HPLC content determination method for cortex lycii radicis medicinal material

Abstract

The invention belongs to the field of traditional Chinese medicine detection, and particularly relates to an HPLC (High Performance Liquid Chromatography) content determination method of a cortex lycii radicis medicinal material, which comprises the following steps: taking a phosphoric acid aqueous solution-acetonitrile-methanol mixed solution as a mobile phase, and taking a solution containing lycium barbarum B as a reference substance solution; in a liquid chromatograph, octadecyl silane bonded silica gel is used as a filling agent; and carrying out gradient elution. According to the method, the content of the lycium barbarum B in the cortex lycii radicis medicinal material is measured by adopting an HPLC (High Performance Liquid Chromatography) method, a target peak and an impurity peak can be effectively separated, a map baseline is stable, and a peak shape is relatively good After the cortex lycii radicis medicinal material is cleaned and dried, the loss of kukoamine B in the decoction pieces is large, and the transfer rate is uncontrollable; the content of the lycium barbarum B is not obviously reduced, and the index component of the lycium barbarum B is more stable and is stable in a neutral solution, so that the risk that the effective components in the decoction containing the cortex lycii radicis medicinal material cannot be monitored does not exist when the method is used for monitoring the effective components in the decoction containing the cortex lycii radicis medicinal material.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicine detection, and particularly relates to an HPLC content determination method for a Chinese medicinal material. Background Art

[0002] Currently, most of the research on the content index components of Radix Lycii is focused on lycidin, such as the study by Hao Yihuan et al., "Quality evaluation of Radix Lycii and roasted Radix Lycii based on fingerprint and content determination."

[0003] Disadvantages of the existing technology: By analyzing the chemical structure of cefotaxime, it was found that cefotaxime is a water-soluble spermine alkaloid. The o-dihydrocaffeine group in the structure is easily oxidized to quinone and is unstable in a neutral solution (pH = 7). Therefore, there is a risk that the decoction containing this medicinal flavor cannot be monitored.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] Because the content of Lycium barbarum B is unstable and cannot be monitored in subsequent studies, a HPLC method for determining the content of Lycium barbarum B in Lycium barbarum B was invented. The present invention belongs to the field of traditional Chinese medicine detection technology and relates to the construction of a method for determining the content of Lycium barbarum B in Lycium barbarum B, which can effectively separate the target peak from the impurity peak, and the chromatogram baseline is stable and the peak shape is good.

[0006] A HPLC method for determining the content of the medicinal material of Lycium bark, the detection method steps comprising:

[0007] Preparation of mobile phase: A mixture of phosphoric acid aqueous solution, acetonitrile and methanol was used as the mobile phase;

[0008] Preparation of reference solution: dissolve Lycium barbarum B in methanol to prepare Lycium barbarum B mother solution, and dilute the mixed solution of Lycium barbarum B mother solution, acetonitrile and phosphoric acid aqueous solution;

[0009] Preparation of test solution: Mix the sample to be tested, ethanol and acetic acid, and filter to obtain the solution;

[0010] Inject the reference solution into the liquid chromatograph, then inject the test solution into the liquid chromatograph and record the chromatogram;

[0011] Wherein, in the liquid chromatograph, octadecylsilane bonded silica gel is used as a filler; gradient elution is performed.

[0012] As a further improvement, the concentration of Lycium barbarum B in the reference solution is 0.01 mg / ml, the volume ratio of acetonitrile to phosphoric acid aqueous solution in the mixed solution of acetonitrile and phosphoric acid aqueous solution is 10:90, and the mass fraction of phosphoric acid aqueous solution is 0.1%.

[0013] As a further improvement, the method for preparing the test solution comprises the following steps:

[0014] Take 0.5±0.05g of the sample to be tested, add 25ml of ethanol-0.5% acetic acid solution with a volume ratio of 1:1, ultrasonically treat for 60 minutes, filter, and take the filtrate to obtain; wherein, the ethanol-0.5% acetic acid solution is obtained by mixing ethanol with a mass fraction of 0.5% acetic acid solution.

[0015] As a further improvement, the mobile phase is a 0.1% by mass phosphoric acid aqueous solution as mobile phase A, acetonitrile as mobile phase B, and methanol as mobile phase C.

[0016] In a further improvement, the conditions of the gradient elution are:

[0017] Time (minutes) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0～50 76 22 2 50～51 76→20 22→80 2→0 51～54 20 80 0 54～55 20→76 80→22 0→2 55～60 76 22 2

[0018] As a further improvement, the column temperature was set to 25°C.

[0019] A further improvement is the flow rate of 0.5 ml per minute.

[0020] As a further improvement, the detection wavelength is 218nm.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention adopts HPLC method to measure the content of Lycium barbarum B in the medicinal material of Lycium bark, which can achieve effective separation of target peak and impurity peak, stable chromatogram baseline and good peak shape.

[0023] 2. Compared with the Chinese wolfberry bark bark medicinal materials, after washing and drying, the loss of Chinese wolfberry bark bark ethylene in the medicinal pieces is relatively large, and the transfer rate is uncontrollable; however, the content of wolfberry bark bark B has no obvious downward trend, and the indicator component of wolfberry bark bark B is more stable and stable in a neutral solution. Therefore, the present invention is used to monitor the effective ingredients in the decoction containing the Chinese wolfberry bark medicinal materials, and there is no risk of being unable to be monitored. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the chromatogram when the mobile phase is "0.1% phosphoric acid-acetonitrile = 90:10";

[0025] Figure 2 This is the chromatogram when the mobile phase is "0.1% phosphoric acid-acetonitrile = 85:15";

[0026] Figure 3 This is the chromatogram when the mobile phase is "0.1% phosphoric acid-acetonitrile = 76:24";

[0027] Figure 4 This is the chromatogram when the mobile phase is "0.1% phosphoric acid-methanol = 76:24";

[0028] Figure 5 This is the chromatogram when the mobile phase is "flow rate 0.8ml / min";

[0029] Figure 6 This is the chromatogram when the mobile phase is "flow rate 0.6ml / min";

[0030] Figure 7 This is the chromatogram when the mobile phase is "flow rate 0.5ml / min";

[0031] Figure 8 is a chromatogram of the assay method of the present invention;

[0032] Figure 9 This is a comparison chart of specific chromatograms;

[0033] Figure 10 is the linear regression equation. DETAILED DESCRIPTION

[0034] The present invention will be further described in detail below through specific embodiments in conjunction with the accompanying drawings.

[0035] Definitions of abbreviations and key terms (definition of custom abbreviations and non-standard terms)

[0036] RSD: relative standard deviation, RD: relative deviation.

[0037] Example 1

[0038] 1. Determination of detection method

[0039] After comparing and exploring the methods, it was found that ordinary liquid phase conditions could not achieve effective separation of the target peaks. The comparison results are as follows:

[0040] Compared with the test conditions determined in the subsequent "Chromatographic Conditions and System Suitability Test", the only difference in this group of tests was the mobile phase, while the other conditions and parameters were the same; the results of the different mobile phase tests are shown in Table 1:

[0041] Table 1

[0042] Chromatographic conditions result Mobile phase: 0.1% phosphoric acid-acetonitrile = 90:10 See Figure 1 Mobile phase: 0.1% phosphoric acid-acetonitrile = 85:15 See Figure 2 Mobile phase: 0.1% phosphoric acid-acetonitrile = 76:24 See Figure 3 Mobile phase: 0.1% phosphoric acid-methanol = 76:24 See Figure 4

[0043] Compared with the test conditions determined in the subsequent "Chromatographic Conditions and System Suitability Test", the only difference in this group of tests was the mobile phase, while the other conditions and parameters were the same. The results of the investigation at different flow rates are shown in Table 2:

[0044] Table 2

[0045] Chromatographic conditions result Flow rate: 0.8ml / min See Figure 5 Flow rate: 0.6ml / min See Figure 6 Flow rate: 0.5ml / min See Figure 7

[0046] In summary, by investigating and comparing different mobile phase gradients, organic phase types, and flow rates, we found that when using two solvents as elution mobile phases, the target peak in the chromatogram could not be completely separated from the surrounding impurities. Adjusting other chromatographic conditions also failed to achieve this. Therefore, we invented this method, replacing the mobile phase with 0.1% phosphoric acid, acetonitrile, and methanol. The results are as follows:

[0047] like Figure 8 As shown, it can be found that when the three solvents are used as mobile phases for detection, the separation effect of the target peak and the subsequent impurity peak is significantly better than that of other chromatographic conditions. Therefore, the chromatographic conditions of the present invention are determined to be:

[0048] Determined according to high performance liquid chromatography (General Rule 0512).

[0049] Chromatographic conditions and system suitability test

[0050] Octadecylsilane bonded silica gel was used as the filler; 0.1% phosphoric acid (0.1% by mass phosphoric acid aqueous solution) was used as mobile phase A, acetonitrile was used as mobile phase B, and methanol was used as mobile phase C. Gradient elution was performed according to the requirements in Table 3; the column temperature was 25°C, the flow rate was 0.5 ml per minute, and the detection wavelength was 218 nm.

[0051] Table 3

[0052]

[0053] Preparation of reference solution

[0054] Accurately weigh an appropriate amount of Lycium barbarum B reference substance and add methanol to prepare a Lycium barbarum B mother solution at a concentration of 0.1 mg / ml, i.e., each 1ml of solution contains 0.1 mg of Lycium barbarum B. Accurately pipette 1ml of Lycium barbarum B mother solution into a 10ml volumetric flask and dilute with a mixture of acetonitrile and 0.1% phosphoric acid solution (10:90 by volume) to obtain a solution containing 0.01 mg of Lycium barbarum B per 1ml.

[0055] Preparation of test solution

[0056] Take about 0.5g of the powder of this product (passed through No. 3 sieve), accurately weigh it, place it in a 150ml conical flask, accurately add 25ml of ethanol-0.5% acetic acid solution (mixed with 1 volume of ethanol and 1 volume of 0.5% acetic acid solution) in a volume ratio of 1:1, weigh the weight, and ultrasonically treat (power 250W, frequency 40kHz) for 60 minutes. Take it out, let it cool, weigh it again, make up the lost weight with ethanol-0.5% acetic acid solution in a volume ratio of 1:1, shake it evenly, filter it, and take the filtrate to obtain the product.

[0057] Assay

[0058] Accurately pipette 10 μl of reference solution and test solution respectively, inject into liquid chromatograph, and measure to obtain the result.

[0059] 2. Method Validation

[0060] 2.1) Specificity

[0061] The specificity of the method was investigated using samples, mobile phase blanks, and solvent blanks as controls. The results are as follows:

[0062] Table 4 Results of specificity investigation

[0063] Sample type Retention time Peak area Peak height Symmetry Factor Theoretical plate number Solvent blank / / / / / Mobile phase blank / / / / / Reference solution 41.663 745.038 17.9 0.99 22494 Test solution 41.888 802.70 18.789 0.99 21551

[0064] Figure 9 The curves in the figure are as follows from top to bottom: mobile phase blank, solvent blank, reference solution, test solution;

[0065] Conclusion: From the above results, it can be seen that the solvent and mobile phase of the present invention have no interference in the detection of Lycium barbarum B, indicating that the present invention has specificity.

[0066] 2.2) System adaptability

[0067] The same reference solution was tested in parallel 6 times and the test solution was tested once, and the system adaptability parameters were recorded. The results are as follows:

[0068] Table 5 System adaptability results

[0069]

[0070] Conclusion: From the above results, it can be seen that the system adaptability of the present invention is good in repeatability (RSD of retention time and peak area of ​​reference substance in 6 parallel assays are less than 2%), the number of theoretical plates is greater than 4000, the signal-to-noise ratio is greater than 10, the tailing factor and peak height are moderate, indicating that the system adaptability of the present invention is good.

[0071] 2.3) Linearity and range

[0072] Prepare different concentrations of Lycium barbarum B reference solution (concentrations as shown in Table 6), and establish a standard curve with the reference solution concentration or injection volume as the horizontal axis and the peak area as the vertical axis. Figure 10 , and obtain the regression equation. The R value is used to evaluate whether the linear relationship is good and the linear range is obtained.

[0073] Table 6 Linearity results inspection table

[0074] serial number Concentration (μg / ml) Peak area Injection volume (μl) Injection volume 1 100.94 6645685 10 1.0094 2 50.47 3255756 10 0.5047 3 20.19 1318296 10 0.2019 4 10.09 635501 10 0.1009 5 5.05 313811 10 0.0505 6 2.02 124603 10 0.0202

[0075] Conclusion: The Lycium barbarum B reference solution was tested at different concentrations, and the concentration of the reference solution was used as the horizontal axis and the peak area as the vertical axis to establish a standard curve. The regression equation was y=65865.3013x-23156.2298, R 2 =0.9999; it can be seen that the linearity for Lycium barbarum B is good in the range of 2.02μg / ml to 100.94μg / ml.

[0076] 2.4) Precision

[0077] ①Instrument precision

[0078] The same test solution was injected in parallel for 6 times, and the RSD of the area and retention time of Lycium barbarum B was used as the evaluation index. The results are shown in Table 7. The RSD ≤ 2% showed that the instrument precision was good.

[0079] Table 7 Instrument precision results

[0080] serial number 1 2 3 4 5 6 RSD% Retention time 42.477 42.134 42.673 42.996 42.879 42.169 0.84 Peak area 838.211 841.228 839.818 841.61 854.777 841.57 0.71

[0081] Conclusion: From the above results, it can be seen that the RSD of the peak area of ​​the 6 test solutions detected in parallel is less than 2%, indicating that the precision of the instrument is good.

[0082] ②Repeatability

[0083] Six test solutions were prepared in parallel using the proposed test solution preparation method and sampled for detection. The content was calculated and the repeatability of the present invention was evaluated using the content RSD. The results are shown in Table 8. The RSD ≤ 2% showed good repeatability.

[0084] Table 8 Repeatability results inspection table

[0085] serial number 1 2 3 4 5 6 mean RSD% content% 0.0568 0.0568 0.0580 0.0580 0.0575 0.0575 0.0574 1.01

[0086] Conclusion: From the above results, it can be seen that the content of 6 test solutions of the same concentration prepared in parallel is 0.0574%, and the RSD is less than 2%, indicating that the present invention has good reproducibility.

[0087] ③Intermediate precision

[0088] The repeatability of this method was investigated by different personnel and different instruments. The results are shown in Tables 9 and 10:

[0089] Table 9 Intermediate precision inspection results (different personnel)

[0090] personnel 1 2 3 RSD% content% 0.061 0.060 0.058 2.56

[0091] Table 10 Intermediate precision inspection results (different instruments)

[0092]

[0093]

[0094] Conclusion: From the above results, it can be seen that the RD or RSD of the content of the same batch of samples detected by different personnel and different instruments are all less than 3%, indicating that the intermediate precision of the present invention is good.

[0095] 5) Accuracy

[0096] Accurately weigh 6 samples of known content (equivalent to 100% concentration level), accurately add an equal amount of reference substance, measure according to the specified conditions, and calculate the recovery rate. The results are shown in Table 11:

[0097] Table 11 Statistics of sample recovery test results

[0098]

[0099] Conclusion: The recovery rate of this product is 95.6% and the RSD is less than 2%. It can be seen that the accuracy and precision of this method meet the requirements.

[0100] 6) Durability

[0101] ①Solution stability

[0102] The test solution was sampled and tested at 0, 2, 4, 8, 16, 24, 36, and 43 hours after preparation, and its peak area and content RSD were used as evaluation indicators. The results are shown in Table 12. RSD ≤ 2% shows that the solution has good stability within 43 hours.

[0103] Table 12 Solution stability test results

[0104]

[0105] Conclusion: From the above results, it can be seen that the peak area RSD of the test solution is less than 2% 43 hours after preparation, indicating that the solution has good stability within 43 hours after preparation.

[0106] ②Durability / different column temperature investigation

[0107] The methodological validation of this method was conducted at a column temperature of 25°C. It was planned to increase the column temperature to investigate the durability of the present invention. The results are shown in Tables 13 and 14:

[0108] Table 13 Durability / column temperature investigation results (content)

[0109]

[0110] Table 14 Summary of system adaptability results for durability investigation (column temperature)

[0111]

[0112] Conclusion: From the above results, it can be seen that the influence of different column temperatures on the chromatographic peak separation and content determination results is relatively small, and the adaptability parameters of each system under different column temperatures are relatively moderate, which shows that the column temperature durability of the present invention is good.

[0113] ③Durability / different flow rate inspection

[0114] The present invention was verified by conducting experiments at a flow rate of 0.5 ml / min. It was intended to increase the flow rate to investigate the durability of the method. The results are shown in Tables 15 and 16:

[0115] Table 15 Durability / flow rate investigation results (content)

[0116]

[0117] Table 16 Summary of durability test system adaptability results (flow rate)

[0118]

[0119]

[0120] Conclusion: From the above results, it can be seen that different flow rates of the present invention have little effect on the separation and content determination results, and the adaptability parameters of each system under different flow rates are relatively moderate, which shows that the flow rate durability of the present invention is good.

[0121] ④ Durability / Durability inspection of different chromatographic columns

[0122] The present invention was verified by using an Agilent brand chromatographic column InfinityLab Poroshell 120SB-C18 (150*4.6mm, 2.7μm). It is intended to add chromatographic columns of different brands to investigate the durability of the present invention. The results are shown in Tables 17 and 18:

[0123] Table 17 Durability / Inspection Results of Different Chromatographic Columns (System Suitability Parameters)

[0124]

[0125] Table 18 Durability / Inspection Results of Different Chromatographic Columns

[0126]

[0127]

[0128] Conclusion: From the above results, it can be seen that the RSD of the content determination results of different chromatographic columns is less than 2%, which is well separated from the impurity peaks, and the system adaptability parameters are relatively moderate, indicating that the durability of the different chromatographic columns of the present invention is good.

[0129] ⑤Durability / different mobile phase gradient investigation

[0130] The method validation experiment was conducted with a mobile phase gradient of 0.1% phosphoric acid-acetonitrile-methanol = 76:22:2. It is planned to increase different gradients to investigate the robustness of the method. The results are shown in Tables 19 and 20:

[0131] Table 19 Durability / mobile phase gradient investigation results (content)

[0132]

[0133] Table 20 Summary of system adaptability results for durability study (mobile phase gradient)

[0134]

[0135] Conclusion: From the above results, it can be seen that different mobile phase gradients of the present invention have little effect on the separation and content determination results, and the adaptability parameters of each system under different gradients are relatively moderate. It can be seen that the mobile phase gradient of the present invention has good durability.

[0136] Example 2

[0137] The transfer rates of two component indicators after the same batch of medicinal materials were processed into slices were used as evaluation. The data are shown in Tables 21 and 22:

[0138] Table 21 Correlation study results of lycosides

[0139]

[0140]

[0141] Table 22 Lycium barbarum B content value transfer

[0142]

[0143]

[0144] By comparison, it was found that the content of Lycium barbarum B in the medicinal slices of Rehmannia glutinosa had no obvious downward trend after washing and drying, while the loss of Lycium barbarum E was large and the transfer rate was uncontrollable, indicating that the Lycium barbarum B indicator component was more stable, and it would be more reliable to evaluate this component in subsequent preparation research.

[0145] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A HPLC content determination method for the medicinal material of Lycium bark, characterized in that: The detection method steps include: Preparation of mobile phase: A mixture of phosphoric acid aqueous solution, acetonitrile and methanol was used as the mobile phase; Preparation of reference solution: dissolve Lycium barbarum B in methanol to prepare Lycium barbarum B mother solution, and dilute the mixed solution of Lycium barbarum B mother solution, acetonitrile and phosphoric acid aqueous solution; Preparation of test solution: Mix the sample to be tested, ethanol and acetic acid, and filter to obtain the solution; Inject the reference solution into the liquid chromatograph, then inject the test solution into the liquid chromatograph and record the chromatogram; Wherein, in the liquid chromatograph, octadecylsilane bonded silica gel is used as a filler; gradient elution is performed.

2. The HPLC content determination method of a Chinese medicinal material of Lycium bark according to claim 1, wherein: The concentration of Lycium barbarum B in the reference solution was 0.01 mg / ml, the volume ratio of acetonitrile to the phosphoric acid aqueous solution in the mixed solution of acetonitrile and phosphoric acid aqueous solution was 10:90, and the mass fraction of the phosphoric acid aqueous solution was 0.1%.

3. The HPLC content determination method of a Chinese medicinal material of Lycium bark according to claim 1, wherein: The preparation method of the test solution comprises the following steps: Take 0.5±0.05g of the sample to be tested, add 25ml of ethanol-0.5% acetic acid solution with a volume ratio of 1:1, ultrasonically treat for 60 minutes, filter, and take the filtrate to obtain; wherein, the ethanol-0.5% acetic acid solution is obtained by mixing ethanol with a mass fraction of 0.5% acetic acid solution.

4. The HPLC content determination method of a Chinese medicinal material of Lycium bark according to claim 1, wherein: The mobile phase is a phosphoric acid aqueous solution with a mass fraction of 0.1% as mobile phase A, acetonitrile as mobile phase B, and methanol as mobile phase C.

5. The HPLC content determination method of a Chinese medicinal material of Lycium bark according to claim 1, wherein: The conditions of the gradient elution are: 。 6. The HPLC content determination method of a Chinese medicinal material of Lycium bark according to claim 1, wherein: The column temperature was 25°C.

7. The HPLC content determination method of a Chinese medicinal material of Lycium bark according to claim 1, wherein: The flow rate was 0.5 ml per minute.

8. The HPLC content determination method of a Chinese medicinal material of Lycium bark according to claim 1, wherein: The detection wavelength is 218 nm.

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