Compound levamisole oral liquid as well as preparation method and content determination method thereof

Abstract

The invention discloses a compound levamisole oral liquid. Each 100ml of the compound levamisole oral liquid contains 0.5 to 4.5 g of levamisole hydrochloride, 0.5 to 4.5 g of hydroxyclozanide, 5 to 20g of niclosamide, 0.5 to 4.5 g of albendazole, 0.5 to 2.5 g of sodium carboxymethyl cellulose, 0.05 to 0.1 g of sodium hydrogen sulfite, 0.001 to 0.01 g of ethylparaben, 0.01 to 0.1 g of propylene glycol and the balance of water. The compound levamisole oral liquid is advanced and reasonable in prescription design, has the effects of increasing the insect-resistant spectrum, reducing the administration dosage and the like, only needs to repel insects twice a year, and greatly reduces the labor cost of animal husbandry. The invention also provides a content determination method of the compound levamisole oral liquid, the method is strong in specificity, good in precision and high in accuracy, the contents of levamisole hydrochloride, hydroxyclozanide, niclosamide and albendazole can be simultaneously determined, a rapid detection method can be provided for veterinary drug production, and development of animal husbandry is jointly promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparations and drug detection, and relates to a compound levamisole oral liquid, and also relates to a preparation method and a content determination method thereof. Background Art

[0002] China's livestock farming industry is becoming more and more large-scale. Due to the complexity of the breeding environment in the vast pastoral areas, economic animals such as cattle and sheep often suffer from mixed infections of multiple parasites during the breeding process. However, due to the lack of compound preparations of anthelmintics, they can only choose to combine multiple single anthelmintics or choose broad-spectrum anthelmintics for treatment. However, the number of cattle and sheep raised in some pastoral areas is large, and most of them are managed by families. It is unrealistic to give farmed animals multiple and repeated medications, and multiple gavages consume excessive manpower and time costs.

[0003] At present, the domestic single-function anthelmintic oral products include hydroxyclozanide suspension, albendazole tablets, albendazole oral solution, niclosamide tablets, levamisole powder, levamisole tablets, etc., with a narrow anthelmintic spectrum. Foreign compound preparations include hydroxyclozanide hydrochloride levamisole suspension, while domestic compound preparations only have albendazole ivermectin premix. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a compound levamisole oral solution, which has an advanced and reasonable prescription design, can increase the anti-insect spectrum, reduce the dosage, etc., and only needs to deworm twice a year, which greatly reduces the labor cost of animal husbandry.

[0005] After research, the present invention provides the following technical solutions:

[0006] 1. Compound levamisole oral solution. Each 100 ml of the oral solution contains 0.5-4.5 g of levamisole hydrochloride, 0.5-4.5 g of hydroxyclozanide, 5-20 g of niclosamide, 0.5-4.5 g of albendazole, 0.5-2.5 g of sodium carboxymethyl cellulose, 0.05-0.1 g of sodium bisulfite, 0.001-0.01 g of ethylparaben, 0.01-0.1 g of propylene glycol, and the balance is water.

[0007] Preferably, each 100 ml of the oral solution contains 2.5 g of levamisole hydrochloride, 3.5 g of hydroxyclozanide, 5 g of niclosamide, 3.5 g of albendazole, 1 g of sodium carboxymethylcellulose, 0.05 g of sodium bisulfite, 0.003 g of ethylparaben, 0.1 g of propylene glycol, and the balance is water.

[0008] Levamisole is an imidazothiazole antinematode drug that is active against most nematodes of cattle, sheep, pigs, dogs and chickens. Its anthelmintic mechanism is to excite the parasympathetic and sympathetic ganglia of worms, showing a nicotinic effect; at high concentrations, levamisole interferes with the sugar metabolism of nematodes by blocking the reduction of fumarate and the oxidation of succinate, and ultimately paralyzes the worms, causing the live worms to excrete the product. In addition to its anthelmintic activity, levamisole can also significantly enhance the immune response. It can restore the cell-mediated immune function of peripheral T lymphocytes and excite the phagocytosis of monocytes, and its effect is more obvious on animals with impaired immune function.

[0009] Hydroxyclozanide is a salicylanilide antiparasitic drug. It mainly inhibits the oxidative phosphorylation process in the parasite, reduces the production of ATP, and reduces the glycogen content, causing the accumulation of succinic acid, thereby affecting the energy metabolism process of the parasite and causing the parasite to die.

[0010] Niclosamide is a tapeworm killer that has a good expelling effect on naked head tapeworms, leaf-shaped naked head tapeworms and dwarf naked head tapeworms in horses; it is effective against Moniezia, yolkless gland tapeworms and striped tapeworms in cattle and sheep, and has the same effect on the head and body segments of tapeworms; it is effective against large abdominal tapeworms, bean-shaped tapeworms, alveolar tapeworms and tapeworms in dogs and cats, and has a poor effect on canine granulosus tapeworms; it has an expelling effect on Riley's tapeworms and funnel-shaped tapeworms in poultry; it also has the effect of killing snails and schistosome cercariae and miracidia. Its mechanism of action is to inhibit the absorption of glucose by tapeworms, uncouple the oxidative phosphorylation process in tapeworm mitochondria, block the tricarboxylic acid cycle, lead to lactic acid accumulation, and thus kill tapeworms. The anthelmintic effect of niclosamide may also be related to its overstimulation of the activity of ATPase in mitochondria.

[0011] Albendazole is a benzimidazole with a broad-spectrum anthelmintic effect. Nematodes are sensitive to it, and it also has a strong effect on tapeworms and flukes (but requires a larger dose), but is ineffective against schistosomes. Its mechanism of action is mainly to bind to the microtubules of nematodes. After albendazole binds to β-tubulin, it prevents it from polymerizing with a-tubulin to assemble into microtubules. Microtubules are the basic structural units of many organelles and are necessary for cell reproduction processes such as mitosis, protein assembly and energy metabolism. Albendazole has a significantly higher affinity for nematode microtubules than mammalian microtubules, so it is very toxic to mammals. Albendazole not only has a strong effect on adults, but also has a strong effect on immature worms and larvae, and also has an insecticidal effect on eggs.

[0012] The invention rationally combines levamisole hydrochloride, hydroxyclozanide, niclosamide and albendazole to prepare a compound levamisole oral liquid, which can increase the anti-insect spectrum, reduce the dosage and the like, and only requires deworming twice a year, thereby greatly reducing the labor cost of animal husbandry.

[0013] 2. The preparation method of the compound levamisole oral solution comprises the following steps: mixing part of water and propylene glycol, adding sodium carboxymethyl cellulose to fully swell it, then adding sodium bisulfite, ethyl hydroxybenzoate and levamisole hydrochloride, stirring, then adding hydroxyclozanide, niclosamide and albendazole, stirring, making up to volume with water, stirring evenly, repeating homogenization 2 to 3 times under 15 to 25 MPa conditions, and obtaining the compound levamisole oral solution.

[0014] 3. The content determination method of the compound levamisole oral solution is to determine the content of levamisole hydrochloride, hydroxyclozanide, niclosamide and albendazole by high performance liquid chromatography;

[0015] (1) Chromatographic conditions

[0016] Octadecylsilane bonded silica gel was used as filler; a methanol-0.05 mol / L ammonium acetate mixture with a volume ratio of 7:3 was used as mobile phase; the detection wavelength was 250 nm;

[0017] (2) Preparation of mixed reference solution

[0018] Accurately weigh levamisole hydrochloride reference substance, hydroxyclozanide reference substance, niclosamide reference substance, and albendazole reference substance, add glacial acetic acid-acidified methanol, dissolve by ultrasound, cool, and dilute with glacial acetic acid-acidified methanol to prepare a mixed reference substance solution;

[0019] (3) Preparation of test solution

[0020] Accurately measure the compound levamisole oral solution and dilute it with methanol acidified with glacial acetic acid as the test solution;

[0021] (4) Determination method

[0022] Accurately pipette the mixed reference solution and test solution separately, inject into liquid chromatograph, record the chromatogram, and calculate the contents of levamisole hydrochloride, hydroxyclozanide, niclosamide, and albendazole in the test sample by peak area according to the external standard method.

[0023] The beneficial effects of the present invention are as follows: the present invention provides a compound levamisole oral liquid, the prescription design is advanced and reasonable, and can play the role of increasing the anti-insect spectrum, reducing the dosage, etc., and only needs to deworm twice a year, which greatly reduces the human cost of animal husbandry. The present invention also provides a content determination method of the compound levamisole oral liquid, which has strong specificity, good precision and high accuracy, and can simultaneously determine the contents of levamisole hydrochloride, hydroxyclozanide, niclosamide and albendazole, and can provide a rapid detection method for veterinary drug production, and jointly promote the development of animal husbandry. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1This is the chromatogram of compound levamisole oral solution. The chromatographic peak No. 1 is niclosamide, the chromatographic peak No. 2 is hydroxyclozanide, the chromatographic peak No. 3 is levamisole hydrochloride, and the chromatographic peak No. 4 is albendazole. DETAILED DESCRIPTION

[0025] In order to make the objectives, technical solutions and beneficial effects of the present invention more clear, the preferred embodiments of the present invention are described in detail below.

[0026] Example 1 Preparation of compound levamisole oral solution

[0027] Every 1000ml of compound levamisole oral solution contains 25g of levamisole hydrochloride, 35g of hydroxyclozanide, 50g of niclosamide, 35g of albendazole, 10g of sodium carboxymethyl cellulose, 0.5g of sodium bisulfite, 0.03g of ethylparaben, 1g of propylene glycol, and the balance is water.

[0028] Preparation method: Grind levamisole hydrochloride, hydroxyclozanide, niclosamide, albendazole and ethylparaben separately with a ball mill and set aside; add 500 mL of water and propylene glycol in a beaker, stir evenly, add sodium carboxymethyl cellulose to fully swell, then add sodium bisulfite, ethylparaben and levamisole hydrochloride, stir, then add hydroxyclozanide, niclosamide and albendazole, stir, make up to 1000 mL with water, stir evenly, use a homogenizer to repeat homogenization 3 times under 20 MPa to obtain the compound levamisole oral solution.

[0029] Example 2 Determination of the content of compound levamisole oral solution

[0030] The contents of niclosamide, hydroxyclozanide, levamisole and albendazole in compound levamisole oral solution were determined simultaneously by HPLC.

[0031] Chromatographic conditions: The chromatographic column was a C18 column (250 mm × 4.6 mm, 5 μm), the mobile phase was a methanol-0.05 mol / L ammonium acetate mixture in a volume ratio of 7:3, the detection wavelength was 250 nm, the flow rate was 1 ml / min, and the column temperature was 35 °C.

[0032] Preparation of mixed reference solution: accurately weigh 25 mg of levamisole hydrochloride reference substance, 35 mg of hydroxyclozanide reference substance, 50 mg of niclosamide reference substance, and 35 mg of albendazole reference substance into a 100 ml volumetric flask, add glacial acetic acid to acidify methanol, sonicate to dissolve, cool, dilute to scale with glacial acetic acid to acidify methanol, shake well, and use as mixed reference solution;

[0033] Preparation of test solution: accurately measure 1 ml of compound levamisole oral solution and place it in a 100 ml volumetric flask, dilute to the mark with methanol acidified with glacial acetic acid, and shake well to serve as the test solution;

[0034] Determination method: Accurately pipette 10 μl of the mixed reference solution and test solution respectively, inject into liquid chromatograph, record the chromatogram, and calculate the contents of levamisole hydrochloride, hydroxyclozanide, niclosamide, and albendazole in the test sample by peak area according to the external standard method.

[0035] The chromatogram of compound levamisole oral solution is shown in Figure 1 , among which the chromatographic peak No. 1 is niclosamide, the chromatographic peak No. 2 is hydroxyclozanide, the chromatographic peak No. 3 is levamisole hydrochloride, and the chromatographic peak No. 4 is albendazole.

[0036] Example 3 Methodological verification of content determination

[0037] 1. Exclusivity

[0038] Prepare the following 4 negative samples: negative sample lacking levamisole hydrochloride, negative sample lacking hydroxyclozanide, negative sample lacking niclosamide, and negative sample lacking albendazole. Take the 4 negative samples and prepare negative test solutions according to the preparation method of the test solution in Example 2. Accurately pipette 10 μl of each of the mixed reference solution and the 4 negative test solutions, inject and measure according to the chromatographic conditions in Example 2, and record the chromatogram.

[0039] By comparing the chromatograms of the mixed reference solution and the four negative test solutions, the Figure 1 In the chromatogram of the compound levamisole oral solution shown, chromatographic peak No. 1 is niclosamide, chromatographic peak No. 2 is hydroxyclozanide, chromatographic peak No. 3 is levamisole hydrochloride, and chromatographic peak No. 4 is albendazole.

[0040] By comparing the chromatograms of the mixed reference solution and the four negative test solutions, it was found that the retention time of each component was consistent, proving that the negative test solution had no obvious interference in the retention time position of each component, further proving that under the chromatographic conditions of the present invention, each component can be perfectly separated and the method has strong specificity.

[0041] 2. Linear Relationship

[0042] Accurately weigh 25 mg of levamisole hydrochloride reference substance, 35 mg of hydroxyclozanide reference substance, 50 mg of niclosamide reference substance, and 35 mg of albendazole reference substance, respectively, and place them in a 25 ml volumetric flask, dilute to the mark with glacial acetic acid-acidified methanol, and shake well to use as a mixed reference substance stock solution; accurately measure 1, 5, 10, 15, and 25 ml of the mixed reference substance stock solution, respectively, and place them in a 50 ml volumetric flask, dilute to the mark with glacial acetic acid-acidified methanol, and shake well to prepare a series of mixed reference substance dilutions, and perform sample injection and determination according to the chromatographic conditions in Example 2, and record the chromatogram. With the injection amount X of the mixed reference substance dilution as the abscissa and the peak area Y as the ordinate, a standard curve is drawn to obtain a regression equation.

[0043] The results are shown in Table 1. The four active ingredients of niclosamide, hydroxyclozanide, levamisole hydrochloride, and albendazole showed good linear relationships with the peak area in the concentration ranges of 0.02-0.5, 0.028-0.7, 0.04-1.0, and 0.028-0.7 mg / ml, respectively. The correlation coefficient R 2 All are greater than 0.999.

[0044] Table 1 Linear relationship investigation results

[0045]

[0046]

[0047] 3. Precision

[0048] The mixed reference solution in Example 2 was taken, and the sample was continuously injected and measured 6 times according to the chromatographic conditions in Example 2, and the chromatogram was recorded. The relative standard deviation (RSD) was calculated based on the 6 peak areas of the chromatographic peaks of each active ingredient.

[0049] The results showed that the RSDs of the peak areas of the four active ingredients, niclosamide, hydroxyclozanide, levamisole hydrochloride and albendazole, were all less than 1.2%, and were 0.34%, 0.56%, 0.78% and 0.56% respectively, proving that the method of the present invention has good precision.

[0050] 4. Sample recovery rate

[0051] Accurately measure 1 ml of compound levamisole oral solution of known content in a 100 ml volumetric flask, a total of 6 portions, and accurately add mixed reference solution according to low, medium and high concentrations, prepare the test solution according to the method described in Example 2, and perform sampling and measurement according to the chromatographic conditions in Example 2, record the chromatogram, calculate the content of each measured component, and calculate the average sample recovery rate of the measured component based on the amount of reference substance added.

[0052] The results are shown in Table 2. The average recoveries of the four active ingredients were all greater than 98%, and RSD was ≤ 1.2%, which proved that the method of the present invention has high accuracy.

[0053] Table 2 Results of sample recovery investigation

[0054] Tested ingredients Niclosamide Hydroxyclozanide Levamisole hydrochloride Albendazole Average recovery rate (%) 99.80 98.94 99.42 99.84 RSD(%) 0.35 0.42 0.54 0.46

[0055] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. Compound levamisole oral solution, characterized in that: Each 100 ml of oral solution contains 0.5-4.5 g of levamisole hydrochloride, 0.5-4.5 g of hydroxyclozanide, 5-20 g of niclosamide, 0.5-4.5 g of albendazole, 0.5-2.5 g of sodium carboxymethyl cellulose, 0.05-0.1 g of sodium bisulfite, 0.001-0.01 g of ethylparaben, 0.01-0.1 g of propylene glycol, and the balance is water.

2. The compound levamisole oral solution according to claim 1, characterized in that: Each 100 ml of oral solution contains 2.5 g of levamisole hydrochloride, 3.5 g of hydroxyclozanide, 5 g of niclosamide, 3.5 g of albendazole, 1 g of sodium carboxymethyl cellulose, 0.05 g of sodium bisulfite, 0.003 g of ethylparaben, 0.1 g of propylene glycol, and the balance is water.

3. The method for preparing the compound levamisole oral solution according to claim 1 or 2, characterized in that: The following steps are involved: Mix part of the water and propylene glycol, add sodium carboxymethyl cellulose to make it fully swell, then add sodium bisulfite, ethylparaben and levamisole hydrochloride, stir, then add hydroxyclozanide, niclosamide and albendazole, stir, make up to volume with water, stir evenly, repeat homogenization at 15-25 MPa for 2-3 times to obtain the compound levamisole oral solution.

4. The method for determining the content of the compound levamisole oral solution according to claim 1 or 2, characterized in that: The contents of levamisole hydrochloride, hydroxyclozanide, niclosamide and albendazole were determined by HPLC; (1) Chromatographic conditions Octadecylsilane bonded silica gel was used as filler; a methanol-0.05 mol / L ammonium acetate mixture with a volume ratio of 7:3 was used as mobile phase; the detection wavelength was 250 nm; (2) Preparation of mixed reference solution Accurately weigh levamisole hydrochloride reference substance, hydroxyclozanide reference substance, niclosamide reference substance, and albendazole reference substance, add glacial acetic acid-acidified methanol, dissolve by ultrasound, cool, and dilute with glacial acetic acid-acidified methanol to prepare a mixed reference substance solution; (3) Preparation of test solution Accurately measure the compound levamisole oral solution and dilute it with methanol acidified with glacial acetic acid as the test solution; (4) Determination method Accurately pipette the mixed reference solution and test solution separately, inject into liquid chromatograph, record the chromatogram, and calculate the contents of levamisole hydrochloride, hydroxyclozanide, niclosamide, and albendazole in the test sample by peak area according to the external standard method.

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