36%, 99.36%, and 101.30%, respectively, and the RSDs are 2.16%, 2.89%, and 1.72%, respectively. The results show that the method has good accuracy.

[0102] Establishment of relative correction factor (f):

[0103] Under the chromatographic conditions of Example 1, 2 μl, 4 μl, 6 μl, 8 μl, 10 μl, and 12 μl of the mixed reference solution prepared in Example 1 were injected respectively, and the peak areas of the three effective components were recorded. With naringin as the internal standard, the relative correction factor (f) of the component to be tested and the internal standard was calculated. f = (Ai × Cr) / (Ar × Ci) [Ai is the peak area of ​​the internal standard; Ar is the peak area of ​​the component to be tested; Ci is the concentration of the internal standard; Cr is the concentration of the component to be tested]. The results show that when naringin is used as the internal standard, the RSDs of f of the other components, caffeic acid and erythroside, are all less than 2%. The results are shown in Table 2.

[0104] Table 2

[0105]

[0106] Durability evaluation of f:

[0107] The influence of different chromatographic columns and different chromatographs on f:

[0108] The mixed reference solution prepared in Example 1 was injected in an amount of 10 μl, and Shimadzu LC-2030C 3D Plus and Shimadzu LC-20AD high performance liquid chromatographs were used to investigate the effects of EclipsePlus C18 columns, ZORBAX SB-C18 columns, and TC-C18 (2) columns of the same specifications (4.6 mm × 250 mm, 5 μm) on the components f, respectively. The results are shown in Table 3. The results show that different high performance liquid chromatographs and chromatographic columns have no significant effect on the components f.

[0109] Table 3

[0110]

[0111] Investigation of extraction solvent and extraction time:

[0112] The number and size of peak areas when 50% methanol, anhydrous methanol, 50% ethanol, anhydrous ethanol, and water were used as extraction solvents were investigated respectively. The results are shown in Figure 6-Figure 10 The results showed that when anhydrous ethanol was used as the extraction solvent, the chromatographic peak showed tailing and bifurcation; the chromatographic peak area was larger when anhydrous methanol was used as the extraction solvent than when 50% methanol was used as the extraction solvent; when water was used as the extraction solvent, there were more impurity peaks; the situation was basically the same when 50% ethanol was used as the extraction solvent and anhydrous methanol was used as the extraction solvent. Considering its simplicity and availability, methanol was determined to be the best extraction solvent.

[0113] The time of ultrasonic extraction was investigated, and ultrasonic extraction was carried out for 10, 20, 30 and 45 minutes respectively. The peak areas of the three test components, caffeic acid, neoerythrin and naringin, were compared. The results showed that the peak areas were consistent when ultrasonic extraction was carried out for 30 minutes and 45 minutes, and the peak areas were smaller when extracted for 10 minutes and 20 minutes than that at 30 minutes. Therefore, the extraction was complete after 30 minutes of ultrasonic extraction.

[0114] The influence of different column temperatures on f:

[0115] The peak separation and peak shape were investigated at four different column temperatures of 25, 30, 35 and 40°C. The results showed that the separation effect and peak shape of each component were good at a column temperature of 30°C.

[0116] Investigation of detection wavelength:

[0117] The test solution was scanned at full wavelength. The results showed that when the detection wavelength was ≤240nm, the baseline of the chromatographic peak of total flavonoids in Drynaria was unstable, the number of chromatographic peaks decreased, and the main components were difficult to separate; there were more chromatographic peaks at 250-260nm, with less interference and a flat baseline, which could comprehensively reflect the quality of total flavonoids in Drynaria, and the determined components to be tested had maximum absorption near 250-260nm; when ≥270nm, the response of caffeic acid was weak. Therefore, combined with the ultraviolet absorption curves of each component and the wavelength-peak area curve at 250-260nm, the detection wavelength for determining the content of total flavonoids in Drynaria by the "one-measurement-multiple-evaluation method" was determined to be 256nm.

[0118] Investigation of mobile phase elution conditions:

[0119] Different mobile phase elution conditions were investigated respectively, as shown in Table 4. The chromatographic conditions were optimized based on the number of chromatographic peaks, chromatographic peak separation, and chromatographic peak area. Figure 11-Figure 16 . The results showed that in method 1, some components were not separated; in method 2, the distribution range of each chromatographic peak was large and not concentrated; in method 3, each chromatographic peak appeared concentratedly, and the separation was poor; in method 4, some peaks had tailing; in method 5, the separation effect was good and baseline separation was achieved; in method 6, each chromatographic peak appeared concentratedly, and the separation was poor. Therefore, the final chromatographic conditions were determined as follows: mobile phase methanol (A)-0.1% phosphoric acid water (B), gradient elution 0-50min, 20-60% A, 50-60min, 80% A, 60-70min, 20% A; flow rate 1.0mL / min; detection wavelength 256nm; column temperature 30℃; injection volume 10μl.

[0120] Table 4

[0121]

[0122] Investigation of chromatographic peak positioning parameters:

[0123] The reproducibility of two parameters, relative retention time and retention time difference, on different instruments (LC-2030C 3D Plus HPLC, LC-20AD HPLC) and different chromatographic columns (Eclipse Plus C18 column, ZORBAX SB-C18 column, TC-C18 column) was investigated. The mixed reference solution was taken and the relative retention time (RRT) and retention time difference (ΔRT) of naringin to caffeic acid and erythrin were calculated using two HPLC systems and three chromatographic columns. The results showed that the relative retention time of each component fluctuated slightly under different instruments and chromatographic columns, with RSDs of 2.02% and 1.32%, respectively, as shown in Table 5, and the relative retention time difference fluctuated greatly, with RSDs of 4.76% and 8.59%, respectively, as shown in Table 6. Therefore, it is considered reasonable to use the relative retention time parameter as the positioning index of the above target chromatographic peaks.

[0124] Table 5

[0125]

[0126] Table 6

[0127]

[0128]

[0129] Comparison of the results between the QAMS method and the external standard method (ESM):

[0130] Take the total flavonoids sample of Drynariae Rhizoma, prepare the test solution according to the method of Example 1, inject and measure according to the chromatographic conditions in Example 1, record the peak area of ​​3 effective components, first calculate the content of each component according to the standard curve (external standard method), then use naringin as the reference of QAMS method, and calculate the content of 3 components in the total flavonoids of Drynariae Rhizoma by the f average value of each component to be measured and the internal reference, and calculate the relative error (RE) of the measurement results of the two methods, and the results are shown in Table 7. The P value was determined by the paired two-sided t test, and the results showed that there was no significant difference in the contents measured by the two methods (P>0.05), and the relative error (RE) was <5%.

[0131]

[0132] Table 7

[0133]

[0134] Comparative Example 1

[0135] The difference from Example 1 is that the extraction solvent is cyclohexane, 0.1 g of total flavonoids from Drynariae Rhizoma is accurately weighed, 50 ml of cyclohexane is added, and Soxhlet extraction is performed for 3 h. After evaporation, a small amount of tetrahydrofuran is used to dissolve, and then a tetrahydrofuran-acetone (2:1) mixed solution is added and transferred to a 10 mL volumetric flask, filtered with a 0.45 μm microporous filter membrane, and the filtrate is obtained. The rest is the same as in Example 1. The results are shown in Fig.17 The results show that Fig.17 An unknown large peak appears in the image, making other peaks difficult to observe, and the target peak is too small.

[0136] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining the contents of three components in a bone strengthening capsule by a one-test-multiple-evaluation method, characterized in that: Naringin is used as an internal standard for one-test-multiple-evaluation, a relative correction factor between naringin, caffeic acid and erythroside is established, the contents of caffeic acid and erythroside in Qianggu Capsules are calculated by the relative correction factor, and the determination is carried out by liquid chromatography, comprising the following steps: 1) Preparation of reference solution: dissolve caffeic acid, neoericin and naringin in a solvent to prepare a reference stock solution, fix the volume, mix well, and obtain a reference solution; 2) Preparation of test solution: The Qianggu capsule sample and the solvent are mixed, ultrasonically treated to dissolve, cooled, fixed to volume, mixed, filtered, and the filtrate is taken to obtain the test solution; 3) Chromatographic conditions: Mobile phase A: methanol; Mobile phase B: phosphoric acid aqueous solution; Elution conditions: From 0min to 50min, the volume fraction of mobile phase A increased from 10-30% to 50-70%; 50min-60min, the volume fraction of mobile phase A is 70-90%; 60min-70min, the volume fraction of mobile phase A is 10-30%; Detection wavelength: 250nm-260nm; flow rate: 0.8-1.2mL / min; column temperature: 25-40℃; Chromatographic column: C18 column; 4) Determination: Inject the test solution into the HPLC, use the internal standard method to calculate the content of caffeic acid and neoericin by the relative correction factor with naringin; The calculation formula of the relative correction factor in step (4) is (f)=(Ai×Cr) / (Ar×Ci); Where Ai is the peak area of ​​the internal standard, Ar is the peak area of ​​the component to be measured, Ci is the concentration of the internal standard, and Cr is the concentration of the component to be measured.

2. The method according to claim 1, characterized in that The solvent in step (1) and step (2) is methanol.

3. The method according to claim 1, characterized in that In step (1), the concentration of the reference solution is 36.64-306.3 µg / mL.

4. The method according to claim 1, characterized in that: In step (2), the ultrasonic time is 20-40 minutes, the power is 300-800W, and the frequency is 30-50KHz.

5. The method according to claim 4, characterized in that The ultrasonic time in step (2) is 30 minutes, the power is 500W, and the frequency is 40KHz.

6. The method according to claim 1, characterized in that The detection wavelength in step (3) is 256 nm, the flow rate is 1.0 mL / min, and the column temperature is 30°C.

7. The method according to claim 1, characterized in that In step (3), the gradient elution conditions are: 0 min-50 min, the volume fraction of mobile phase A increases from 20% to 60%; 50min-60min, the volume fraction of mobile phase A is 80%; 60min-70min, the volume fraction of mobile phase A is 20%.

8. The method according to claim 1, characterized in that In step (3), the mobile phase B is a phosphoric acid aqueous solution with a mass fraction of 0.01-1%.

9. The method according to claim 8, characterized in that In step (3), the mobile phase B is a 0.1% by mass phosphoric acid aqueous solution.

10. The method according to claim 1, characterized in that In step (3), the C18 column is selected from at least one of Eclipse Plus C18 column, Aglient ZORBAX SB-C18 column, Aglient TC-C18 (2) column, and 4.6 mm × 250 mm, 5 μm chromatographic column.

11. The method according to claim 10, characterized in that In step (3), the C18 column is an Aglient ZORBAXSB-C18 column.

12. Use of the method according to any one of claims 1 to 11 in detecting the contents of caffeic acid, neoeriocin and naringin in bone-strengthening capsules.

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