Detection method of antiviral components and its application

Abstract

The present invention discloses a method for detecting an antiviral component and an application thereof. The detection method can detect five antiviral components, namely, monolavir, oseltamivir, daclatasvir, nematevir and ritonavir, at one time, with high sensitivity, strong specificity and good repeatability. The detection limit concentration of the five representative components is about 0.3 μg / ml, and the quantitative limit concentration is about 1 μg / ml. In addition, the recovery rate and the recovery of the spiked product are good. The method is simple and suitable for the rapid detection of antiviral components in medicines, is beneficial to the supervision and inspection of antiviral medicines, ensures the drug safety of consumers, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of drug analysis and detection, and in particular relates to a detection method for antiviral components and an application thereof. Background Art

[0002] The only existing standards for nematevir and monoclavir are the National Medical Products Administration's drug registration standard JX20220014. Liquid chromatography is used to detect all other ingredients. Ritonavir can also be detected by liquid chromatography-mass spectrometry, while oseltamivir can also be detected by liquid chromatography-mass spectrometry, Raman spectroscopy, and nuclear magnetic resonance. To ensure consumer medication safety, the development of multi-component rapid testing methods suitable for grassroots organizations is imperative. Therefore, this paper uses the lower-cost and more universal liquid chromatography method to develop rapid testing methods for five antiviral ingredients. Summary of the Invention

[0003] The purpose of the present invention is to provide a method and application thereof for detecting five antiviral ingredients, namely, mononavir, oseltamivir, daclatasvir, namatevir and ritonavir, at one time.

[0004] The technical solution adopted by the present invention is:

[0005] The first aspect of the present invention provides a method for detecting antiviral components, comprising the following steps: preparing a test solution and a reference solution, and detecting the test solution and the reference solution by high performance liquid chromatography; the antiviral drug comprises at least one of monolavir, oseltamivir, daclatasvir, nematevir and ritonavir.

[0006] Preferably, the solvent of the test sample is methanol.

[0007] Preferably, the elution mode in the high performance liquid chromatography is gradient elution, the mobile phase A is a formic acid aqueous solution, and the mobile phase B is acetonitrile.

[0008] Preferably, the pH of the formic acid aqueous solution is 2.5 to 3.5.

[0009] Preferably, the gradient elution procedure is:

[0010]

[0011] Preferably, the column temperature during the HPLC detection process is 33-38°C.

[0012] Preferably, the flow rate during the high performance liquid chromatography detection process is 1 to 2 mL / min.

[0013] Preferably, the detection wavelength during the high performance liquid chromatography detection process is 200 to 230 nm.

[0014] Preferably, the scanning wavelength range during the high performance liquid chromatography detection process is 200-400 nm.

[0015] Preferably, the chromatographic column used in the high performance liquid chromatography detection process is a C18 chromatographic column.

[0016] Preferably, the chromatographic column used in the high performance liquid chromatography detection process is an AGILENT Poroshell 120 EC-C18 chromatographic column.

[0017] Preferably, the preparation method of the test sample is: adding methanol to the test sample, ultrasonically extracting, fixing the volume, and filtering.

[0018] Preferably, the ratio of the mass of the test sample to the volume of methanol is (0.1-0.3) g / 10 ml.

[0019] Preferably, the frequency of the ultrasonic extraction is 30 to 40 kHz, and the time of the ultrasonic extraction is 10 to 20 minutes.

[0020] Preferably, the pore diameter of the filter membrane during filtration is 0.2 to 0.25 μm.

[0021] The second aspect of the present invention provides the use of the detection method described in the first aspect of the present invention in the quality detection and / or quality control of antiviral drugs; the antiviral drugs include at least one of mononavir, oseltamivir, daclatasvir, nematevir and ritonavir.

[0022] The beneficial effects of the present invention are:

[0023] The present invention provides a liquid chromatography method with higher universality, which can detect five antiviral ingredients, namely, monolavir, oseltamivir, daclatasvir, namatevir and ritonavir, at one time, with high sensitivity, strong specificity and good repeatability. The detection limit concentration of the five representative ingredients is about 0.3 μg / ml, and the quantification limit concentration is about 1 μg / ml. In addition, the recovery rate and the recovery of the spiked substance are good. The method is simple and suitable for the rapid detection of antiviral ingredients in medicines, is beneficial to the supervision and inspection of antiviral drugs, ensures the drug safety of consumers, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The following are the spectra of 5 reference substances: 1 is mononavir, 2 is oseltamivir, 3 is daclatasvir, 4 is namatevir, and 5 is ritonavir (25µg / ml).

[0025] Figure 2 This is the representative sample spectrum of sample No. 11, Monolavin.

[0026] Figure 3This is a representative sample spectrum of sample No. 2, oseltamivir.

[0027] Figure 4 This is a representative sample spectrum of sample No. 4, daclatasvir.

[0028] Figure 5 This is the representative sample spectrum of sample No. 6, Namatevir.

[0029] Figure 6 This is a representative sample spectrum of sample No. 6, ritonavir.

[0030] Figure 7 for Figure 1 UV spectrum of Monovac (25 μg / ml).

[0031] Figure 8 for Figure 1 UV spectrum of oseltamivir in 25μg / ml.

[0032] Figure 9 for Figure 1 UV spectrum of daclatasvir in 25μg / ml.

[0033] Figure 10 for Figure 1 UV spectrum of Nematevir (25 μg / ml).

[0034] Figure 11 for Figure 1 UV spectrum of ritonavir in 25μg / ml. DETAILED DESCRIPTION

[0035] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0036] Experimental instruments and materials

[0037] Mononavir, oseltamivir, daclatasvir, nematevir and ritonavir were determined by high performance liquid chromatography (General Chapter 0512 of the 2020 edition of the Chinese Pharmacopoeia).

[0038] High-performance liquid chromatograph: Shimadzu LC-20AT (with DAD detector); electronic balance: METTLER XPE205; pH meter: Sartorius PB-10; sonicator: Elma TI-H 10MF3.

[0039] Chromatographic column: AGILENT Poroshell 120 EC-C18 (50 mm × 4.6 mm, 2.7 μm or equivalent); diode array detector, detection wavelength: 210 nm, scanning wavelength range: 200–400 nm; column temperature: 35°C; flow rate: 1.5 ml / min.

[0040] Reference substances: Information on the reference substances of the five ingredients is shown in Tables 1 and 2.

[0041] Reagents: methanol (chromatographic grade, Merck), acetonitrile (chromatographic grade, Merck), formic acid (chromatographic grade, Maclean).

[0042] Test samples: 11 batches of samples were collected for supervisory inspection. Detailed information on the test samples is shown in Table 3.

[0043] Table 1 Reference substance information

[0044]

[0045] Table 2

[0046]

[0047] Table 3 Test sample information

[0048]

[0049] Example 1 Determination of chromatographic conditions

[0050] 1. Selection of mobile phase

[0051] To better ensure the quality control of nematevir and monoclavir, this study optimized the target component analysis with reference to the existing nematevir registration standard JX20220014. The analytical conditions for nematevir content determination in this standard were similar to those for its degradation products. The reference and test samples were extracted with acetonitrile-water (50:50), using a phenyl column (150 mm × 2.1 mm, 1.8 μm or equivalent) and a mobile phase of 0.05% formic acid in 0.05% formic acid-acetonitrile at a flow rate of 0.475 ml / min and a column temperature of 75°C. These conditions were primarily intended to better separate nematevir from its degradation products. However, the column pressure was high, the column temperature approached the instrument limit, and the analysis time was long, making them unsuitable for the separation, inspection, and content determination of multiple components in this study.

[0052] To make the method more universally applicable at the grassroots level, this experiment directly modified the chromatographic column to a core-shell C18 column (50 mm × 4.6 mm, 2.7 μm or equivalent), more suitable for rapid liquid phase analysis. The column temperature was adjusted to the more commonly used 35°C. An attempt to optimize the mobile phase using the simplest acetonitrile-water mobile phase resulted in poor peak shapes for oseltamivir and daclatasvir, making them unsuitable for quantitative analysis. An attempt to optimize the mobile phase by adjusting the pH of the water to 3.0 with formic acid significantly altered the peak shapes, and adjusting the gradient enabled complete separation of the five components. Therefore, mobile phase A was selected for this experiment as a pH 3.0 formic acid-water mobile phase, and mobile phase B was acetonitrile.

[0053] 2. Optimization of gradient conditions

[0054] The gradient conditions in the registration standard are relatively complicated and the analysis time is long. This experiment attempts to optimize the gradient conditions. Under the selected mobile phase conditions, monolavir elutes earlier, while namatevir and ritonavir require a larger organic phase for elution, requiring a larger ratio change in the middle. The initial conditions use a smaller organic phase ratio to ensure that monolavir can be well retained and separated from the solvent peak, then use a slower ratio change to ensure that oseltamivir and daclatasvir can be completely separated, and finally use a larger ratio change to make namatevir and ritonavir elute as early as possible; the gradient elution program is shown in Table 4;

[0055] Table 4 Gradient elution

[0056]

[0057] 3. Solution preparation

[0058] Reference solution: Accurately weigh approximately 10 mg of each of the five reference substances into a 10 ml volumetric flask, dilute to the mark with methanol, and shake well to obtain approximately 1000 μg / ml reference substance stock solution. Accurately measure 1 ml of each reference substance stock solution into the same 10 ml volumetric flask, dilute to the mark with methanol, and shake well to obtain approximately 100 μg / ml mixed reference substance solution.

[0059] The spectra of oseltamivir and nematevir suggest terminal absorption only around 200 nm. Initially, extraction with the registered standard 50% acetonitrile was considered to reduce interference from terminal absorption, but ritonavir was found to be poorly soluble in 50% acetonitrile. An attempt to optimize the extraction solvent to methanol revealed that all components were soluble, and the detection limits for oseltamivir and nematevir met experimental requirements. Therefore, methanol was selected as the extraction solvent for this experiment.

[0060] Test solution: Based on the clinical dosage, tablet weight, uniformity, and solubility of each component, accurately weigh approximately 0.2 g of the test sample into a 10 ml volumetric flask. Add an appropriate amount of methanol and sonicate for 15 minutes. Cool to room temperature, dilute to the mark with methanol, shake well, and filter. The solution may be diluted with methanol to within the linear range based on the actual concentration.

[0061] 4. Determination of detection wavelength

[0062] Of the five antiviral ingredients, oseltamivir and nematevir only exhibit terminal absorption near 200 nm, while the other ingredients all exhibit characteristic absorption. Considering the effects of baseline and detection limit, 210 nm was selected as the detection wavelength in this experiment.

[0063] 5. Determination: 10 μl of each reference solution and test solution were injected into the liquid chromatograph and the chromatogram and DAD spectrum were recorded. The integrated peak area of ​​the liquid chromatogram was substituted into the fitting equation to calculate the content of the target component in the test sample.

[0064] 6. Result judgment: If a chromatographic peak appears in the chromatogram of the test sample with the same retention time and DAD spectrum (220-400nm) as the reference sample chromatogram, it can be determined that the corresponding ingredient has been added to the test sample.

[0065] Effect Example 1

[0066] 1. Linear range

[0067] Accurately weigh approximately 20 mg of each reference substance into a 20 ml volumetric flask. Dissolve and dilute to the mark with methanol, shake well, and prepare the reference solution (concentration approximately 1000 μg / ml). Accurately measure 10 ml of the reference solution into a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain a 100 μg / ml linear reference solution. Accurately measure 5 ml, 5 ml, 1 ml, and 1 ml of the 100 μg / ml reference solution into 10 ml, 20 ml, 10 ml, and 20 ml volumetric flasks, respectively, dilute to the mark with methanol, and shake well to obtain 50 μg / ml, 25 μg / ml, 10 μg / ml, and 5 μg / ml linear reference solutions. Accurately measure 1 ml of the 10 μg / ml reference solution into a 10 ml volumetric flask, dilute to the mark with methanol, and shake well to obtain a 1 μg / ml linear reference solution. Inject the reference solution and linear reference solution and measure the peak area. Linear regression was performed using the peak area y versus the injected concentration x. The regression equation and concentration range are shown in the table below. The peak area and concentration showed good linearity (see Table 5).

[0068] Table 5 Regression equations and concentration ranges of the five components

[0069]

[0070] 2. Repeatability

[0071] The 100 μg / ml reference solution was injected six times continuously. The peak areas, retention times and RSD% of the five components are shown in Table 6, indicating good repeatability.

[0072] Table 6 Repeatability test results of 5 representative ingredients

[0073]

[0074] 3. Solution stability

[0075] A portion of the reference solution was taken and injected at 0, 2, 4, 8, 12, 16 and 24 hours. The peak areas and RSDs of the five components are shown in Table 7, indicating that the solutions of each component were stable for at least 24 hours.

[0076] Table 7 8 representative component stability test results

[0077]

[0078] 4. Limit of detection and limit of quantification

[0079] 0.2 g of starch was placed in a 10 ml volumetric flask, and an appropriate amount of the reference solution was added. The sample was serially diluted with methanol. Based on a signal-to-noise ratio of 3:1, the detection limit for the five representative components was approximately 0.3 μg / ml. The detection limits were as follows: monolavir (approximately 2.9 ng), oseltamivir (approximately 3.0 ng), daclatasvir (approximately 3.0 ng), nematevir (approximately 3.0 ng), and ritonavir (approximately 3.0 ng). The quantification limits for the five components were approximately 1 μg / ml.

[0080] 5. Recovery rate and spike recovery

[0081] Because sample collection was difficult, available quantities were limited, and the matrices varied, starch was used as a blank matrix to investigate the recoveries of each component. Two samples of each of the four components with significant sample quantities (oseltamivir, daclatasvir, namatevir, and ritonavir) were spiked. The recoveries and spike recovery rates were good.

[0082] About 0.2 g of starch was accurately weighed and placed in 18 10 ml volumetric flasks. Six portions of the appropriate reference solution were added according to the limit of quantification, 2 times the limit of quantification, and 10 times the limit of quantification. An appropriate amount of methanol was added first, and the mixture was ultrasonically treated for 15 minutes. The mixture was cooled to room temperature, diluted to the mark with methanol, shaken, and filtered. The content was determined according to the proposed method. The recovery rates of the five components were as follows: the average recoveries of the three concentrations of monoclavir were 104.3%, 100.8%, and 100.5%, respectively, with RSDs of 0.5%, 0.5%, and 0.4% (n=6); the average recoveries of the three concentrations of oseltamivir were 98.4%, 97.6%, and 101.1%, respectively, with RSDs of 0.6%, 0.9%, and 0.5% (n=6); and the average recoveries of the three concentrations of daclatasvir were 10. The average recoveries of the three concentrations of namatevir were 101.0%, 100.5%, and 100.1%, with RSDs of 1.0%, 0.9%, and 0.4%, respectively (n=6); the average recoveries of the three concentrations of ritonavir were 96.6%, 96.1%, and 100.4%, with RSDs of 1.3%, 1.5%, and 0.6%, respectively (n=6). See Tables 8 to 12 for details.

[0083] Table 8 Monolavine recovery test results

[0084]

[0085] Table 9 Oseltamivir recovery test results

[0086]

[0087] Table 10 Daclatasvir recovery test results

[0088]

[0089] Table 11 Results of the recovery test of Namatevir

[0090]

[0091] Table 12 Ritonavir recovery test results

[0092]

[0093] Accurately weigh approximately 0.1 g of each of the Namatevir tablets (actually containing oseltamivir) labeled in test sample No. 2, the Namatevir tablets (actually containing daclatasvir) labeled in test sample No. 4, the Namatevir tablets (actually containing daclatasvir) labeled in test sample No. 6, and the ritonavir tablets labeled in test sample No. 6, and place them in 10 ml volumetric flasks respectively. Add appropriate amount of reference solution according to the ratio of test sample content to preparation concentration of approximately 1:1, and prepare according to the selected test sample preparation method. The content of the four components was determined according to the proposed method. The results of the spiked samples were as follows: the average spiked sample recovery for oseltamivir was 104.5% with an RSD of 0.5% (n=4); the average spiked sample recovery for daclatasvir was 101.4% with an RSD of 0.7% (n=4); the average spiked sample recovery for namatevir was 100.7% with an RSD of 0.5% (n=4); and the average spiked sample recovery for ritonavir was 96.8% with an RSD of 1.0% (n=4). See Tables 13 to 16 for details.

[0094] Table 13 Results of oseltamivir spiked test

[0095]

[0096] Table 14 Daclatasvir spiked test results

[0097]

[0098] Table 15 Results of the Namatevir spiked test

[0099]

[0100] Table 16 Results of ritonavir spiked test

[0101]

[0102] 6. Exclusivity

[0103] In the liquid chromatogram, the blank solvent has no interference and the components can be basically completely separated.

[0104] 7. Spectra of reference substances and representative samples

[0105] The sample test results are shown in Table 17, and the reference samples of the five components are shown in Figure 1 , Figure 7-11 It is the 25μg / ml mixed standard solution in the "Preparation of Reference Solution" in the standard text (i.e. Figure 1 ) The 200-400nm UV spectrum of each component chromatographic peak corresponding to the chromatographic peak is used together with the retention time of the chromatographic peak to determine whether the suspicious sample contains the target component. The representative positive sample spectrum is shown in Figures 2 to 6 .

[0106] Table 17 Sample test results

[0107]

[0108] The above specific embodiments provide a detailed description of the present invention. However, the present invention is not limited to the above embodiments. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with each other unless there is a conflict.

Claims

1. A method for detecting an antiviral component, comprising the following steps: Prepare test solution and reference solution, and detect the test solution and reference solution by high performance liquid chromatography; The elution mode in the high performance liquid chromatography is gradient elution, the mobile phase A is formic acid aqueous solution, and the mobile phase B is acetonitrile; The antiviral ingredients include mononavir, oseltamivir, daclatasvir, namatevir and ritonavir, and methanol is used as the extraction solvent; the pH of the formic acid aqueous solution is 2.5-3.5; the detection wavelength during the high-performance liquid chromatography detection process is 200-230nm; the chromatographic column during the high-performance liquid chromatography detection process is a 120 EC-C18 chromatographic column; The procedure of the gradient elution is: 。 2. The detection method according to claim 1, wherein The column temperature during the high performance liquid chromatography detection process is 33-38°C.

3. The detection method according to claim 1, wherein The flow rate during the high performance liquid chromatography detection process is 1 to 2 mL / min.

4. The detection method according to claim 1, wherein The scanning wavelength range of the high performance liquid chromatography detection process is 200-400 nm.

5. The detection method according to claim 1, wherein The preparation method of the test sample is as follows: adding methanol to the test sample, ultrasonically extracting, fixing the volume, and filtering.

6. The detection method according to claim 5, characterized in that The ratio of the mass of the test sample to the volume of methanol is (0.1-0.3) g / 10 ml.

7. The detection method according to claim 5, characterized in that The frequency of the ultrasonic extraction is 30 to 40 kHz, and the time of the ultrasonic extraction is 10 to 20 minutes.

8. The detection method according to claim 5, characterized in that The pore diameter of the filter membrane during the filtration is 0.2 to 0.25 μm.

9. Use of the detection method according to any one of claims 1 to 8 in the quality detection and / or quality control of antiviral drugs; the antiviral ingredients include mononavir, oseltamivir, daclatasvir, nematevir and ritonavir.

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