Method for detecting content of compound selamectin drops

Abstract

The invention provides a method for detecting the content of compound selamectin drops, which adopts high performance liquid chromatography for detection, and comprises the following specific steps: sequentially preparing a blank solution, a blank auxiliary material solution, a contrast solution and a test solution, sequentially and respectively injecting the blank solution, the blank auxiliary material solution, the contrast solution and the test solution into a high performance liquid chromatograph, an octadecyl silane bonded silica gel column is used as a chromatographic column, a formic acid solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and gradient elution is carried out. According to the method disclosed by the invention, the three active ingredients, namely the selamectin, the fluralan and the praziquantel, in the compound selamectin drops can be effectively detected, the quality detection result is accurate and reliable, and the three active ingredients in the compound selamectin drops can be accurately quantified, so that the product quality of the compound selamectin drops can be better controlled.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, and in particular relates to a method for detecting the content of compound seracridine drops. Background Technology

[0002] Selamectin is a new generation of macrolide antiparasitic drugs, belonging to the same class as ivermectin, doramectin, and acetaminophen. It is an antibiotic specifically for animals, primarily used to treat infections of roundworms, hookworms, scabies mites, fleas, and lice in dogs and cats, and can also prevent heartworm. Selamectin can also activate glutamate-gated chloride channels in neurons and pharyngeal muscles to prevent infections of filariasis, lymphatic filariasis, and nematodes. It is also an effective substrate and inhibitor of P-glycoprotein, with an IC50 of 120 nM. Its safety profile has been greatly improved, with good efficacy both orally and by injection. It is an in vitro and in vivo insecticide primarily targeting adult fleas, filarial worms, and scabies in pet cats and dogs.

[0003] Fluranar primarily exerts its effects by interfering with GABA-gated chloride ion channels, similar to the target mechanisms of cyclopentadiene, phenylpyrazole, and macrolide insecticides. Fluranar is a broad-spectrum insecticide with good insecticidal activity against ticks, fleas, lice, hemiptera, and dipterans, exhibiting toxicity higher than or comparable to commonly used insecticides. Fluranar not only shows no significant cross-resistance with existing insecticides but also demonstrates good insecticidal activity against some resistant pests.

[0004] Fluranar is a gamma-aminobutyric acid (GABA)-gated chloride channel blocker that primarily acts on the nervous system of insects. It causes symptoms of hyperexcitability and convulsions in insects by blocking GABA-activated chloride channels. GABA plays a crucial role in the transmission of nerve impulses in insects, acting as a major inhibitory neurotransmitter and widely distributed throughout the central nervous system. GABA receptors are ligand-gated ion channel receptors and are also the primary targets of insecticides such as cyclopentadienes and avermectins. Unlike traditional insecticides, fluranar not only kills pests through stomach poisoning but also has contact action and a longer residual effect on pests.

[0005] Currently, the content detection methods in the quality standards for selamectin raw materials and preparations listed in pharmacopoeias of various countries are only applicable to selamectin raw materials. The content detection method in the announcement of selamectin solutions published by the Chinese Ministry of Agriculture and Rural Affairs is only applicable to the determination of content in selamectin single-component solutions. These methods cannot effectively detect components such as fluranal and praziquantel contained in selamectin compound preparations, and therefore cannot achieve rapid quantification. No pharmacopoeias of any country include methods for detecting the content of this selamectin compound preparation, and no relevant literature has been reported in the existing technology. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art and provide a rapid detection method for the content of compound seracramide drops. Formic acid solution is used as mobile phase A, acetonitrile as mobile phase B, and gradient elution is applied by changing the ratio of the mobile phases to detect the content of the three components in the compound seracramide drops.

[0007] The technical solution of this invention is as follows:

[0008] One technical solution of the present invention is as follows:

[0009] A method for detecting the content of compound selamectin drops, wherein the compound preparation contains selamectin, fluranal, and praziquantel.

[0010] The compound selamectin drops contain one or more of selamectin, fluranal, and praziquantel, and their structures are shown in Table 1 below:

[0011] The specific steps for detecting the content of the compound seracridine drops are as follows:

[0012] (1) Prepare blank solution, blank excipient solution, control solution and test solution;

[0013] (2) Inject the blank solution, blank excipient solution, control solution and test solution into the high performance liquid chromatograph and record the chromatograms.

[0014] The conditions for the high-performance liquid chromatography are as follows:

[0015] Injection volume: 1–30 μl;

[0016] Chromatographic column: KeKao C18, 4.6mm*150mm, 3μm.

[0017] Flow rate: 0.2–1.5 ml / min;

[0018] Column temperature: 20~60℃;

[0019] Detection wavelength: 210–280 nm;

[0020] Mobile phase A is a 0.01-0.2% formic acid solution (take 0.1-2 ml of formic acid, dissolve in water and dilute to 1000 ml); mobile phase B is acetonitrile.

[0021] In the chromatogram of the mixed reference solution obtained under the chromatographic conditions described in step (2), the resolution between the peaks of selamectin, flerastatin and praziquantel should be greater than 1.5.

[0022] The gradient elution conditions are shown in Table 2:

[0023] Preferably, the flow rate is 1.5 ml / min.

[0024] Preferably, the column temperature is 40°C.

[0025] Preferably, the detection wavelength is 210 nm.

[0026] Preferably, the chromatographic column is a KeKao C18 (4.6mm*150mm, 3μm).

[0027] Preferably, the concentration of formic acid solution in the mobile phase A is 0.1%.

[0028] Preferably, the mobile phase B contains acetonitrile.

[0029] Preferably, the gradient elution conditions are as shown in Table 3:

[0030] Table 3 Gradient elution conditions

[0031] The method for detecting the content of compound selamectin drops is applied in the quality control of compound preparations containing selamectin, flurana, and praziquantel.

[0032] The compound seracramide drops include, but are not limited to, solutions, pour-over solutions, drops, tablets, suspensions, granules, and capsules.

[0033] Preferably, the compound seracromycin drops are in the form of drops.

[0034] This invention utilizes high-performance liquid chromatography (HPLC) to analyze and detect the content of compound ceramsite drops, with the following beneficial effects:

[0035] 1. These chromatographic conditions allow for the simultaneous separation of three components: selamectin, fluranal, and praziquantel, with each component well separated.

[0036] 2. The mobile phase gradient, flow rate, and column temperature of this invention can cause three main components with inconsistent maximum absorption wavelengths and large polarity differences to elute simultaneously on the chromatographic column, effectively separating them, saving detection costs, and improving detection efficiency.

[0037] 3. The instrument uses a high-performance liquid chromatography DAD detector, which can perform detection across the entire wavelength range.

[0038] 4. Select KeKao C18 (4.6mm*150mm, 3μm) chromatographic column. This particle size column has strong separation effect, good separation of each component of the sample, and good column stability.

[0039] 5. Choosing 210 nm as the detection wavelength allows for the simultaneous detection of selamectin, fluranal, and praziquantel, which have significantly different absorption wavelengths, ensuring effective separation of all components. At this wavelength, each component exhibits high sensitivity and can be accurately quantified. Attached Figure Description

[0040] Figure 1 UV absorption spectrum of selamectin solution in Example 1;

[0041] Figure 2 UV absorption spectrum of fluranar solution in Example 1;

[0042] Figure 3 UV absorption spectrum of praziquantel solution in Example 1;

[0043] Figure 4 HPLC chromatogram of the sample solution (265 nm) in Example 1;

[0044] Figure 5 HPLC chromatogram of the sample solution (244 nm) in Example 1;

[0045] Figure 6 HPLC chromatogram of the sample solution (210 nm) in Example 1; Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described clearly and completely below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0047] Method establishment

[0048] Detection wavelength determined

[0049] The reference solutions of selamectin, fluranal, and praziquantel were scanned in the full wavelength range under a DAD detector. Figures 1-3 The results analysis is shown in Table 4.

[0050] Table 4

[0051] Inconsistent responses and large measurement errors

[0052] Based on existing publicly available information, experiments were conducted to screen and optimize the results, as detailed in Table 5.

[0053] Table 5

[0054] Blank solution: Acetonitrile is the blank solution.

[0055] Blank excipient solution: Accurately weigh an appropriate amount of blank excipient and quantitatively dilute it with acetonitrile to prepare a solution containing 0.15 mg / ml.

[0056] Mixed reference solution: appropriate amounts of selamectin, flurana, and praziquantel reference standards were dissolved and diluted with acetonitrile to prepare a solution containing approximately 0.15 mg of each of the selamectin, flurana, and praziquantel reference standards per 1 ml.

[0057] Sample solution: Take an appropriate amount of the test sample, dissolve and dilute it with acetonitrile to prepare a solution containing approximately 0.15 mg of selamectin, flerostatin and praziquantel reference standards per 1 ml.

[0058] Accurately measure blank solution, blank excipient solution, control solution, and test solution and inject them into the high-performance liquid chromatograph for detection using the methods in Table 2. Finally, after optimization, the following parameters and methods are adopted.

[0059] The determination was performed according to the high performance liquid chromatography method described in Appendix 0512 of the 2020 edition of the Chinese Veterinary Pharmacopoeia.

[0060] Reversed-phase high-performance liquid chromatography conditions:

[0061] A KeKao C18 (4.6 mm * 150 mm, 3 μm) column was used. The flow rate was 1.5 ml / min; the column temperature was 40℃; the detection wavelength was 210 nm; and the gradient elution conditions are shown in Table 6.

[0062] Table 6 Gradient elution conditions

[0063] Mobile phase A: 0.1% formic acid solution (take 1 ml of formic acid, dissolve in water and dilute to 1000 ml);

[0064] Mobile phase B: Acetonitrile;

[0065] Injection volume: 20 μl.

[0066] Conclusion: Under the chromatographic conditions of this detection method, the main peaks of each component can be separated well.

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting the content of active ingredient in compound seracramide drops, characterized in that, High-performance liquid chromatography (HPLC) with a DAD detector was used to sequentially elute peaks from a mixed solution of three active ingredients at specific wavelengths and in a specific mobile phase. Although the three active ingredients have different maximum absorption wavelengths and different compound polarities, they can share a single acquisition method, which can save acquisition time and reduce detection costs. The chromatographic conditions are as follows: Column: Octadecylsilane-bonded silica gel column; Mobile phase A: 0.01–0.2% formic acid solution; Mobile phase B: Acetonitrile; Flow rate: 0.2–1.5 ml / min; Column temperature: 20~60℃; Detection wavelength: 210–280 nm; The gradient elution conditions are as follows:

2. The detection method according to claim 1, characterized in that, The gradient elution conditions are as follows:

3. The detection method according to claim 1 or 2, characterized in that, The active ingredients selamectin, fluranal, and praziquantel in the compound selamectin drops have the following structures:

4. The detection method according to claim 1 or 2, characterized in that, The formic acid solution in the mobile phase A is prepared by taking 0.1-2 ml of formic acid, dissolving it in water, and diluting it to 1000 ml.

5. The detection method according to claim 1 or 2, characterized in that, The chromatographic column was a KeKao C18, 4.6mm*150mm, 3μm.

6. The detection method according to claim 1 or 2, characterized in that, The concentration of formic acid solution in the mobile phase A is 0.1%.

7. The detection method according to claim 1 or 2, characterized in that, The acetonitrile content in the mobile phase B is 45-65.

8. The detection method according to claim 7, characterized in that, The acetonitrile content in mobile phase B is 55.

9. The detection method according to claim 1 or 2, characterized in that, The high-performance liquid chromatography method is performed under the following conditions: Flow rate: 1.5 ml / min; Column temperature: 40℃; Detection wavelength: 210nm.

10. The application of the detection method according to any one of claims 1-9 in the quality control of compound seracramide drops.

Images