Compound enrofloxacin injection as well as preparation method and content determination method thereof

Abstract

The invention discloses a compound enrofloxacin injection, and each 100ml of the compound enrofloxacin injection contains 10 to 25g of enrofloxacin, 2 to 10g of sulfamonomethoxine sodium, 5 to 12g of florfenicol, 0.5 to 2.5 g of bromhexine hydrochloride, 1 to 20g of glacial acetic acid, 2 to 5g of Tween 80, 5 to 15g of alpha-pyrrolidone, 5 to 20g of N, N-dimethylformamide and the balance of water for injection. The compound enrofloxacin injection provided by the invention is advanced and reasonable in prescription design, stable and reliable in preparation process, good in preparation stability and small in irritation, and can achieve the effect of treating both symptoms and root causes when being used for respiratory system diseases of livestock and poultry.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and relates to a compound enrofloxacin injection, and also relates to a preparation method and a content determination method thereof. Background Art

[0002] Enrofloxacin is a synthetic third-generation quinolone antibiotic, also known as ethyl ciprofloxacin. It is a quinolone antibiotic for livestock, poultry and aquatic products, with broad-spectrum antibacterial activity. Its metabolite ciprofloxacin still has antibacterial effects. The enrofloxacin injection used in veterinary clinics has problems such as low concentration, insufficient stability, high irritation, and pain during injection. The chemical structure of enrofloxacin is acidic and alkaline, and it has high solubility in dilute alkaline solutions. However, high-concentration solutions can only be stable in a high pH environment (pH about 12), which will cause strong irritation and injection pain. In practical application, it has also been found that injection irritation and pain cause severe discomfort and disability in piglets, poultry, and pets.

[0003] Bromhexine is a synthetic drug with optimized structure of chloranthine, containing structural groups such as benzyl bromide and cyclohexylamine, and has poor water solubility. Its water solubility is improved by salting with hydrochloric acid, but the increase in pH causes the acid radical to be neutralized, and the water-poorly soluble bromhexine monomer will be released, causing the injection to become turbid.

[0004] Sulfadimethoxine sodium is the strongest sulfonamide with antibacterial effect in vivo and in vitro. It has strong inhibitory effect on most Gram-positive and Gram-negative bacteria. It is mainly used for various diseases caused by sensitive bacteria, Toxoplasma, Eperythrozoon (such as high fever, streptococcal disease, Haemophilus parasuis disease, toxoplasmosis, swine edema disease, Eperythrozoon disease, infectious pleuropneumonia, Pasteurella pneumonia, etc.) and mixed infections. The absorption rate of intramuscular injection is as high as 99.5%, the blood drug concentration is high, and the duration of action is as long as 48 hours. This drug develops drug resistance slowly.

[0005] Florfenicol belongs to the amide class of broad-spectrum antibiotics and is an antibacterial agent. It works by binding to the 50S subunit of the ribosome to inhibit the synthesis of bacterial proteins. It has strong antibacterial activity against a variety of Gram-positive and Gram-negative bacteria. It is mainly used for bacterial diseases of pigs, chickens, and fish caused by sensitive bacteria (such as respiratory diseases of cattle and pigs caused by hemolytic Pasteurella, multocida Pasteurella, and Actinobacillus pleuropneumoniae); typhoid and paratyphoid fever caused by Salmonella, chicken cholera, pullorum, Escherichia coli, etc.; fish bacterial septicemia, enteritis, red skin disease, etc. caused by fish Pasteurella, Vibrio, Staphylococcus aureus, Hydromonas, Enteritis, etc. Florfenicol is rapidly absorbed after oral administration, and the therapeutic concentration can be reached in the blood after about 1 hour, and the peak blood drug concentration can be reached in 1-3 hours, with a bioavailability of more than 80%. Florfenicol is widely distributed in animals and can pass through the blood-brain barrier. It is mainly excreted unchanged in the urine, and a small amount is excreted in the feces.

[0006] Currently, there is no compound preparation on the market that can cure both the symptoms and the root cause of animal respiratory diseases. Summary of the invention

[0007] In view of this, the object of the present invention is to provide a compound enrofloxacin injection, which has an advanced and reasonable prescription design, a stable and reliable preparation process, good preparation stability and low irritation, and can be used for respiratory diseases in livestock and poultry to achieve the effect of curing both the symptoms and the root causes.

[0008] After research, the present invention provides the following technical solutions:

[0009] 1. Compound enrofloxacin injection. Each 100 ml of injection contains 10-25 g of enrofloxacin, 2-10 g of sulfamethoxazole sodium, 5-12 g of florfenicol, 0.5-2.5 g of bromhexine hydrochloride, 1-20 g of glacial acetic acid, 2-5 g of Tween 80, 5-15 g of α-pyrrolidone, 5-20 g of N,N-dimethylformamide, and the balance is water for injection.

[0010] Preferably, each 100 ml injection contains 20 g of enrofloxacin, 5 g of sulfamethoxazole sodium, 10 g of florfenicol, 1.5 g of bromhexine hydrochloride, 10 g of glacial acetic acid, 3 g of Tween 80, 10 g of α-pyrrolidone, and 10 g of N,N-dimethylformamide, and the balance is water for injection.

[0011] When animals have respiratory diseases, in addition to lung infection, they are often accompanied by coughing and sputum. Enrofloxacin and florfenicol are the first choice drugs for lung flora infection. Sulfadimethoxine sodium is effective for mixed infections, and bromhexine hydrochloride has an expectorant effect. Several antibiotics combined with bromhexine hydrochloride can achieve the effect of curing both the symptoms and the root cause of respiratory diseases in livestock and poultry.

[0012] 2. The preparation method of the compound enrofloxacin injection comprises the following steps:

[0013] 1) Mix α-pyrrolidone and Tween-80, add part of glacial acetic acid and water for injection and mix well, then add bromhexine hydrochloride, heat to dissolve, and obtain liquid A;

[0014] 2) Mix part of glacial acetic acid and water for injection, add enrofloxacin and sulfamethoxazole sodium, stir to dissolve, and obtain liquid B;

[0015] 3) Add florfenicol to N,N-dimethylformamide and stir to dissolve to obtain liquid C;

[0016] 4) Combine solutions a, b and c, mix well, adjust the pH to 4.0-6.0 with glacial acetic acid, make up to volume with water for injection, filter, fill and sterilize to obtain compound enrofloxacin injection.

[0017] 3. The content determination method of the compound enrofloxacin injection is to determine the content of enrofloxacin, sulfamethoxazole sodium, florfenicol and bromhexine hydrochloride by high performance liquid chromatography;

[0018] (1) Chromatographic conditions

[0019] Octadecylsilane bonded silica gel is used as filler;

[0020] Use 0.1% (ml / ml) trifluoroacetic acid solution as mobile phase A, methanol as mobile phase B, and acetonitrile as mobile phase C, and perform gradient elution according to the following procedure:

[0021] 0-18min: the volume fraction of mobile phase A changes linearly from 75% to 10%, the volume fraction of mobile phase B changes linearly from 5% to 40%, and the volume fraction of mobile phase C changes linearly from 20% to 50%;

[0022] 18-21min: the volume fraction of mobile phase A changes linearly from 10% to 75%, the volume fraction of mobile phase B changes linearly from 40% to 5%, and the volume fraction of mobile phase C changes linearly from 50% to 20%;

[0023] 21-26min: the volume fraction of mobile phase A is 75%, the volume fraction of mobile phase B is 5%, and the volume fraction of mobile phase C is 20%;

[0024] The detection wavelength for enrofloxacin, sulfamethoxazole sodium, and bromhexine hydrochloride is 254 nm, and the detection wavelength for florfenicol is 224 nm;

[0025] (2) Preparation of reference solution

[0026] Accurately weigh enrofloxacin reference substance, sulfamethoxine sodium reference substance, florfenicol reference substance, and bromhexine hydrochloride reference substance, dissolve and dilute them with methanol respectively, then accurately measure the obtained dilution, dilute it with a mixed solution of mobile phase A, B, and C in a volume ratio of 75:5:20, to prepare enrofloxacin reference substance solution, sulfamethoxine sodium reference substance solution, florfenicol reference substance solution, and bromhexine hydrochloride reference substance solution respectively;

[0027] (3) Preparation of test solution

[0028] Accurately measure the compound enrofloxacin injection, dilute it with methanol, then accurately measure the resulting dilution, dilute it with a mixture of mobile phases A, B, and C in a volume ratio of 75:5:20 to prepare the test solution;

[0029] (4) Determination method

[0030] Accurately pipette 4 kinds of reference solution and test solution respectively, inject into liquid chromatograph, record chromatogram, calculate the content of enrofloxacin, sulfamethoxazole sodium, florfenicol and bromhexine hydrochloride in the test sample by peak area according to external standard method.

[0031] The beneficial effects of the present invention are as follows: the present invention provides a compound enrofloxacin injection, which has an advanced and reasonable prescription design, a stable and reliable preparation process, good preparation stability and low irritation, and can be used for respiratory diseases of livestock and poultry to achieve the effect of curing both the symptoms and the root causes. This patent also provides a method for determining the content of the compound enrofloxacin injection, which can simultaneously detect the contents of enrofloxacin, sulfamethoxazole sodium, florfenicol and bromhexine hydrochloride, has a wide linear range, good accuracy and repeatability, and good method durability. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the chromatogram of enrofloxacin reference substance at a wavelength of 254 nm.

[0033] Figure 2 It is the chromatogram of sulfamethoxazole sodium reference substance at a wavelength of 254nm.

[0034] Figure 3 This is the chromatogram of bromhexine hydrochloride reference substance at a wavelength of 254nm.

[0035] Figure 4 This is the chromatogram of the florfenicol reference substance at a wavelength of 224 nm.

[0036] Figure 5 These are the chromatograms of compound enrofloxacin injection at wavelengths of 254nm and 224nm respectively.

[0037] Figure 6 The chromatograms of the blank preparation at wavelengths of 254 nm and 224 nm respectively.

[0038] Figure 7 This is the anatomical diagram of the rabbit muscle irritation test of compound enrofloxacin injection. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and beneficial effects of the present invention more clear, preferred embodiments of the present invention are described in detail below.

[0040] Example 1 Preparation of compound enrofloxacin injection

[0041] Each 100 ml of compound enrofloxacin injection contains 20 g of enrofloxacin, 5 g of sulfamethoxazole sodium, 10 g of florfenicol, 1.5 g of bromhexine hydrochloride, 10 g of glacial acetic acid, 3 g of Tween 80, 10 g of α-pyrrolidone, and 10 g of N,N-dimethylformamide, and the balance is water for injection.

[0042] The preparation method comprises the following steps:

[0043] 1) Mix α-pyrrolidone and Tween-80, add part of glacial acetic acid and water for injection and mix well, then add bromhexine hydrochloride, heat to dissolve, and obtain liquid A;

[0044] 2) Mix part of glacial acetic acid and water for injection, add enrofloxacin and sulfamethoxazole sodium, stir to dissolve, and obtain liquid B;

[0045] 3) Add florfenicol to N,N-dimethylformamide and stir to dissolve to obtain liquid C;

[0046] 4) Combine solutions a, b and c, mix well, adjust the pH to 4.0-6.0 with glacial acetic acid, make up to volume with water for injection, filter, fill and sterilize to obtain compound enrofloxacin injection.

[0047] Example 2 Determination of the content of compound enrofloxacin injection

[0048] 1. Determination method

[0049] The contents of enrofloxacin, sulfamethoxazole sodium, florfenicol and bromhexine hydrochloride were determined by HPLC.

[0050] Chromatographic conditions: the chromatographic column was a C18 column (250 mm × 4.6 mm, 5 μm), the mobile phase A was a 0.1% (ml / ml) trifluoroacetic acid solution, the mobile phase B was methanol, the mobile phase C was acetonitrile, and gradient elution was performed according to the program shown in Table 1, the flow rate was 1.0 ml / min, the column temperature was 35°C, the detection wavelength for enrofloxacin, sulfamethoxine sodium, and bromhexine hydrochloride was 254 nm, and the detection wavelength for florfenicol was 224 nm.

[0051] Table 1 Gradient elution program

[0052]

[0053] Preparation of reference solution: Accurately weigh appropriate amount of enrofloxacin reference substance, sulfamethoxazole sodium reference substance, florfenicol reference substance, and bromhexine hydrochloride reference substance, dissolve them in methanol and dilute to 100 ml, shake well, then accurately measure 1 ml into a 50 ml volumetric flask, add a mixture of mobile phases A, B, and C in a volume ratio of 75:5:20 to dilute to the scale, shake well, and prepare 4 single reference solutions.

[0054] Preparation of test solution: Accurately measure 1 ml of compound enrofloxacin injection into a 100 ml volumetric flask, add methanol to dilute to the scale, shake well, then accurately measure 1 ml into a 50 ml volumetric flask, add a mixture of mobile phases A, B, and C in a volume ratio of 75:5:20 to dilute to the scale, shake well, and obtain.

[0055] Determination method: Accurately pipette 20 μl of each of the four single reference solutions and the test solution, inject into the liquid chromatograph, and record the chromatogram; calculate the contents of enrofloxacin, sulfamethoxazole sodium, florfenicol, and bromhexine hydrochloride in the test sample by the peak area using the external standard method.

[0056] The chromatograms of enrofloxacin reference substance, sulfamethoxazole sodium reference substance, and bromhexine hydrochloride reference substance at a wavelength of 254 nm are shown in Figures 1 to 3 The chromatogram of the florfenicol reference substance at a wavelength of 224 nm is shown in Figure 4 The chromatograms of compound enrofloxacin injection at wavelengths of 254 nm and 224 nm are shown in Figure 5 .

[0057] 2. Methodological investigation

[0058] 2.1 Exclusivity

[0059] A blank preparation without enrofloxacin, sulfamethoxazole sodium, florfenicol, or bromhexine hydrochloride was prepared and sampled and measured under the above chromatographic conditions. The chromatograms at wavelengths of 254 nm and 224 nm were shown in FIG. Figure 6 No obvious interference was observed at the corresponding retention times of enrofloxacin, sulfamethoxazole sodium, florfenicol, and bromhexine hydrochloride.

[0060] 2.2 Linear Relationship

[0061] Take appropriate amount of enrofloxacin reference substance, sulfamethoxine sodium reference substance, florfenicol reference substance, bromhexine hydrochloride reference substance, accurately weigh, add methanol to dissolve and prepare reference substance stock solution. Accurately measure appropriate amount of reference substance stock solution respectively, add mobile phase A, B, C mixed solution with volume ratio of 75:5:20 to dilute and prepare reference substance solution of different concentrations, inject and measure according to the above chromatographic conditions, record chromatogram, and perform linear regression with concentration (Y) on peak area (X). The results are shown in Table 2, and enrofloxacin, bromhexine hydrochloride, sulfamethoxine sodium, and florfenicol have good linear relationship with peak area in the concentration range of 1-80, 1-30, 1-20, and 1-40 μg / ml, respectively.

[0062] Table 2 Results of investigation on the linear relationship of main components

[0063] drug Linear <![CDATA[Correlation coefficient (R 2 )]]> Linear range (μg / ml) Enrofloxacin y=33.201x+9.9806 0.9997 1-80 Bromhexine Hydrochloride y=23.074x-10.49 0.9998 1-30 Sulfadimethoxine Sodium y=59.19x-40.367 0.9996 1-20 Florfenicol y=22.34x+277.87 0.9995 1-40

[0064] 2.3 Accuracy and repeatability

[0065] The solutions of low, medium and high concentrations of enrofloxacin, bromhexine hydrochloride, sulfamethoxine sodium and florfenicol were prepared respectively, and 3 samples were prepared for each concentration. The samples were injected and measured according to the above chromatographic conditions, and each sample was injected 3 times to calculate the recovery rate and coefficient of variation. The results are shown in Table 3. The average recovery rates of low, medium and high concentrations of enrofloxacin, bromhexine hydrochloride, sulfamethoxine sodium and florfenicol were between 99.74% and 100.41%, and the coefficient of variation was ≤1.15%, with good accuracy and repeatability.

[0066] Table 3 Results of investigation on recovery rate of main components

[0067]

[0068] 2.4 Durability

[0069] The same compound enrofloxacin injection sample was taken and the same high performance liquid chromatograph was used. The chromatographic columns were Waters C 18 (5μm, 4.6mm×250mm) and Waters XBridge-C 18 (5μm, 4.6mm×250mm), and the sample was injected and measured under the condition that other chromatographic conditions remained unchanged. The results showed that there was no obvious difference in the peak time of the main components of different chromatographic columns, and the difference in the calculated content was less than 0.5%, indicating that the method had good durability.

[0070] Example 3 Stability Study of Compound Enrofloxacin Injection

[0071] According to the "Guidelines for Stability Testing of Veterinary Drugs", the stability of the compound enrofloxacin injection prepared in Example 1 was investigated.

[0072] 1. High temperature test

[0073] The compound enrofloxacin injection was placed in a forced air drying oven at 60°C for 10 days, and samples were taken for testing on the 5th and 10th days.

[0074] The results are shown in Table 4. After being placed at 60° C. for 10 days, the properties, pH value and main component content of the compound enrofloxacin injection prepared in Example 1 did not change significantly.

[0075] Table 4 High temperature test results

[0076]

[0077]

[0078] 2. High humidity test

[0079] The compound enrofloxacin injection was placed in a constant temperature and humidity chamber at a temperature of 25±2°C and a relative humidity of 70±5% for 10 days, and samples were taken for testing on the 5th and 10th days.

[0080] The results are shown in Table 5. After being placed under the conditions of 25±2°C and 70±5% for 10 days, the properties, pH value and main component content of the compound enrofloxacin injection prepared in Example 1 did not change significantly.

[0081] Table 5 High humidity test results

[0082]

[0083] 3. Accelerated testing

[0084] The compound enrofloxacin injection was placed in a constant temperature and humidity chamber at a temperature of 40±2°C and a relative humidity of 75±5% for 6 months, and samples were taken for testing at the end of the 1st, 2nd, 3rd and 6th month.

[0085] The results are shown in Table 6. After being placed at 40±2°C and 75±5% for 6 months, the properties, pH value and main component content of the compound enrofloxacin injection prepared in Example 1 did not change significantly.

[0086] Table 6 Accelerated test results

[0087]

[0088] Example 4 Investigation of muscle irritation of compound enrofloxacin injection

[0089] According to the "Guidelines for Irritant, Allergic and Hemolytic Properties of Chemical Drugs", 3 rabbits weighing 2.5-3.5kg were selected and fed for two weeks before the experiment to confirm that the rabbits were in good health and had no skin diseases, and were weighed and numbered. Using the self-left and right control method, each rabbit was injected with 1 ml of the compound tylosin tartrate injection prepared in Example 1 at the right thigh quadriceps, and the same dose of normal saline was injected at the same site on the other side. After 48 hours of administration, the rabbits were killed, the skin was peeled off to expose the quadriceps, and the longitudinal incision was made to observe whether the muscles at the injection site showed irritation reactions such as congestion, edema, deformation or necrosis. The reaction level was obtained according to the muscle irritation reaction classification standard in Table 7, and the average reaction level was calculated. When the average reaction level is below 2, it can be used for intramuscular injection; when the average reaction level exceeds 3, intramuscular injection cannot be performed; when the average reaction level is between 2 and 3, repeated tests can be performed or combined with other indicators for comprehensive consideration.

[0090] Rabbit muscle anatomy diagram Figure 7 , it can be seen that the rabbit muscle texture is clear, there is a certain amount of unabsorbed drug solution, and the muscle is slightly degenerated. The muscle irritation reaction grading results show that the reaction level of the three rabbits is level 3, and the average reaction level is 3. Since in actual clinical applications, the dose of the compound enrofloxacin injection is about 0.0125ml / (kg·bw), which is much lower than the above-mentioned muscle irritation test dose (1ml), therefore, the compound enrofloxacin injection of the present invention has low irritation and can be used for intramuscular injection.

[0091] Table 7 Classification criteria for muscle stimulation response

[0092]

[0093] 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 enrofloxacin injection, characterized in that: Each 100 ml of injection contains 10-25 g of enrofloxacin, 2-10 g of sulfamethoxazole sodium, 5-12 g of florfenicol, 0.5-2.5 g of bromhexine hydrochloride, 1-20 g of glacial acetic acid, 2-5 g of Tween 80, 5-15 g of α-pyrrolidone, 5-20 g of N,N-dimethylformamide, and the balance is water for injection.

2. The compound enrofloxacin injection according to claim 1, characterized in that: Each 100 ml of injection contains 20 g of enrofloxacin, 5 g of sulfamethoxazole sodium, 10 g of florfenicol, 1.5 g of bromhexine hydrochloride, 10 g of glacial acetic acid, 3 g of Tween 80, 10 g of α-pyrrolidone, and 10 g of N,N-dimethylformamide, with the balance being water for injection.

3. The method for preparing the compound enrofloxacin injection according to claim 1 or 2, characterized in that: The following steps are involved: 1) Mix α-pyrrolidone and Tween-80, add part of glacial acetic acid and water for injection and mix well, then add bromhexine hydrochloride, heat to dissolve, and obtain liquid A; 2) Mix part of glacial acetic acid and water for injection, add enrofloxacin and sulfamethoxazole sodium, stir to dissolve, and obtain liquid B; 3) Add florfenicol to N,N-dimethylformamide and stir to dissolve to obtain liquid C; 4) Combine solutions a, b and c, mix well, adjust the pH to 4.0-6.0 with glacial acetic acid, make up to volume with water for injection, filter, fill and sterilize to obtain compound enrofloxacin injection.

4. The method for determining the content of the compound enrofloxacin injection according to claim 1 or 2, characterized in that: The contents of enrofloxacin, sulfamethoxazole sodium, florfenicol, and bromhexine hydrochloride were determined by HPLC; (1) Chromatographic conditions Octadecylsilane bonded silica gel is used as filler; Use 0.1% (ml / ml) trifluoroacetic acid solution as mobile phase A, methanol as mobile phase B, and acetonitrile as mobile phase C, and perform gradient elution according to the following procedure: 0-18min: the volume fraction of mobile phase A changes linearly from 75% to 10%, the volume fraction of mobile phase B changes linearly from 5% to 40%, and the volume fraction of mobile phase C changes linearly from 20% to 50%; 18-21min: the volume fraction of mobile phase A changes linearly from 10% to 75%, the volume fraction of mobile phase B changes linearly from 40% to 5%, and the volume fraction of mobile phase C changes linearly from 50% to 20%; 21-26min: the volume fraction of mobile phase A is 75%, the volume fraction of mobile phase B is 5%, and the volume fraction of mobile phase C is 20%; The detection wavelength for enrofloxacin, sulfamethoxazole sodium, and bromhexine hydrochloride is 254 nm, and the detection wavelength for florfenicol is 224 nm; (2) Preparation of reference solution Accurately weigh enrofloxacin reference substance, sulfamethoxine sodium reference substance, florfenicol reference substance, and bromhexine hydrochloride reference substance, dissolve and dilute them with methanol respectively, then accurately measure the obtained dilution, dilute it with a mixed solution of mobile phase A, B, and C in a volume ratio of 75:5:20, to prepare enrofloxacin reference substance solution, sulfamethoxine sodium reference substance solution, florfenicol reference substance solution, and bromhexine hydrochloride reference substance solution respectively; (3) Preparation of test solution Accurately measure the compound enrofloxacin injection, dilute it with methanol, then accurately measure the resulting dilution, dilute it with a mixture of mobile phases A, B, and C in a volume ratio of 75:5:20 to prepare the test solution; (4) Determination method Accurately pipette 4 kinds of reference solution and test solution respectively, inject into liquid chromatograph, record chromatogram, calculate the content of enrofloxacin, sulfamethoxazole sodium, florfenicol and bromhexine hydrochloride in the test sample by peak area according to external standard method.

Images