Compound enrofloxacin injection and preparation method and content determination method thereof

Abstract

The application discloses a compound enrofloxacin injection, which contains 10-25g of enrofloxacin, 2-10g of sulfadimoxine sodium, 5-12g of florfenicol, 0.5-2.5g of bromhexine hydrochloride, 1-20g of glacial acetic acid, 2-5g of Tween 80, 5-15g of alpha-pyrrolidone and 5-20g of N, N-dimethylformamide per 100ml of the injection, and the rest is water for injection. The compound enrofloxacin injection has advanced and reasonable prescription design, stable and reliable preparation process, good preparation stability and small irritation, and can achieve the effect of treating all samples for respiratory system diseases of livestock and poultry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical preparations, and relates to a compound enrofloxacin injection, and a preparation method and content determination method thereof. BACKGROUND

[0002] Enrofloxacin is a synthetic third-generation quinolone antibacterial drug, also known as ethyl cyclopropylcinoxacin, which is a quinolone antibacterial drug specially used for livestock and aquatic products, and has broad-spectrum antibacterial activity. Its metabolite cyclopropylcinoxacin still has antibacterial effect. Enrofloxacin injection used in veterinary clinics has problems such as low concentration, insufficient stability, strong irritation, and pain during injection. The chemical structure of enrofloxacin is amphoteric, which has high solubility in dilute alkaline solution, but high-concentration solution can only be stable in a high-pH (pH about 12) environment, which will cause strong irritation and injection pain. It is also found in application practice that pigs, poultry and pets appear serious discomfort and disability due to injection irritation and pain.

[0003] Bromhexine is a synthetic drug optimized from psilocine, containing bromobenzyl, cyclohexylamine and other structural groups, and has poor water solubility. The water solubility is improved by salifying with hydrochloric acid, but the increase of pH causes the neutralization of acid radical, and the poor water solubility of bromhexine monomer will be free to cause the turbidity of injection.

[0004] Sulfadimoxine sodium is the strongest sulfonamide drug against bacteria in vivo and in vitro, and has strong inhibitory effect on most gram-positive and negative bacteria, and is mainly used for various diseases (such as high fever, streptococcosis, parahemophilus disease, toxoplasmosis, edema disease, eperythrozoa disease, contagious pleuropneumonia, pasteurella multocida pneumonia, etc.) and mixed infections caused by sensitive bacteria, eperythrozoa and hematoxylin. Intramuscular injection has a high absorption rate of more than 99.5%, high blood drug concentration, and a long maintenance effect of more than 48 hours. This drug produces resistance slowly.

[0005] Florfenicol is a broad-spectrum antibiotic belonging to the amamide alcohol class. It is a bacteriostatic agent that exerts its effect by binding to the 50S subunit of the ribosome and inhibiting bacterial protein synthesis. It has strong antibacterial activity against a variety of Gram-positive and Gram-negative bacteria. It is mainly used for bacterial diseases in pigs, chickens, and fish caused by susceptible bacteria (such as respiratory diseases in cattle and pigs caused by Pasteurella multocida, Pasteurella multocida, and Actinobacillus pleuropneumoniae); typhoid and paratyphoid fever caused by Salmonella; fowl cholera, pullorum disease, and Escherichia coli infection; and bacterial septicemia, enteritis, and red spot disease in fish caused by Pasteurella, Vibrio, Staphylococcus aureus, Hydrophila, and Enterobacteriaceae. Florfenicol is rapidly absorbed orally, reaching therapeutic concentrations in the blood in about 1 hour and peak plasma concentrations in 1-3 hours, with a bioavailability of over 80%. Florfenicol is widely distributed in animals and can cross the blood-brain barrier. It is mainly excreted unchanged in urine, with a small amount excreted in feces.

[0006] Currently, there are no compound preparations on the market that can treat both the symptoms and the root cause of respiratory diseases in animals. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a compound enrofloxacin injection with an advanced and reasonable formulation design, a stable and reliable preparation process, good formulation stability and low irritation, which can achieve the effect of treating both the symptoms and the root cause of respiratory diseases in livestock and poultry.

[0008] Based on research, the present invention provides the following technical solution:

[0009] 1. Compound Enrofloxacin Injection: Each 100ml of injection contains 10-25g of enrofloxacin, 2-10g of sulfamethoxypyrimidine sodium, 5-12g of florfenicol, 0.5-2.5g of bromhexine hydrochloride, 1-20g of glacial acetic acid, 2-5g of Tween 80, 5-15g of α-pyrrolidone, 5-20g of N,N-dimethylformamide, with the remainder being water for injection.

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

[0011] When animals develop respiratory diseases, in addition to lung infection, they often experience coughing and phlegm. Enrofloxacin and florfenicol are the first-line drugs for pulmonary bacterial infections, sulfamethoxypyrimidine sodium is effective for mixed infections, and bromhexine hydrochloride has an expectorant effect. The combined use of several antibiotics with bromhexine hydrochloride can achieve a comprehensive cure for respiratory diseases in livestock and poultry.

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

[0013] 1) Mix α-pyrrolidone and Tween-80, add some glacial acetic acid and water for injection, mix well, then add bromhexine hydrochloride, heat to dissolve, and obtain solution a.

[0014] 2) Take a portion of glacial acetic acid and water for injection, mix them well, add enrofloxacin and sulfamethoxypyrimidine sodium, stir to dissolve, and obtain solution b;

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

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

[0017] 3. The method for determining the content of the compound enrofloxacin injection is to use high performance liquid chromatography to determine the content of enrofloxacin, sulfamethoxypyrimidine sodium, florfenicol, and bromhexine hydrochloride;

[0018] (1) Chromatographic conditions

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

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

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

[0022] 18–21 min: 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] 21–26 min: Mobile phase A volume fraction 75%, mobile phase B volume fraction 5%, mobile phase C volume fraction 20%;

[0024] Enrofloxacin, sulfamethoxypyrimidine sodium, and bromhexine hydrochloride are detected at a wavelength of 254 nm, while florfenicol is detected at a wavelength of 224 nm.

[0025] (2) Preparation of reference solution

[0026] Accurately weigh enrofloxacin reference standard, sulfamethoxypyrimidine sodium reference standard, florfenicol reference standard, and bromhexine hydrochloride reference standard, dissolve and dilute them separately in methanol, and then accurately measure the resulting diluted solutions and dilute them with a mobile phase mixture of A, B, and C with a volume ratio of 75:5:20 to prepare enrofloxacin reference standard solution, sulfamethoxypyrimidine sodium reference standard solution, florfenicol reference standard solution, and bromhexine hydrochloride reference standard solution, respectively.

[0027] (3) Preparation of the test solution

[0028] Accurately measure the compound enrofloxacin injection, dilute it with methanol, and then accurately measure the resulting diluted solution. 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 four reference solutions and the test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the contents of enrofloxacin, sulfamethoxypyrimidine sodium, florfenicol, and bromhexine hydrochloride in the test sample by peak area using the external standard method.

[0031] The beneficial effects of this invention are as follows: This invention provides a compound enrofloxacin injection with an advanced and reasonable formulation design, a stable and reliable preparation process, good formulation stability, and low irritation. It can achieve both symptomatic and root-cause treatment for respiratory diseases in livestock and poultry. This patent also provides a method for determining the content of compound enrofloxacin injection, which can simultaneously detect the content of enrofloxacin, sulfamethoxypyrimidine sodium, florfenicol, and bromhexine hydrochloride. The method has a wide linear range, good accuracy and repeatability, and good robustness. Attached Figure Description

[0032] Figure 1 The chromatogram of enrofloxacin reference standard at a wavelength of 254 nm is shown.

[0033] Figure 2 The chromatogram of sulfamethoxypyrimidine sodium reference standard is shown at a wavelength of 254 nm.

[0034] Figure 3 The chromatogram of bromhexine hydrochloride reference standard at a wavelength of 254 nm is shown.

[0035] Figure 4 The chromatogram of florfenicol reference standard at a wavelength of 224 nm is shown.

[0036] Figure 5 The chromatograms of compound enrofloxacin injection at wavelengths of 254 nm and 224 nm are shown.

[0037] Figure 6 The chromatograms of the blank formulation are shown at wavelengths of 254 nm and 224 nm, respectively.

[0038] Figure 7 Anatomical diagram of rabbit muscle irritation test for compound enrofloxacin injection. Detailed Implementation

[0039] To make the objectives, technical solutions, and beneficial effects of the present invention clearer, the preferred embodiments of the present invention will be described in detail below.

[0040] Example 1: Preparation of Compound Enrofloxacin Injection

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

[0042] The preparation method includes the following steps:

[0043] 1) Mix α-pyrrolidone and Tween-80, add some glacial acetic acid and water for injection, mix well, then add bromhexine hydrochloride, heat to dissolve, and obtain solution a.

[0044] 2) Take a portion of glacial acetic acid and water for injection, mix them well, add enrofloxacin and sulfamethoxypyrimidine sodium, stir to dissolve, and obtain solution b;

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

[0046] 4) Combine solutions a, b, and c, mix well, adjust the pH to 4.0-6.0 with glacial acetic acid, bring the volume to a final 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. Measurement Method

[0049] The contents of enrofloxacin, sulfamethoxypyrimidine sodium, florfenicol, and bromhexine hydrochloride were determined by high performance liquid chromatography.

[0050] Chromatographic conditions: A C18 column (250 mm × 4.6 mm, 5 μm) was used. Mobile phase A was 0.1% (ml / ml) trifluoroacetic acid solution, mobile phase B was methanol, and mobile phase C was acetonitrile. 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, and the detection wavelengths for enrofloxacin, sulfamethoxypyrimidine sodium, and bromhexine hydrochloride were 254 nm, while the detection wavelength for florfenicol was 224 nm.

[0051] Table 1 Gradient elution program

[0052]

[0053] Preparation of reference solutions: Accurately weigh appropriate amounts of enrofloxacin reference standard, sulfamethoxypyrimidine sodium reference standard, florfenicol reference standard, and bromhexine hydrochloride reference standard, 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 with a volume ratio of 75:5:20 and dilute to the mark, shake well, and obtain four single reference solutions.

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

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

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

[0057] 2. Methodological Examination

[0058] 2.1 Specificity

[0059] A blank formulation was prepared that did not contain enrofloxacin, sulfamethoxypyrimidine sodium, florfenicol, or bromhexine hydrochloride. The sample was injected and analyzed under the chromatographic conditions described above. The chromatograms at wavelengths of 254 nm and 224 nm are shown below. Figure 6 No significant interference was observed at the corresponding retention times of enrofloxacin, sulfamethoxypyrimidine sodium, florfenicol, and bromhexine hydrochloride.

[0060] 2.2 Linear Relationship

[0061] Accurately weigh appropriate amounts of enrofloxacin reference standard, sulfamethoxypyrimidine sodium reference standard, florfenicol reference standard, and bromhexine hydrochloride reference standard, and dissolve them in methanol to prepare reference standard stock solutions. Accurately measure appropriate amounts of each reference standard stock solution and dilute them with a 75:5:20 mixture of mobile phases A, B, and C to prepare reference standard solutions of different concentrations. Inject and determine the solutions under the chromatographic conditions described above, record the chromatograms, and perform linear regression on the peak area (X) against the concentration (Y). The results are shown in Table 2. Enrofloxacin, bromhexine hydrochloride, sulfamethoxypyrimidine sodium, and florfenicol showed good linear relationships with peak area in concentration ranges of 1-80, 1-30, 1-20, and 1-40 μg / ml, respectively.

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

[0063] Drug Linear Correlation coefficient (R 2 )]]> Linear range (pg / ml) Enrofloxacin y = 33.201 x + 9.9806 0.9997 1-80 Bromhexine hydrochloride y = 23.074 x - 10.49 0.9998 1-30 Sulfadimoxine sodium y = 59.19 x - 40.367 0.9996 1-20 Florfenicol y = 22.34 x + 277.87 0.9995 1-40

[0064] 2.3 Accuracy and Repeatability

[0065] Solutions of enrofloxacin, bromhexine hydrochloride, sulfamethoxypyrimidine sodium, and florfenicol at low, medium, and high concentrations were prepared. Three samples were prepared for each concentration, and the samples were injected and analyzed under the chromatographic conditions described above. Each sample was injected three times, and the recovery rate and coefficient of variation were calculated. The results are shown in Table 3. The average recoveries of enrofloxacin, bromhexine hydrochloride, sulfamethoxypyrimidine sodium, and florfenicol at low, medium, and high concentrations ranged from 99.74% to 100.41%, with a coefficient of variation ≤1.15%, indicating good accuracy and repeatability.

[0066] Table 3 Results of the recovery rate of major components

[0067]

[0068] 2.4 Durability

[0069] Take the same compound enrofloxacin injection sample and use the same high-performance liquid chromatograph with Waters column. C 18 (5μm, 4.6mm × 250mm) and Waters XBridge-C 18 (5μm, 4.6mm×250mm) Under the condition that other chromatographic conditions remain unchanged, the injection and determination of the main components of different chromatographic columns showed no significant difference in elution time, and the calculated content difference was less than 0.5%, indicating that the method has good robustness.

[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 solution was placed in a forced-air drying oven and stored at 60°C for 10 days. 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 solution was placed in a constant temperature and humidity chamber and kept at a temperature of 25±2℃ and a relative humidity of 70±5% for 10 days. Samples were taken for testing on the 5th and 10th days.

[0080] The results are shown in Table 5. After being placed at 25±2℃ 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 Results of High Humidity Test

[0082]

[0083] 3. Accelerated testing

[0084] Compound enrofloxacin injection was placed in a constant temperature and humidity chamber and stored at a temperature of 40±2℃ and a relative humidity of 75±5% for 6 months. Samples were taken and tested at the end of the 1st, 2nd, 3rd and 6th months.

[0085] The results are shown in Table 6. After being placed at 40±2℃ 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 Irritation, Allergenicity, and Hemolytic Properties of Chemical Drugs," three rabbits weighing 2.5-3.5 kg were selected. They were fed for two weeks prior to the experiment to ensure good health and absence of skin diseases. The rabbits were weighed and numbered. Using a self-controlled left-right method, each rabbit received an injection of 1 ml of the compound tylosin tartrate injection prepared in Example 1 into the quadriceps femoris muscle of its right thigh, and the same volume of physiological saline was injected into the same location on the other side. Forty-eight hours after administration, the rabbits were euthanized, the skin was peeled off to expose the quadriceps femoris muscle, and it was longitudinally incised. The injection site was observed for signs of congestion, edema, deformation, or necrosis. The reaction grade was determined according to the muscle stimulation reaction grading standard in Table 7, and the average reaction grade was calculated. An average reaction grade below 2 was acceptable for intramuscular injection; an average reaction grade exceeding 3 was not acceptable; and an average reaction grade between 2 and 3 allowed for repeated trials or consideration of other indicators.

[0090] See rabbit muscle anatomy diagram. Figure 7 The rabbits showed clear muscle texture, some unabsorbed drug solution, and slight muscle degeneration. The muscle stimulation response grading results showed that all three rabbits had a response grade of 3, with an average grade of 3. Since the actual clinical application dose of compound enrofloxacin injection is approximately 0.0125 ml / (kg·bw), far lower than the dose in the muscle stimulation test (1 ml), the compound enrofloxacin injection described in this invention has low irritation and can be used for intramuscular injection.

[0091] Table 7 Grading 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 and are not intended 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 to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. Compound Enrofloxacin Injection, characterized in that: Each 100ml of injection contains 10-25g of enrofloxacin, 2-10g of sulfamethoxypyrimidine sodium, 5-12g of florfenicol, 0.5-2.5g of bromhexine hydrochloride, 1-20g of glacial acetic acid, 2-5g of Tween 80, 5-15g of α-pyrrolidone, 5-20g of N,N-dimethylformamide, with the remainder being water for injection.

2. The compound enrofloxacin injection as described in claim 1, characterized in that: Each 100ml of injection contains 20g of enrofloxacin, 5g of sulfamethoxypyrimidine sodium, 10g of florfenicol, 1.5g of bromhexine hydrochloride, 10g of glacial acetic acid, 3g of Tween 80, 10g of α-pyrrolidone, 10g of N,N-dimethylformamide, and the remainder is water for injection.

3. The preparation method of the compound enrofloxacin injection according to claim 1 or 2, characterized in that: Includes the following steps: 1) Mix α-pyrrolidone and Tween-80, add some glacial acetic acid and water for injection, mix well, then add bromhexine hydrochloride, heat to dissolve, and obtain solution a. 2) Take a portion of glacial acetic acid and water for injection, mix them well, add enrofloxacin and sulfamethoxypyrimidine sodium, stir to dissolve, and obtain solution b; 3) Add florfenicol to N,N-dimethylformamide and stir to dissolve, to obtain solution c; 4) Combine solutions a, b, and c, mix well, adjust the pH to 4.0-6.0 with glacial acetic acid, bring the volume to a final volume with water for injection, filter, fill, and sterilize to obtain compound enrofloxacin injection.

4. The method for determining the content of compound enrofloxacin injection according to claim 1 or 2, characterized in that: The contents of enrofloxacin, sulfamethoxypyrimidine sodium, florfenicol, and bromhexine hydrochloride were determined by high performance liquid chromatography. (1) Chromatographic conditions Octadecylsilane-bonded silica gel is used as a filler; Using 0.1% (ml / ml) trifluoroacetic acid solution as mobile phase A, methanol as mobile phase B, and acetonitrile as mobile phase C, gradient elution was performed according to the following procedure: 0–18 min: The volume fraction of mobile phase A changed linearly from 75% to 10%, the volume fraction of mobile phase B changed linearly from 5% to 40%, and the volume fraction of mobile phase C changed linearly from 20% to 50%. 18–21 min: 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%. 21–26 min: Mobile phase A volume fraction 75%, mobile phase B volume fraction 5%, mobile phase C volume fraction 20%; Enrofloxacin, sulfamethoxypyrimidine sodium, and bromhexine hydrochloride are detected at a wavelength of 254 nm, while florfenicol is detected at a wavelength of 224 nm. (2) Preparation of reference solution Accurately weigh enrofloxacin reference standard, sulfamethoxypyrimidine sodium reference standard, florfenicol reference standard, and bromhexine hydrochloride reference standard, dissolve and dilute them separately in methanol, and then accurately measure the resulting diluted solutions and dilute them with a mobile phase mixture of A, B, and C with a volume ratio of 75:5:20 to prepare enrofloxacin reference standard solution, sulfamethoxypyrimidine sodium reference standard solution, florfenicol reference standard solution, and bromhexine hydrochloride reference standard solution, respectively. (3) Preparation of the test solution Accurately measure the compound enrofloxacin injection, dilute it with methanol, and then accurately measure the resulting diluted solution. 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 four reference solutions and the test solution, inject them into the liquid chromatograph, record the chromatograms, and calculate the contents of enrofloxacin, sulfamethoxypyrimidine sodium, florfenicol, and bromhexine hydrochloride in the test sample by peak area using the external standard method.

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