Method for detecting nucleoside components in cornu cervi pantotrichum preparation

Abstract

The invention discloses a method for detecting nucleoside components in a cornu cervi pantotrichum preparation, and belongs to the technical field of detection, the detection method comprises the following steps: S1, preparing a test solution; s2, preparing a reference substance solution, wherein reference substances comprise uracil, hypoxanthine, uridine, guanosine and inosine; s3, preparing a blank solvent; and S4, respectively taking the test solution, the reference solution and the blank solvent, and injecting into high performance liquid chromatography. According to the method, the five components in the antler essence injection can be simultaneously determined in a short time, and the detection method is stable, reliable and simple to operate. Compared with the prior art, the method can be used for comprehensively detecting main effective components in the cornu cervi pantotrichum and the preparation thereof, provides a basis for quality evaluation of the cornu cervi pantotrichum and the preparation thereof, and can be used for effectively monitoring the quality of the cornu cervi pantotrichum and the preparation thereof.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a method for detecting nucleoside components in velvet antler preparations. Background Art

[0002] Deer antler injection is a traditional Chinese medicine preparation whose main ingredient is deer antler. It can enhance body vitality and promote cell metabolism. It is used for neurasthenia, loss of appetite, malnutrition, sexual dysfunction and amnesia.

[0003] At present, the quality control of deer antler essence injection mainly focuses on pH value, protein, heavy metals and absorbance. There is a lack of testing items for the content of its main active ingredients, and the quality of the preparation cannot be fully and effectively controlled.

[0004] CN104173379A discloses a method for processing and extracting velvet antler and its application in the field of medicine and food, wherein a method for determining the hypoxanthine content in velvet antler by HPLC is disclosed. CN108152382A discloses a HPLC standard fingerprint spectrum of ultra-thin velvet antler slices and a construction method thereof, wherein the construction method comprises the preparation of a test solution and a reference solution, and adopts a linear gradient elution, wherein the mobile phase A is a 0.144% trifluoroacetic acid aqueous solution, and the mobile phase B is a 0.1% trifluoroacetic acid acetonitrile solution; the flow rate is 0.8-1.0 mL / min; the detection wavelength is 265-275 nm, and the standard fingerprint spectrum of ultra-thin velvet antler slices is obtained by high performance liquid chromatography, wherein the fingerprint spectrum has 20 common characteristic peaks, wherein the peak No. 3 is uracil, the peak No. 13 is adenosine, and the peak No. 16 is tyrosine.

[0005] At present, there are few studies on the effective ingredients of velvet antler and its processed products, and it is not possible to comprehensively and effectively control the product quality. Therefore, providing a more comprehensive method for detecting nucleoside components is very meaningful for the quality control of velvet antler and its processed products. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a method for detecting nucleoside components in velvet antler preparations.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a method for detecting nucleoside components in a deer antler preparation, comprising the following steps: S1. Prepare the test solution: S2. preparing a reference solution, wherein the reference solution includes uracil, hypoxanthine, uridine, guanosine and inosine; S3, preparing blank solvent; S4. Take the test solution prepared in step S1, the reference solution prepared in step S2 and the blank solvent prepared in step S3 respectively, and inject them into the high performance liquid chromatography.

[0008] The antler preparations of the present invention include antler and processed products thereof, wherein the processed products are preparations containing antler, such as antler essence injection.

[0009] In some embodiments, when the antler preparation is pilose antler, the preparation method of the test solution is: crushing the pilose antler, adding water for ultrasonic treatment, separating the solid and liquid, and filtering the supernatant.

[0010] Preferably, the ultrasonic treatment time is 20-40 min, more preferably 25-35 min, and even more preferably 30 min.

[0011] Preferably, the solid-liquid separation is a conventional solid-liquid separation method in the art, including at least one of centrifugation, filtration and filter pressing.

[0012] In some embodiments, the test solution is prepared by crushing the antler, adding water and ultrasonically treating it for 20-40 minutes, centrifuging it, and filtering the supernatant.

[0013] Preferably, the preparation method of the test solution is: crush the antler, take 0.05-2g, add 1-4mL of water, ultrasonically treat for 20-40min, centrifuge, take the supernatant and filter.

[0014] In some embodiments, when the velvet antler preparation is velvet antler essence injection, the preparation method of the test solution is: filtering the velvet antler essence injection.

[0015] Preferably, the method for preparing the test solution is: filtering the pilose antler essence injection solution through a 0.22 μm filter membrane.

[0016] In some embodiments, the reference solution is prepared by dissolving uracil, hypoxanthine, uridine, guanosine and inosine in methanol.

[0017] Preferably, the volume fraction of methanol is 5-15%, more preferably 8-12%.

[0018] Preferably, the concentration of uracil in the reference solution is 85-96 mg / mL, the concentration of hypoxanthine is 70-90 mg / mL, the concentration of uridine is 65-76 mg / mL, the concentration of guanosine is 45-60 mg / mL, and the concentration of inosine is 30-40 mg / mL.

[0019] Further preferably, the concentration of uracil in the reference solution is 90-91 mg / mL, the concentration of hypoxanthine is 79-80 mg / mL, the concentration of uridine is 70-71 mg / mL, the concentration of guanosine is 51-52 mg / mL, and the concentration of inosine is 34-35 mg / mL.

[0020] More preferably, the concentration of uracil in the reference solution is 90.89 mg / mL, the concentration of hypoxanthine is 79.92 mg / mL, the concentration of uridine is 70.20 mg / mL, the concentration of guanosine is 51.08 mg / mL, and the concentration of inosine is 34.99 mg / mL.

[0021] In some embodiments, the blank solvent is commercially available purified water, such as Wahaha purified water.

[0022] In some embodiments, the chromatographic conditions of the high performance liquid chromatography are: Mobile phase: Mobile phase A and mobile phase B were used, wherein mobile phase A was 0.1-0.3% formic acid water and mobile phase B was acetonitrile; Elution: Gradient elution, elution program see Table 1.

[0023] Table 1 Gradient elution program

[0024] Preferably, the chromatographic conditions of the high mobile phase effective liquid chromatography also include: Column: Ultimate AQ-C 18 、Uenusil MP-C 18 or Sepax HP-C 18 ; Detection wavelength: 254nm; Flow rate: 0.6-1mL·min -1 ; Column temperature: 35-45°C; Injection volume: 8-12μL.

[0025] Further preferably, the chromatographic conditions of the high performance liquid chromatography are: Column: Ultimate AQ-C 18 (4.6×250 mm, 5 μm); Mobile phase: Mobile phase A is 0.2% formic acid water, mobile phase B is acetonitrile; Detection wavelength: 254nm; Flow rate: 0.8 mL min -1 ; Column temperature: 45°C; Injection volume: 10 μL; The beneficial effects of the present invention are: The invention can simultaneously measure five components in the velvet antler essence injection in a short time. Meanwhile, the detection method of the invention is stable and reliable, and is easy to operate, thus providing an experimental basis for quickly and comprehensively controlling the quality of traditional Chinese medicine injections.

[0026] Compared with the prior art, the present invention can comprehensively detect the main effective ingredients in pilose antler and its preparations, provide a basis for the quality evaluation of pilose antler and its preparations, and can effectively monitor the quality of pilose antler and its preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is the chromatogram of Example 1, wherein 1 is uracil, 2 is hypoxanthine, 3 is uridine, 4 is guanosine, 5 is inosine, A is velvet antler injection, B is reference solution, C is velvet antler medicinal material, and D is blank solution. Figure 2 It is the chromatogram of Comparative Example 1.

[0028] Figure 3 It is the chromatogram of Comparative Example 2.

[0029] Figure 4 It is the chromatogram of Comparative Example 3. DETAILED DESCRIPTION

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the invention belongs. The present invention is described in more detail below in conjunction with specific examples, but the present invention is not limited thereto. Unless otherwise specified, the temperatures described are all room temperature (15-35°C), the solvents used to prepare the solutions are all water, and the percentages involved are all volume percentages.

[0031] 1. Instrument LC-30AD high performance liquid chromatograph, Shimadzu Co., Ltd., Japan; SPD-M20A diode array detector, Shimadzu Co., Ltd., Japan; LC-Solution chromatography workstation, Shimadzu Co., Ltd., Japan; BSA124S electronic analytical balance and BT25S electronic analytical balance, Sartorius Scientific Instruments (Beijing) Co., Ltd., China; SHZ-S(Ⅲ) circulating water multi-purpose vacuum pump, Shanghai Lichen Bangxi Instrument Technology Co., Ltd., China; KQ-100DE CNC ultrasonic cleaner, Kunshan Ultrasonic Instrument Co., Ltd., China.

[0032] 2. Reagents Methanol (chromatographic grade) and acetonitrile (chromatographic grade) were obtained from Concord Technology Co., Ltd., Tianjin, China; formic acid (chromatographic grade) was obtained from Damao Chemical Reagent Factory, Tianjin, China; phosphoric acid (chromatographic grade) was obtained from Kelong Chemical Reagent Factory, Chengdu, China; purified water was obtained from Wahaha Group Co., Ltd., Hangzhou, China.

[0033] 3. Reference substances Uracil (batch number: 110469-201302, content ≥99%), hypoxanthine (batch number: 140661-202005, purity ≥99%), guanosine (batch number: 111977-201501, purity ≥99%), inosine (batch number: 140669-202007, purity ≥99%), and uridine (batch number: 110887-202104, purity ≥99%) were all provided by China Food and Drug Inspection Institutes.

[0034] Example 1 A method for detecting nucleoside components in velvet antler preparations, comprising the following steps: S1. Prepare the test solution: Deer antler medicinal material: crush the deer antler through a 60-mesh sieve, accurately weigh 0.1 g, add 2 mL of water, ultrasonically treat for 30 min, centrifuge, take the supernatant and filter it through a 0.22 μm filter membrane for later use; Deer antler essence injection: filter the deer antler essence injection through a 0.22μm filter membrane and set aside.

[0035] S2. Prepare reference solution, which includes uracil, hypoxanthine, uridine, guanosine and inosine. Take appropriate amount of uracil, hypoxanthine, uridine, guanosine and inosine reference solution in 5 mL volumetric flask, weigh accurately, dissolve and dilute with 10% methanol, and make the mass concentrations of uracil, hypoxanthine, uridine, guanosine and inosine into 1.245, 1.11, 1.17, 1.125 and 1.215 mg·mL -1 Prepare the reference stock solution for future use.

[0036] Then accurately pipette 365, 360, 300, 225 and 144 μL of the reference substance stock solution into a 5 mL volumetric flask to prepare mixed reference substance stock solutions with mass concentrations of 90.89, 79.92, 70.20, 51.08 and 34.99 mg / L, respectively.

[0037] S3. Prepare blank solvent: take Wahaha purified water.

[0038] S4, respectively take the test solution prepared in step S1, the reference solution prepared in step S2 and the blank solvent prepared in step S3, inject them into the HPLC, and record the chromatogram. Figure 1 .

[0039] The chromatographic conditions of the high performance liquid chromatography are: Column: Ultimate AQ-C 18 (4.6×250 mm, 5 μm); Mobile phase: Mobile phase A is 0.2% formic acid water, mobile phase B is acetonitrile; Elution: gradient elution, elution program see Table 2; Detection wavelength: 254nm; Flow rate: 0.8 mL min -1 ; Column temperature: 45°C; Injection volume: 10 μL.

[0040] Table 2 Gradient elution program

[0041] The results showed that the theoretical plate number was no less than 5000 based on uridine, the separation between adjacent peaks was greater than 1.5, the tailing factor was within 0.95-1.05, and the blank solution had no interference with the determination; the retention times of the main peaks of velvet antler injection were consistent with those of velvet antler reference medicinal materials, uracil, hypoxanthine, uridine, guanosine and inosine, respectively, and could be used for sample identification.

[0042] 1.1 Investigation of linear relationship Precisely measure the mixed reference solution in multiples of 1, 2, 4, 6, 8 and 10 in a 2 mL volumetric flask, dilute with water, and shake well to prepare a series of mixed reference standard solutions. Inject and analyze according to the chromatographic conditions of Example 1, and record the chromatogram. Draw a standard curve with the mass concentration (ρ) of each mixed reference standard solution as the abscissa and the peak area (A) as the ordinate, and perform linear regression analysis. The results are shown in Table 3.

[0043] Table 3 Linear regression equation and correlation coefficient

[0044] 1.2 Instrument precision test Take the mixed reference solution and repeat the injection 6 times according to the chromatographic conditions of Example 1, record the chromatographic peak area, and measure the RSD of the chromatographic peak area of ​​uracil, hypoxanthine, uridine, guanosine and inosine to be 0.2%, 0.6%, 0.5%, 0.1% and 0.2% (n=6), respectively, indicating that the instrument has good precision.

[0045] 1.3 Repeatability test From the same batch (SB210601) of velvet antler injection, 6 test solutions were prepared in parallel according to the preparation method of the test solution in Example 1. The samples were injected and analyzed according to the chromatographic conditions, and the chromatographic peak areas were recorded. The RSDs of the contents of uracil, hypoxanthine, uridine, guanosine and inosine were measured to be 0.1%, 0.6%, 0.3%, 0.5% and 0.3% (n = 6), respectively, indicating that the method had good repeatability.

[0046] 1.4 Stability test The same batch of pilose antler injection (SB210601) was used to prepare the test solution according to the preparation method of Example 1. According to the chromatographic conditions, the samples were injected and analyzed at 0, 2, 4, 6, 8, 10, 12, and 24 h, and the chromatographic peak areas were recorded. The RSDs of the chromatographic peak areas of uracil, hypoxanthine, uridine, guanosine, and inosine were 0.1%, 0.2%, 0.5%, 0.4%, and 0.5%, respectively, indicating that the above five components in the test solution were stable within 24 h at room temperature.

[0047] 1.5 Durability test The same batch of pilose antler essence injection (SB210601) was used to prepare the test solution according to the preparation method of the test solution in Example 1, and the flow rate was changed (0.6 mL·min -1 、1mL·min -1 ), injection volume (8μL, 12μL), column temperature (35 ℃, 40 ℃), mobile phase pH (2.40, 2.55), column (Ultimate AQ-C 18 , 4.6×250 mm, 5μm; Uenusil MP-C 18 , 4.6×250 mm, 5μm; Sepax HP-C 18 The sample was injected and analyzed, and the chromatographic peak areas were recorded. The RSDs of the chromatographic peak areas of uracil, hypoxanthine, uridine, guanosine and inosine under each condition were all below 3% (n = 6), indicating that the method had good durability.

[0048] 1.6 Recovery test Take 9 portions of the sample (SB210601) of deer antler essence injection with known content, accurately draw 500 μL of each portion, accurately add appropriate amount of mixed reference substance stock solution equivalent to 50%, 100% and 150% of uracil, hypoxanthine, uridine, guanosine and inosine content, 3 portions for each concentration, prepare sample solutions according to the preparation method of the test solution in Example 1, shake well, filter with 0.22 μm microporous filter membrane, inject and analyze according to the chromatographic conditions in Example 1, and calculate the recovery rates of uracil, hypoxanthine, guanosine, guanosine and inosine respectively. The results are shown in Table 4. The average recovery rates of uracil, hypoxanthine, uridine, guanosine and inosine are 95.5%, 94.1%, 101.3%, 101.3% and 98.9%, respectively, and the RSDs are 1.0%, 1.4%, 0.8%, 1.5% and 1.7% (n = 9), respectively, indicating that the method has good accuracy.

[0049] Table 4 Recovery rate test results (n = 9)

[0050] Comparative Example 1 The difference between this comparative example and Example 1 is that mobile phase A is acetonitrile, mobile phase B is 0.2% formic acid water, and other conditions are the same as in Example 1.

[0051] Separation results are shown in Figure 2 , the five component peaks are inseparable.

[0052] Comparative Example 2 The difference between this comparative example and Example 1 is that the gradient elution program is shown in Table 5, and the other conditions are the same as Example 1.

[0053] Table 5 Gradient elution program

[0054] Separation results are shown in Figure 3 , the five component peaks are inseparable.

[0055] Comparative Example 3 The difference between this comparative example and Example 1 is that the gradient elution program is shown in Table 6, and the other conditions are the same as Example 1.

[0056] Table 6 Gradient elution program

[0057] Separation results are shown in Figure 4 , the five component peaks are inseparable.

[0058] Example 2 Sample content determination Accurately weigh deer antler samples 1-1 (0.1051 g), 1-2 (0.1023 g), 1-3 (0.1049 g), 2 (0.1085 g), and 3 (0.1020 g) to prepare test solutions according to the preparation method of the test solution in Example 1. The samples were injected and analyzed under the chromatographic conditions of Example 1. The contents of uracil, hypoxanthine, uridine, guanosine, and inosine were calculated by peak area using the external standard method. The results are shown in Table 7.

[0059] Table 7 Contents of five nucleosides in velvet antler (n = 3, mg g -1 )

[0060] Twelve batches of velvet antler injection were taken, and test solutions were prepared according to the preparation method of the test solution in Example 1. The samples were injected and analyzed under the chromatographic conditions of Example 1. The contents of uracil, hypoxanthine, uridine, guanosine and inosine were calculated by peak area using the external standard method. The results are shown in Table 8.

[0061] Table 8 Contents of five nucleosides in Lutongjing injection (n = 3, mg mL -1 )

[0062] Through the experiment of the present invention, it is known that the contents of each nucleoside component of batch number SB210601 in the above 12 batches of injection are relatively high. It is recommended that the content of uracil in the deer antler essence injection shall not be less than 0.0191 mg / mL, the content of hypoxanthine shall not be less than 0.0130 mg / mL, the content of uridine shall not be less than 0.0141 mg / mL, the content of guanosine shall not be less than 0.0076 mg / mL, and the content of inosine shall not be less than 0.0118 mg / mL, which is more conducive to exerting the corresponding effects.

[0063] The above is a further description of the present invention in conjunction with specific embodiments, but these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Claims

1. A method for detecting nucleoside components in velvet antler preparations, characterized in that: The steps include: S1. Prepare the test solution: S2. preparing a reference solution, wherein the reference solution includes uracil, hypoxanthine, uridine, guanosine and inosine; S3, preparing blank solvent; S4, respectively taking the test solution prepared in step S1, the reference solution prepared in step S2 and the blank solvent prepared in step S3, and injecting them into a high performance liquid chromatograph; The chromatographic conditions of the high performance liquid chromatography are: Mobile phase: Mobile phase A and mobile phase B were used, wherein mobile phase A was 0.1-0.3% formic acid water and mobile phase B was acetonitrile; Elution: Gradient elution, the elution program is: Detection wavelength: 254nm.

2. The detection method according to claim 1, characterized in that: The velvet antler preparation includes velvet antler and velvet antler essence injection; The preparation method of the test solution in step S1 is: crushing the antler, adding water for ultrasonic treatment, separating the solid and liquid, and filtering the supernatant; Or you can filter the velvet antler essence injection.

3. The detection method according to claim 2, characterized in that: The ultrasonic treatment time is 20-40 min.

4. The detection method according to claim 2, characterized in that: The preparation method of the test solution is as follows: crush the antler, take 0.05-2g, add 1-4mL of water, perform ultrasonic treatment for 20-40min, centrifuge, take the supernatant and filter.

5. The detection method according to claim 1, characterized in that: The preparation method of the reference solution in step S2 is: dissolving uracil, hypoxanthine, uridine, guanosine and inosine in methanol.

6. The detection method according to claim 5, characterized in that: The volume fraction of the methanol is 5-15%.

7. The detection method according to claim 5, characterized in that: The concentration of uracil in the reference solution is 85-96 mg / mL, the concentration of hypoxanthine is 70-90 mg / mL, the concentration of uridine is 65-76 mg / mL, the concentration of guanosine is 45-60 mg / mL, and the concentration of inosine is 30-40 mg / mL.

8. The detection method according to claim 1, characterized in that: The blank solvent in step S3 is pure water.

9. The detection method according to any one of claims 1 to 8, characterized in that: The chromatographic conditions in step S4 further include: Column: Ultimate AQ-C 18 、Uenusil MP-C 18 or Sepax HP-C 18 ; Flow rate: 0.6-1mL·min -1 ; Column temperature: 35-45°C; Injection volume: 8-12μL.

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