Method for detecting doxycycline monohydrate and related substances thereof

Abstract

The invention belongs to the field of detection, and particularly relates to a method for detecting doxycycline monohydrate and related substances thereof. A mobile phase and a chromatographic column are improved, the mobile phase is prepared from an acetate buffer solution and acetonitrile according to the volume ratio of 90: 10, the acetate buffer solution is prepared from 0.20 mol / L ammonium acetate and 0.1 mol / L disodium ethylene diamine tetraacetate-triethylamine according to the volume ratio of 90: 10: 1, and the pH value is adjusted to 9.0 with ammonia water; a chromatographic column adopts a C18 column with the size of 250 mm * 4.6 mm and the size of 5 microns. The detection method is high in specificity, good in solution stability, high in sensitivity, high in accuracy and good in linearity and range, and can be used for quantitative and qualitative analysis of doxycycline monohydrate and related substances thereof.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and particularly relates to a method for detecting doxycycline monohydrate and related substances. Background Art

[0002] Doxycycline monohydrate, English name Doxycycline, CAS number 17086-28-1, its molecular formula is C 22 H 26 N2O9 has a molecular weight of 462.450. It appears as a yellow powder, and its unique chemical structure endows it with specific physical and chemical properties. The antibacterial mechanism of doxycycline monohydrate is exquisite. It precisely binds to the A position of the bacterial ribosome's 30S subunit. This binding, like precise "targeting," blocks the aminoacyl-tRNA binding at that position, thereby inhibiting peptide chain growth and forcing a halt to bacterial protein synthesis. Without protein, a crucial "material foundation" for life, bacterial growth and reproduction are inhibited, achieving its antibacterial effect.

[0003] Doxycycline monohydrate plays a vital role in the field of veterinary medicine. For example, in the case of ringworm, a common skin disease caused by fungi, doxycycline monohydrate can help pets restore skin health by inhibiting the co-infection of related bacteria. When cats suffer from cat colds and flu, doxycycline monohydrate can effectively control secondary bacterial infections and alleviate respiratory symptoms. In the treatment of canine diseases, whether it is canine distemper, kennel cough, or canine parvovirus infection, doxycycline monohydrate is significantly effective in controlling secondary bacterial infections.

[0004] However, doxycycline monohydrate raw materials and their preparations are not currently available in China. The United States Pharmacopoeia provides a method for detecting doxycycline in doxycycline oral suspension. After chromatographic separation, the separation between doxycycline monohydrate and impurity F is less than 1.5, which is poor. The European Pharmacopoeia provides a method for detecting doxycycline. The separation between the impurities is good and can be used for the detection of the content and related substances of this product, but its mobile phase configuration is complex and has poor fault tolerance, which causes unnecessary errors in subsequent inspections. The chromatographic peaks in the Chinese Pharmacopoeia system suitability solution and the test solution are stacked, and the separation between oxytetracycline and adjacent peaks is less than 1.5, which is poor. The separation between β-doxycycline and adjacent impurity peaks in the test solution is less than 1.5. Due to the poor separation, it cannot be used to detect the related substances and content of this product. CN111443139A discloses a method for simultaneously detecting the relative retention time and peak purity of doxycycline and related substances in doxycycline hydrochloride, and CN118759106A discloses a method for detecting the content of soluble powder of doxycycline hydrochloride by liquid chromatography. Both methods use similar methods to those in the Chinese Pharmacopoeia and cannot overcome the existing defects.

[0005] In summary, it can be seen that there is an urgent need to develop a detection method for doxycycline monohydrate-related substances with good separation effect, simple operation and wide applicability. Summary of the Invention

[0006] In response to the above problems, the present invention combines previous research results, improves the mobile phase and chromatographic column, and provides a method for detecting doxycycline monohydrate and its related substances, overcoming the existing technical problems.

[0007] The present invention adopts the following technical solutions:

[0008] A method for detecting doxycycline monohydrate and its related substances comprises the following steps: using liquid chromatography to detect a test solution; the mobile phase comprises acetate buffer and acetonitrile in a volume ratio of 90:10; the acetate buffer comprises 0.20 mol / L ammonium acetate-0.1 mol / L disodium ethylenediaminetetraacetate-triethylamine in a volume ratio of 90:10:1; and the pH value is adjusted to 9.0 with aqueous ammonia; and the diluent is 0.01 M hydrochloric acid. The chromatographic conditions are: a detection wavelength of 280 nm, a chromatographic column of 250 mm×4.6 mm, 5 μm C18 column (pH applicable range greater than 9), a column temperature of 35° C., and an injection volume of 20 μl.

[0009] Furthermore, the test sample is an oral suspension or oral paste of doxycycline monohydrate.

[0010] Furthermore, the related substances are selected from one or more of oxytetracycline, metacycline, β-doxycycline, and impurities.

[0011] Furthermore, the detection method further comprises preparing a doxycycline monohydrate reference solution, which is fixed to volume using a diluent to a concentration of 200 mg / L.

[0012] Furthermore, the detection method also includes preparing a system suitability solution, which is fixed to volume using a diluent, including 100 mg / L oxytetracycline positioning solution, 100 mg / L metacycline positioning solution, 100 mg / L β-doxycycline positioning solution and 200 mg / L doxycycline monohydrate.

[0013] Furthermore, the detection method includes preparing a test solution, dissolving it with a diluent, and preparing a solution with a theoretical concentration of doxycycline monohydrate of 200 mg / L.

[0014] Compared with the prior art, the beneficial effects of the present invention include at least:

[0015] 1. The present invention adopts an improved mobile phase and pH for the detection of doxycycline monohydrate and its preparations. The components are well separated, the detection method has strong specificity, good solution stability, high sensitivity, high accuracy, good linearity and range, and can be used for the quantitative and qualitative analysis of doxycycline monohydrate and its related substances.

[0016] 2. The use of durable reversed-phase HPLC columns can provide longer service life and stability under extreme pH conditions, making the analysis method more reproducible and efficient. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the standard curve of metacycline;

[0018] Figure 2 This is the standard curve of β-doxycycline;

[0019] Figure 3 is the standard curve of oxytetracycline;

[0020] Figure 4 This is the standard curve of doxycycline monohydrate. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary illustrations of the specific embodiments of the present invention, are intended to be used to explain the present invention, and do not constitute limitations of the present invention. The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include approximate ranges.

[0022] Example 1: Establishment of a doxycycline monohydrate detection method

[0023] Combined with the previous research results, the mobile phase and chromatographic column were improved. The improved method is as follows:

[0024] Mobile phase: acetate buffer [0.20 mol / L ammonium acetate-0.1 mol / L disodium ethylenediaminetetraacetate-triethylamine (90:10:1), pH adjusted to 9.0 with aqueous ammonia]-acetonitrile (90:10).

[0025] Diluent: 0.01M hydrochloric acid

[0026] Chromatographic conditions: detection wavelength 280 nm; chromatographic column 250 mm × 4.6 mm, 5 μm C18 column (pH applicable range greater than 9); column temperature 35°C; injection volume 20 μl.

[0027] Oxytetracycline positioning solution: Take 10 mg of oxytetracycline reference solution, accurately weigh it, put it into a 100 ml volumetric flask, add 0.01 mol / L hydrochloric acid solution to make up the volume, shake well, and use it as the oxytetracycline positioning solution.

[0028] Metacycline localizing solution: Take 10 mg of metacycline reference solution, accurately weigh it, put it into a 100 ml volumetric flask, add 0.01 mol / L hydrochloric acid solution to make up the volume, shake well, and use it as the metacycline localizing solution.

[0029] β-doxycycline localizing solution: Take 10 mg of β-doxycycline reference solution, accurately weigh it, and place it in a 100 ml volumetric flask. Add 0.01 mol / LM0. hydrochloric acid solution to the volume and shake well to prepare the β-doxycycline localizing solution.

[0030] Doxycycline monohydrate reference solution: Take 20 mg of doxycycline monohydrate reference solution, accurately weigh it, put it into a 100 ml volumetric flask, add 0.01 M hydrochloric acid solution to the volume, shake well, and use it as the doxycycline monohydrate standard solution.

[0031] System suitability solution: Weigh accurately 10 mg each of oxytetracycline, metacycline, and β-doxycycline. Then weigh accurately 20 mg of doxycycline monohydrate in a 100 ml volumetric flask, add 0.01 mol / L solution to the volume, and shake well to prepare the system suitability solution.

[0032] Related substance test solution: Take the test sample, add 0.01 mol / L hydrochloric acid solution, dissolve and dilute to make a solution containing approximately 0.2 mg of doxycycline monohydrate per 1 ml, which is used as the test solution.

[0033] The liquid chromatography test results are shown in Table 1. The improved doxycycline monohydrate test method was used to detect the related substances of this product. The separation of each component in the system suitability solution met the requirements. The separation of each component in the test solution also met the requirements.

[0034] Table 1: Summary of liquid chromatography test results

[0035]

[0036] Example 2: Validation of the Doxycycline Monohydrate Detection Method

[0037] (1) Solution stability:

[0038] Test and control solutions were prepared and injected at 0, 3, 6, 9, 12, 15, 22.5, and 29 hours. The stability results of the test and control solutions are shown in Table 2-3. The results indicate that both the test and control solutions were stable within 29 hours.

[0039] Table 2: Stability results of reference solution after injection at different time

[0040]

[0041]

[0042] (2) System adaptability:

[0043] Six consecutive injections of the system suitability solution were measured to examine the retention time, peak area, and resolution of each component. The results, shown in Table 4, show that the RSDs for the retention time and peak area of each component measured over the six consecutive injections were all less than 2.0%, and the resolution between each substance was >1.5, indicating good system suitability of this method.

[0044]

[0045] (3) Sensitivity:

[0046] Reference solutions of metacycline, β-doxycycline, oxytetracycline, and doxycycline monohydrate were prepared at different concentrations, and their peak elution patterns and S / N values were examined. The limits of detection and quantification (LOQs) of each component were calculated based on signal-to-noise ratios (S / N ≥ 3 and S / N ≥ 10, respectively. The results are shown in Table 5. The LOQs for metacycline, β-doxycycline, oxytetracycline, and doxycycline monohydrate were 0.0537 μg / ml, 0.0808 μg / ml, 0.0443 μg / ml, and 0.0415 μg / ml, respectively. The limits of detection for metacycline, β-doxycycline, oxytetracycline, and doxycycline monohydrate were 0.0171 μg / ml, 0.0253 μg / ml, 0.0149 μg / ml, and 0.0108 μg / ml, respectively.

[0047] Table 5: Sensitivity test results

[0048]

[0049] (4) Linearity:

[0050] Accurately weigh an appropriate amount of metacycline reference substance and dilute it with diluent to prepare solutions containing 0.0537 μg / ml, 2.0267 μg / ml, 4.0533 μg / ml, 5.0666 μg / ml, 6.0800 μg / ml, and 8.1066 μg / ml of metacycline as the metacycline standard curve solutions.

[0051] Accurately weigh an appropriate amount of β-doxycycline reference substance and dilute it with diluent to prepare solutions containing 0.088 μg / ml, 2.0200 μg / ml, 4.0400 μg / ml, 5.0500 μg / ml, 6.0600 μg / ml, and 8.0800 μg / ml of β-doxycycline as the β-doxycycline standard curve solutions.

[0052] Accurately weigh an appropriate amount of oxytetracycline reference substance and dilute it with diluent to prepare solutions containing 0.0443μg / ml, 0.8862μg / ml, 1.7724μg / ml, 2.2156μg / ml, 2.6587μg / ml, and 3.5449μg / ml of oxytetracycline as the oxytetracycline standard curve solutions.

[0053] Accurately weigh an appropriate amount of doxycycline monohydrate reference substance and dilute it with diluent to prepare solutions containing 0.1 μg / ml, 4 μg / ml, 0.1 mg / ml, 0.2 mg / ml, and 0.24 mg / ml of doxycycline monohydrate as the doxycycline monohydrate standard curve solutions.

[0054] The results of the linear experiment are shown in Table 6 and Figure 1-4 The results showed that metacycline had good linearity in the range of 0.0537μg / ml to 8.1066μg / ml, with an R value of 0.9999; β-doxycycline had good linearity in the range of 0.0808μg / ml to 8.0800μg / ml, with an R value of 0.9996; oxytetracycline had good linearity in the range of 0.0443μg / ml to 3.5449μg / ml, with an R value of 0.9999; doxycycline monohydrate had good linearity in the range of 0.0415μg / ml to 311.2452μg / ml, with an R value of 1.0000.

[0055] Table 6: Linearity test results

[0056]

[0057] (5) Accuracy:

[0058] Weigh doxycycline monohydrate (80%, 100%, and 120% of the prescribed amount) to prepare three samples with different contents. Prepare three copies of each concentration in parallel to prepare 9 copies of the test solution. Take appropriate amounts of oxytetracycline reference stock solution, β-doxycycline reference stock solution, and metacycline reference stock solution, add them to the test solution and prepare it to contain oxytetracycline 0.9 μg / ml, β-doxycycline 2 μg / ml, metacycline 2 μg / ml (equivalent to 50%), oxytetracycline 0.18 μg / ml, β-doxycycline 4 μg / ml, metacycline 4 μg / ml (equivalent to 100%), oxytetracycline 0.27 μg / ml, β-doxycycline 6 μg / ml, and metacycline 6 μg / ml (equivalent to 150%) (3 copies of each), and prepare 1 copy of the test solution as the background value solution.

[0059] The recovery results for metacycline are shown in Table 7, the recovery results for β-doxycycline are shown in Table 8, and the recovery results for oxytetracycline are shown in Table 9. The recoveries of metacycline, β-doxycycline, and oxytetracycline were all between 92% and 108%, and their RSDs were all less than 2%. This method has good accuracy.

[0060] Table 7: Related substance metacycline recovery results

[0061]

[0062] Table 8: Recovery results of related substances β-doxycycline

[0063]

[0064] Table 9: Related substance oxytetracycline recovery results table

[0065]

[0066] In summary, the detection method of the doxycycline monohydrate oral paste of the present invention has strong specificity, good solution stability, high sensitivity, high accuracy, good linearity and range, and can be used for the quantitative and qualitative analysis of doxycycline monohydrate and its related substances.

[0067] Comparative Example 1: Detection Method for Related Substances of Doxycycline Monohydrate in the Chinese Pharmacopoeia

[0068] Mobile phase: acetate buffer [0.25 mol / L ammonium acetate-0.1 mol / L disodium ethylenediaminetetraacetate-triethylamine (100:10:1), pH adjusted to 8.8 with glacial acetic acid or aqueous ammonia]-acetonitrile (85:15).

[0069] Diluent: 0.01M hydrochloric acid

[0070] Chromatographic conditions: detection wavelength 280 nm; chromatographic column 150 mm × 4.6 mm, 3 μm C18 column (pH applicable range greater than 9); column temperature 35°C; injection volume 20 μl.

[0071] Oxytetracycline positioning solution: Take 10 mg of oxytetracycline reference solution, accurately weigh it, put it into a 100 ml volumetric flask, add 0.01 mol / L hydrochloric acid solution to make up the volume, shake well, and use it as the oxytetracycline positioning solution.

[0072] Metacycline localizing solution: Take 10 mg of metacycline reference solution, accurately weigh it, put it into a 100 ml volumetric flask, add 0.01 mol / L hydrochloric acid solution to make up the volume, shake well, and use it as the metacycline localizing solution.

[0073] β-doxycycline localizing solution: Take 10 mg of β-doxycycline reference solution, accurately weigh it, and place it in a 100 ml volumetric flask. Add 0.01 mol / LM0. hydrochloric acid solution to the volume and shake well to prepare the β-doxycycline localizing solution.

[0074] Doxycycline monohydrate reference solution: Take 20 mg of doxycycline monohydrate reference solution, accurately weigh it, put it into a 100 ml volumetric flask, add 0.01 M hydrochloric acid solution to the volume, shake well, and use it as the doxycycline monohydrate standard solution.

[0075] System suitability solution: Weigh accurately 10 mg each of oxytetracycline, metacycline, and β-doxycycline. Then weigh accurately 20 mg of doxycycline monohydrate in a 100 ml volumetric flask, add 0.01 mol / L solution to the volume, and shake well to prepare the system suitability solution.

[0076] Related substance test solution: Take this product, add 0.01 mol / L hydrochloric acid solution, dissolve and dilute to make a solution containing approximately 0.2 mg of doxycycline monohydrate per 1 ml, as the test solution.

[0077] The results are shown in Table 10. The related substances of this product were detected using the related substance detection method of doxycycline monohydrate in the Chinese Pharmacopoeia. The separation degree of oxytetracycline and the adjacent peaks in the system suitability solution was less than 1.5, which interfered with the elution of oxytetracycline.

[0078] Table 10: Summary of test results of Chinese Pharmacopoeia test methods

[0079]

[0080] Finally, it should be noted that the above embodiments are intended only to illustrate the technical solutions of the present invention and do not constitute a limitation of the present invention. Within the technical concept of the present invention, the technical solutions of the present invention may be subjected to various simple modifications, including combining the various technical features in any other appropriate manner. These simple modifications and combinations should also be considered as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A method for detecting doxycycline monohydrate and its related substances, characterized in that: The test solution was tested by liquid chromatography; The mobile phase was prepared with acetate buffer and acetonitrile in a volume ratio of 90:

10. The acetate buffer was prepared with 0.20 mol / L ammonium acetate, 0.1 mol / L disodium ethylenediaminetetraacetate, and triethylamine in a volume ratio of 90:10:1, and the pH was adjusted to 9.0 with aqueous ammonia. The diluent is 0.01M hydrochloric acid; The chromatographic conditions were as follows: a detection wavelength of 280 nm, a 250 mm × 4.6 mm, 5 μm C18 column (pH applicable range greater than 9), a column temperature of 35° C., and an injection volume of 20 μl.

2. The detection method according to claim 1, wherein The test sample is doxycycline monohydrate oral suspension or oral paste.

3. The detection method according to claim 1, wherein The related substances are selected from one or more of oxytetracycline, metacycline, β-doxycycline and impurities.

4. The detection method according to claim 1, wherein The detection method further includes preparing a doxycycline monohydrate reference solution, which is fixed to volume using a diluent to a concentration of 200 mg / L.

5. The detection method according to claim 1, wherein The detection method also includes preparing a system suitability solution, which is fixed to volume using a diluent, including a 100 mg / L oxytetracycline positioning solution, a 100 mg / L metacycline positioning solution, a 100 mg / L β-doxycycline positioning solution, and a 200 mg / L doxycycline monohydrate.

6. The detection method according to claim 1, wherein The detection method comprises preparing a test solution, dissolving the solution in a diluent, and preparing a solution with a theoretical concentration of doxycycline monohydrate of 200 mg / L.

Images