Method for simultaneously determining contents of two main drugs in tulathromycin and carprofen injection

Abstract

The invention provides a method for simultaneously determining the contents of two main drugs in tulathromycin and carprofen injection. The method comprises the following steps: selecting liquid chromatographic conditions: taking octadecylsilane chemically bonded silica as a chromatographic column of a filler, and taking trifluoroacetic acid and acetonitrile as mobile phases; a proper amount of a tulathromycin reference substance and a proper amount of a carprofen reference substance are taken, the tulathromycin reference substance and the carprofen reference substance are dissolved and diluted with a mobile phase to prepare the reference substance solution containing tulathromycin and carprofen at the same time, and each 1 ml of the reference substance solution contains 1.0 mg of tulathromycin and 0.5 mg of carprofen; preparation of a test solution: precisely weighing a proper amount of tulathromycin and carprofen injection, and adding a mobile phase for dilution until each ml of the test solution contains 1.0 mg of tulathromycin and 0.5 mg of carprofen; and respectively taking the reference solution and the test solution, injecting into a liquid chromatograph for determination, and calculating the concentration of the test solution by peak area according to an external standard method. The contents of the two main components can be accurately measured at the same time under the adopted chromatographic conditions, the operation is simple and convenient, and the efficiency is high.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug content detection, and relates to a method for determining the content of a compound injection, specifically to a method for simultaneously determining the contents of two main drugs in tulathromycin and carprofen injection. Background Art

[0002] Bovine respiratory disease (BRD) caused by pathogens such as bacteria or viruses is a common disease in cattle and one of the main reasons for economic losses in the global cattle industry. The main pathogenic bacteria causing BRD include Pasteurella multocida, Mannheimia haemolytica, Histophilus somni, Streptococcus, Arcanobacterium pyogenes, Mycoplasma, etc., and the viruses include bovine herpesvirus 1 (BHV-1), parainfluenza virus type III (PI-3), bovine respiratory syncytial virus (BRSV), etc.; whether it is primary bacterial infection or secondary bacterial infection caused by the low immunity of cattle due to virus, it will cause pneumonia, and the clinical manifestations are fever, listlessness, cough and runny nose, loss of appetite or even anorexia, body weight loss and other symptoms. If the treatment is not timely, it will affect the growth and development of cattle and even lead to death, causing serious losses to the cattle farm.

[0003] Mastitis in dairy cows is a common inflammatory disease in the process of dairy cow breeding. This disease will not only lead to a decrease in milk production and milk quality of dairy cows, but also the pathogenic bacteria and their secreted toxins will exist in the milk, which has a serious impact on the farm and human health. It is reported that the economic loss caused by subclinical mastitis in China is about 135 million yuan per year, and the clinical incidence of mastitis in large dairy farms can be as high as 3.3% per month, and the total economic loss caused by mastitis per year exceeds 600 million yuan.

[0004] Currently, clinically, antibacterial drugs or a combination of antipyretic, analgesic and anti-inflammatory drugs are often relied on to treat bovine respiratory diseases and mastitis in dairy cows.

[0005] Tulathromycin and Carprofen Injection is a veterinary compound preparation developed by our company, and its main components are tulathromycin and carprofen. Among them, tulathromycin is a semi-synthetic macrolide antibiotic dedicated to animals developed by Pfizer in the United States, mainly used for the treatment and prevention of bovine respiratory diseases caused by Mannheimia haemolytica, Pasteurella multocida, Haemophilus somnus, and Mycoplasma sensitive to tulathromycin; for the treatment and prevention of swine respiratory diseases caused by Actinobacillus pleuropneumoniae, Pasteurella multocida, and Mycoplasma pneumoniae sensitive to tulathromycin. Carprofen is a 2-arylpropionic acid non-steroidal anti-inflammatory and analgesic drug, with strong anti-inflammatory, analgesic, and antipyretic effects, good anti-inflammatory effect, and few side effects. It has been widely used in the United States, Canada, and many European countries. Its effect is significantly stronger than that of aspirin, phenylbutazone, paracetamol, and ibuprofen. It also has the characteristics of rapid absorption, few side effects, and the advantage of no residue in milk. The combination of the two can significantly improve the clinical symptoms of animals and is used for the treatment of various clinical diseases caused by sensitive bacteria.

[0006] There is no standard for the raw materials and preparations of tulathromycin and carprofen in the Chinese Veterinary Pharmacopoeia. The standards for tulathromycin raw materials and preparations are included in the Compilation of Veterinary Drug Quality Standards (2006 - 2011), and the standard for carprofen raw materials is included in the EP foreign pharmacopoeia. However, Tulathromycin and Carprofen Injection is not included in various foreign pharmacopoeias, the Chinese Veterinary Pharmacopoeia, and other national legal standards. Therefore, currently, the content determination methods of the two main drugs use two methods for separate determination, but this determination method requires establishing two different chromatographic conditions and separately preparing reference substance solutions and test solution, with complex operations. Summary of the Invention

[0007] In view of the above defects or deficiencies in the prior art, the present invention provides a method for simultaneously determining the contents of two main drugs in Tulathromycin and Carprofen Injection to solve the problems existing in the above prior art.

[0008] Its main technical solution is: A method for simultaneously determining the contents of two main drugs in Tulathromycin and Carprofen Injection, including the following steps:

[0009] (A) Selection of liquid chromatography conditions

[0010] A chromatographic column filled with octadecylsilane chemically bonded silica gel, with 0.05% trifluoroacetic acid: acetonitrile as the mobile phase; the initial detection wavelength is 230 nm, and the detection wavelength is adjusted to 205 nm after 5.5 minutes; the flow rate is 1.0 mL / min.

[0011] (B) Preparation of solutions

[0012] Preparation of reference solution: Weigh appropriate amounts of tulathromycin reference substance and carprofen reference substance, dissolve and dilute them with the mobile phase to prepare a reference solution containing both tulathromycin and carprofen. Each 1 ml of the reference solution contains 1.0 mg of tulathromycin and 0.5 mg of carprofen.

[0013] Preparation of test solution: Precisely weigh an appropriate amount of tulathromycin and carprofen injection, dilute it with the mobile phase to prepare a test solution containing 1.0 mg of tulathromycin and 0.5 mg of carprofen per 1 ml.

[0014] (C) Determination of samples

[0015] Take 20 μl of the reference solution and the test solution respectively, inject them into the liquid chromatograph for determination, and calculate the concentration of the test solution by the external standard method based on the peak area.

[0016] Furthermore, the ratio of 0.05% trifluoroacetic acid to acetonitrile is 40:60.

[0017] The beneficial effects of the present invention are as follows:

[0018] With the same liquid chromatography conditions, the present invention can simultaneously determine the contents of three components (tulathromycin A, tulathromycin B, carprofen) of two main drugs, tulathromycin and carprofen. The original method requires establishing two chromatographic conditions and preparing four solutions, and the retention times for detecting tulathromycin A, tulathromycin B, and carprofen are relatively long. This method only needs to establish one chromatographic condition and prepare two solutions, shortening the time and operation of liquid chromatography detection and improving the efficiency. The method of the present invention has been verified by a systematic methodology study, with strong specificity, high accuracy, and good reproducibility, and is suitable for adoption in quality control standards, which is conducive to improving the detection efficiency of products.

[0019] In the method of the present invention, there is a good linear relationship between the concentrations of tulathromycin and carprofen and the peak areas. Therefore, the content calculation methods of tulathromycin and carprofen in the method of the present invention can be obtained by simple external standard method calculation, and the calculation method is simple. Description of the drawings

[0020] By reading the detailed description of the non-limiting embodiments with reference to the following drawings, other features, purposes, and advantages of the present application will become more obvious:

[0021] Figure 1 It is the liquid chromatogram of the tulathromycin reference solution in the quality standard compilation method of the present invention;

[0022] Figure 2 It is the liquid chromatogram of the carprofen reference solution in the EP pharmacopoeia method of the present invention;

[0023] Figure 3 It is the liquid chromatogram of the tulathromycin reference solution of the present invention;

[0024] Figure 4 This is the liquid chromatogram of the carprofen reference substance solution of the present invention;

[0025] Figure 5 This is the liquid chromatogram of the mixed reference substance solution of the present invention;

[0026] Figure 6 This is the liquid chromatogram of the test sample (tilmicosin carprofen injection) solution of the present invention;

[0027] Figure 7 This is the linear relationship diagram between the concentration of the tilmicosin solution and the peak area of the present invention;

[0028] Figure 8 This is the linear relationship diagram between the concentration of the carprofen solution and the peak area of the present invention. Detailed implementation manners

[0029] The following further elaborates the present application in conjunction with the drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the relevant invention and not to limit the invention. Additionally, it should be noted that for the sake of description, only the parts related to the invention are shown in the drawings.

[0030] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments can be combined with each other. The following will elaborate on the present application in detail with reference to the drawings and embodiments.

[0031] The present invention can accurately measure the contents of two main components simultaneously under a chromatographic condition, with simple operation and high efficiency.

[0032] Embodiment:

[0033] The present invention provides a high performance liquid chromatography method for simultaneously determining the contents of two main components in tilmicosin carprofen injection. The invention of this method simplifies the operation complexity of solution preparation and determination in the original method for separate determination, making the determination of the content of tilmicosin carprofen injection more accurate, convenient and efficient.

[0034] The technical solution adopted by the present invention is:

[0035] A high performance liquid chromatography method for simultaneously determining the contents of two main drugs in tilmicosin carprofen injection, comprising the following steps:

[0036] Further elaboration on the present invention:

[0037] (A) Selection of liquid chromatography conditions

[0038] A chromatographic column packed with octadecylsilyl silica gel, with a mobile phase of 0.05% trifluoroacetic acid: acetonitrile (40:60); the initial detection wavelength is 230 nm, and the detection wavelength is adjusted to 205 nm after 5.5 minutes; the flow rate is 1.0 mL / min, and the resolution between each component peak should be greater than 1.5.

[0039] (B) Preparation of reference solution

[0040] Weigh appropriate amounts of tulathromycin reference substance and carprofen reference substance respectively, dissolve and dilute with the mobile phase solution to prepare a reference solution containing both tulathromycin and carprofen, a solution containing approximately 1.0 mg of tulathromycin and 0.5 mg of carprofen per 1 mL.

[0041] (C) Preparation of test solution

[0042] Precisely weigh an appropriate amount of tulathromycin and carprofen injection, place it in a brown volumetric flask, dilute with the mobile phase solution to prepare a solution containing approximately 1.0 mg of tulathromycin and 0.5 mg of carprofen per 1 mL, shake well, and you will get it.

[0043] (D) Determination

[0044] Precisely pipette 20 μL each of the reference solution and the test solution, inject them into the liquid chromatograph for determination. Calculate the concentration of the test solution by the external standard method based on the peak area, and you will get it.

[0045] The measured results: The contents of tulathromycin and carprofen are 99.5% and 101.0% respectively.

[0046] The improvement of the present invention lies in that the mobile phase used is 0.05% trifluoroacetic acid: acetonitrile (40:60).

[0047] The further improvement of the present invention lies in that the initial detection wavelength is 230 nm, and the detection wavelength is adjusted to 205 nm after 5.5 minutes; the flow rate is 1.0 mL / min.

[0048] (1) Regarding the specificity of the method of the present invention

[0049] (1) The chromatograms of detecting tulathromycin and carprofen reference substances by the included method are shown in Figure 1 and Figure 2 .

[0050] (2) Preparation of tulathromycin reference solution: Weigh an appropriate amount of tulathromycin reference substance, precisely weigh it, dissolve it with the mobile phase and quantitatively dilute it to prepare a solution containing 1.0 mg of tulathromycin per 1 mL. Precisely pipette 20 μL, inject it into the liquid chromatograph, and record the chromatogram (see Figure 3 ).

[0051] (3) Preparation of ketoprofen reference solution: Weigh an appropriate amount of ketoprofen reference substance accurately, dissolve it in the mobile phase and quantitatively dilute it to prepare a solution containing 0.5 mg of ketoprofen per 1 ml. Precisely pipette 20 μl and inject it into the liquid chromatograph, record the chromatogram (see Figure 4 ).

[0052] (4) Preparation of mixed reference solution: Weigh appropriate amounts of tulathromycin and ketoprofen reference substances respectively, dissolve them in the mobile phase and quantitatively dilute them to prepare a solution containing approximately 1.0 mg of tulathromycin and 0.5 mg of ketoprofen per 1 ml. Precisely pipette 20 μl and inject it into the liquid chromatograph, record the chromatogram (see Figure 5 ).

[0053] (5) Preparation of test solution: Take 1.0 ml of tulathromycin and ketoprofen injection, place it in a 100-ml volumetric flask, dilute it to the mark with the mobile phase, precisely pipette 20 μl and inject it into the liquid chromatograph, record the chromatogram (see Figure 6 ).

[0054] The results of chromatographic detection are shown in Table 1.

[0055] Table 1 Specificity results - retention time

[0056]

[0057] Table 2 Specificity results - resolution

[0058] Solution Name Carprofen Tulathromycin B Tulathromycin A Tulathromycin Reference Solution — — 10.62 Carprofen Reference Solution 15.88 — — Mixed Reference Solution 16.12 5.87 10.57 Test Solution 16.04 5.96 10.55

[0059] As can be seen from Tables 1 - 2, the retention time of the main peak in the chromatogram of the reference solution is consistent with that in the chromatogram of the test solution. The resolution between the main peak and the adjacent peak in the test solution is good, meeting the requirements. The specificity of this method is good.

[0060] (II). Regarding the accuracy and reproducibility of the method

[0061] For tulathromycin and ketoprofen injection (batch number: 240609), the contents of the two main drugs were determined simultaneously by this method. The content of tulathromycin was determined by the content determination method of tulathromycin injection in the Compilation of Veterinary Drug Quality Standards (2006 - 2011), and the content of ketoprofen was determined by the content determination method of ketoprofen in the EP Pharmacopoeia. The determination results are shown in Table 3. The contents of the 6 - time content determination results of this method were 99.58% and 100.48% respectively, and the contents of the 6 - time determination results of the published method were 100.48% and 100.73% respectively. The results determined by the two methods were not significantly different, and the RSD values were 0.18% and 0.47% respectively, proving that the accuracy and reproducibility of this method both meet the requirements.

[0062] Table 3 Results of accuracy and reproducibility experiments

[0063]

[0064] (III) Linear Relationship between Solution Concentration and Peak Area in the Method of the Present Invention

[0065] Appropriately weigh an appropriate amount of tilmicosin and carprofen reference substances, dissolve them accurately in the mobile phase, and quantitatively dilute to prepare solutions containing 10 mg of tilmicosin and 5 mg of carprofen per 1 ml. Accurately measure 0.5 ml, 1 ml, 1.5 ml, 2 ml, 3 ml, and 4 ml of the above solutions respectively and place them in 20-ml volumetric flasks, dilute to the mark with the mobile phase, shake well, inject samples respectively, and record the chromatograms. Plot the peak area as the ordinate y and the concentration as the abscissa x respectively. The regression equations are as follows: For tilmicosin: ytilmicosin = 5.26×106x + 7121.8 (R2 = 0.9999), for carprofen: ycarprofen = 2.82×106x - 11346 (R2 = 0.9993). The results are shown in Table 3 and Figures 5-6 。

[0066] Table 4 Results of Determination of Solution Concentration and Peak Area

[0067]

[0068] From Table 3 and Figures 5-6 it can be seen that in the concentration range of 0.2512 - 2.0092 mg / ml for tilmicosin and 0.1262 - 1.0096 mg / ml for carprofen determined by the method of the present invention, the linear relationships are good.

[0069] The above description is only the preferred embodiment of the present application and the explanation of the applied technical principles. Those skilled in the art should understand that the scope of the invention involved in the present application is not limited to the technical solutions formed by the specific combination of the above technical features, but should also cover other technical solutions formed by any combination of the above technical features or their equivalent features without departing from the inventive concept. For example, the technical solutions formed by mutually replacing the above features with the (but not limited to) technical features with similar functions disclosed in the present application.

Claims

1. A method for simultaneously determining the contents of two main drugs in tulathromycin and carprofen injection, characterized in that, It includes the following steps: (A) Selection of liquid chromatography conditions A chromatographic column filled with octadecylsilyl silica gel, trifluoroacetic acid: acetonitrile as the mobile phase; the initial detection wavelength is 230 nm, and the detection wavelength is adjusted to 205 nm after 5.5 minutes; the flow rate is 1.0 mL / min; (B) Preparation of solutions Preparation of the reference solution: Take appropriate amounts of tulathromycin reference substance and carprofen reference substance, dissolve and dilute with the mobile phase to prepare a reference solution containing both tulathromycin and carprofen. Each 1 ml of the reference solution contains 1.0 mg of tulathromycin and 0.5 mg of carprofen; Preparation of the test solution: Accurately weigh an appropriate amount of tulathromycin and carprofen injection, dilute with the mobile phase to prepare a test solution containing 1.0 mg of tulathromycin and 0.5 mg of carprofen per 1 ml; (C) Determination of samples Respectively take the reference solution and the test solution, inject them into the liquid chromatograph for determination, and calculate the concentration of the test solution by the external standard method based on the peak area.

2. The method for simultaneously determining the contents of two main drugs in tulathromycin and carprofen injection according to claim 1, wherein: The concentration of the trifluoroacetic acid solution is 0.05%.

3. A method for simultaneously determining the contents of two main drugs in tulathromycin and carprofen injection according to claim 2, characterized in that: The ratio of 0.05% trifluoroacetic acid to acetonitrile is 40:

60.

4. A method for simultaneously determining the contents of two main drugs in tulathromycin and carprofen injection according to claim 1, characterized in that: Precisely pipette 20 μl each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine.

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