A method for determining the content of polycarbophil in azithromycin eye drops

Abstract

The present invention discloses a method for determining the content of polycarbophil in azithromycin eye drops, which adopts liquid chromatography to determine the content of polycarbophil in azithromycin eye drops in a size exclusion mode. The present invention provides a method for determining the content of polycarbophil in azithromycin eye drops, which specifically involves the selection of a chromatographic column, the optimization of a mobile phase and the verification of a chromatographic method, and can accurately determine the content of polycarbophil in azithromycin eye drops, with good separation effect, good repeatability, low interference and good quantitative linearity, thus filling the gap in the prior art.

Description

Technical Field

[0001] The invention relates to the field of chemical analysis, and in particular to a method for determining the content of polycarbophil in azithromycin eye drops. Background Art

[0002] Azithromycin is a 15-membered macrolide antibiotic with a broad antibacterial spectrum. In addition to its antibacterial effect on most bacteria, it also has good activity against Chlamydia and Mycoplasma. It is widely used in the clinic for acute pharyngitis and acute tonsillitis caused by Streptococcus pyogenes; sinusitis, otitis media, acute bronchitis, acute exacerbation of chronic bronchitis caused by sensitive bacteria; pneumonia caused by Streptococcus pneumoniae, Haemophilus influenzae and Mycoplasma pneumoniae; urethritis and cervicitis caused by Chlamydia trachomatis and non-multi-drug resistant Neisseria gonorrhoeae; skin and soft tissue infections caused by sensitive bacteria, etc.

[0003] On April 27, 2007, the U.S. FDA approved the marketing of Inspire Pharmaceuticals' 1% azithromycin eye drops (trade name) for the treatment of bacterial conjunctivitis. The preparation uses the sustained-release drug delivery technology DuraSite, which can enhance the time that the antibiotic stays on the tissue surface, thereby achieving a sustained-release effect of once-daily medication. The sustained-release matrix of the currently available 1% azithromycin eye drops is the pharmaceutical excipient polycarbophil. Polycarbophil is a resin cross-linked with polyacrylic acid and 3,4-diol-1,5-hexadiene, which has the characteristics of high water retention. Calcium polycarbophil is the calcium salt of polycarbophil. U.S. Patent (US3297664) describes the use of calcium polycarbophil for the treatment of diarrhea and constipation.

[0004]

[0005] Physical and chemical properties of polycarbophil: Polycarbophil is a white powdery solid with a molecular weight greater than 10 6 , insoluble in water, strong acid and alkali. The pH value of 1% aqueous dispersion is 3.5-4.0. Polycarbophil has water absorption and swelling properties, and the water absorption capacity per gram can reach 62g (USP24); its swelling property is affected by the pH value. Under acidic conditions, it can absorb 10 times its own weight in water, and the swelling rate increases significantly above pH 4; under neutral conditions, it can absorb 70 times its own weight in water. The swelling property of polycarbophil is also affected by ionic strength. As the ionic strength increases, the swelling rate decreases. Divalent metal ions can change this swelling equilibrium, but the effect is small when sodium ions are present.

[0006]

[0007] Existing detection technologies for polycarbophil in azithromycin eye drops include: a method for determining the content of azithromycin in azithromycin sustained-release eye drops (CN102590392B), which discloses an HPLC method for excluding the interference of polycarbophil to accurately determine the content of azithromycin in azithromycin eye drops; a method for detecting related substances of azithromycin for injection (CN103245744A), which introduces the analysis of the injection amount limit of azithromycin B, azithromycin Gx and erythromycin A iminoether in the drug through an abnormal toxicity test test, and evaluates whether the content of related substances in the drug reaches a safe level, thereby achieving the purpose of quantitative evaluation; a method for detecting related substances in azithromycin eye drops (CN104267122A), which provides a method for excluding the interference of ionic compounds (such as preservative benzalkonium chloride) in accurately determining the related substances of azithromycin eye drops; a method for determining polycarbophil calcium oligomers (CN104049059A). However, none of these prior arts discloses a method for determining the content of polycarbophil in azithromycin eye drops. Summary of the invention

[0008] In order to solve the existing technical problems, the present invention provides a method for detecting the content of polycarbophil in azithromycin eye drops. The method can eliminate the interference of other excipients in the detection of polycarbophil in azithromycin eye drops, thereby achieving accurate determination of the content of polycarbophil in azithromycin eye drops.

[0009] To achieve the above object, the technical solution of the present invention is as follows: a method for determining the content of polycarbophil in azithromycin eye drops, using liquid chromatography in size exclusion mode to determine the content of polycarbophil in azithromycin eye drops.

[0010] Wherein, the size exclusion chromatography column filler contains neutral hydrophilic surface functional groups. Preferably, the neutral hydrophilic surface functional groups are ethylene glycol, polyethylene glycol or polyol.

[0011] Wherein, the particle size of the size exclusion chromatography column filler is 1-50 μm. Preferably, the particle size of the size exclusion chromatography column filler is 2, 3, 5 or 10 μm.

[0012] Wherein, the pore size of the size exclusion chromatography column packing is Preferably, the pore size of the size exclusion chromatography column packing is More preferably, the pore size of the size exclusion chromatography column packing is

[0013] Among them, the detection method for determining the polycarbophil content is evaporative light detection, electrospray detection, and ultraviolet detection.

[0014] Wherein, the mobile phase when using evaporative light detection or electrospray detection is: pure water, 5-500 mM ammonium formate or ammonium acetate aqueous solution (pH 2-7), 0.01-2% formic acid or acetic acid aqueous solution.

[0015] Wherein, the mobile phase when ultraviolet detection is adopted is: pure water, 5-200mM phosphate aqueous solution (pH2-7), 0.01-2% formic acid or acetic acid aqueous solution.

[0016] Wherein, during the measurement, the temperature of the chromatographic column is 10-50° C. Preferably, the temperature of the chromatographic column is 30° C.

[0017] The linear velocity during measurement is 0.1-0.5 mm / s. Preferably, the linear velocity is 0.25-0.35 mm / s.

[0018] When using evaporative light detection (ELSD), the preferred chromatographic conditions are: chromatographic column: BioCoreSEC-150 (4.6×300 mm, 5 μm), mobile phase: 10 mM ammonium acetate, pH 5.2 (0.77 g ammonium acetate dissolved in 1000 mL water, pH adjusted to 5.2 with glacial acetic acid), flow rate: 0.35 mL / min, temperature: 30°C, injection volume: 5 μL, detector: ELSD (Evap: 65°C, Neb: 55°C, Gas: 1.6 SLM). Under the preferred chromatographic conditions, polycarbophil (0.1 mg / mL) solution was repeatedly injected, and the retention time RSD of polycarbophil was 0.087%, and the peak area RSD was 0.118%, with good reproducibility; in the concentration range of 50-200 μg / mL, the linear equation was Log(y)=1.494Log(x)-2.3387, and the correlation coefficient R 2 The peak area had a good linear relationship with the concentration. The detection limit was 9 μg / mL and the quantification limit was 30 μg / mL.

[0019] When using ultraviolet detection (UV), the preferred chromatographic conditions are: chromatographic column: BioCore SEC-150 (4.6×300 mm, 5 μm), mobile phase: 10 / 90 v / v acetonitrile / 20 mM KH 2 PO 4 , pH 6.5, flow rate: 0.35 mL / min, temperature: 30°C, UV detection wavelength is 210 nm. Under these conditions, the linear equation for polycarbophil determination in the concentration range of 50-200 μg / mL is y=0.2675x-1.1053, and the correlation coefficient R 2 The peak area had a good linear relationship with the concentration. The detection limit was 6 μg / mL and the quantification limit was 20 μg / mL.

[0020] The present invention provides a liquid chromatography method for determining the content of polycarbophil in an actual sample (drug), including the selection of a chromatographic column, the optimization of a mobile phase and the verification of a chromatographic method. The provided determination method has the following advantages: good separation effect, good repeatability, small interference and good quantitative linearity. The present invention can eliminate the interference of other excipients in the detection of polycarbophil in azithromycin eye drops, accurately determine the content of polycarbophil in azithromycin eye drops, and fill the gap in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the chromatographic result of Example 2.

[0022] Figure 2 This is the chromatographic result of Example 3.

[0023] Figure 3 This is the chromatographic result of Example 4.

[0024] Figure 4 This is the chromatographic result of Example 5.

[0025] Figure 5 It is a schematic diagram of peak area comparison of Example 8.

[0026] Figure 6 It is the ultraviolet detection result figure of Example 9. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to these embodiments.

[0028] The present invention adopts liquid chromatography to determine the content of polycarbophil in azithromycin eye drops in a size exclusion mode.

[0029] Size Exclusion Chromatography Columns:

[0030] 1. Chromatographic column specifications: length: 50-600mm; inner diameter: 2-50mm. Preferred are 4.6×150mm, 4.6×300mm, 7.8×150mm, 7.8×300mm;

[0031] 2.Packing:

[0032] Matrix: silica gel or hybrid silica gel

[0033] Surface functional groups: neutral hydrophilic (ethylene glycol, polyethylene glycol and polyol)

[0034] Particle size: 1-50 μm. 2, 3, 5 and 10 μm are preferred

[0035] Aperture: Best Re-optimization

[0036] 3. Chromatographic conditions:

[0037] Detection methods: Evaporative light detection (ELSD), electrospray ionization detection (CAD), ultraviolet detection (UV@190-210nm)

[0038] Mobile Phase:

[0039] Compatible with ELSD and CAD: pure water, 5-500mM ammonium formate or ammonium acetate aqueous solution (pH2-7), 0.01-2% formic acid or acetic acid aqueous solution;

[0040] UV compatible: pure water, 5-200mM phosphate aqueous solution (pH2-7), 0.01-2% formic acid or acetic acid aqueous solution;

[0041] Temperature: 10-50°C. Preferably: 30°C.

[0042] Linear flow rate: 0.1-0.5 mm / s. Optimum: 0.25-0.35 mm / s.

[0043] Example 1 Preparation of samples

[0044] Azithromycin eye drops (diluted 10 times): Take 1 mL of azithromycin eye drops, place it in a 10 mL centrifuge tube, dilute to the scale with water, and shake well.

[0045] Blank excipient (diluted 10 times): Take 1 mL of blank excipient (without polycarbophil), place it in a 10 mL centrifuge tube, dilute to the scale with water, and shake well.

[0046] Polycarbophil control solution: Take an appropriate amount of polycarbophil powder, dissolve it in water and quantitatively dilute it to make a solution containing approximately 1 mg per 1 mL.

[0047] Poloxamer control solution: Take an appropriate amount of poloxamer powder, dissolve it in water and quantitatively dilute it to make a solution containing approximately 1 mg per 1 mL.

[0048] Example 2: Separation of azithromycin eye drops components using SEC columns with different pore sizes in size exclusion mode

[0049] Chromatographic column:

[0050] Two pore sizes were used: A: BioCoreSEC-300, 5 μm; B: BioCoreSEC-150, 5 μm;

[0051] Specification: 4.6×300mm

[0052] Mobile phase: 10 mM ammonium acetate, pH 5.2

[0053] Flow rate: 0.35mL / min

[0054] Temperature: 30℃

[0055] Injection volume: 5 μL

[0056] Detection: ELSD (Evap: 65℃, Neb: 55℃, Gas: 1.6SLM)

[0057] Sample: Azithromycin eye drops (diluted 10 times)

[0058] Peak name: 1. Polycarbophil

[0059] Test results see Figure 1 , where A is BioCoreSEC-300, 5μm, and B is BioCore SEC-150, 5μm.

[0060] The results show that: The SEC column with a larger pore size has better separation effect, and the peak shape of the main excipient polycarbophil is symmetrical and not interfered by other excipients.

[0061] Example 3: Use in size exclusion mode Study on different flow rates of SEC columns with different pore sizes

[0062] Column: BioCoreSEC-150, 5 μm;

[0063] Specification: 4.6×300mm

[0064] Mobile phase: 50 mM ammonium acetate, pH 5.2

[0065] Flow rate: A 0.35 mL / min, B 0.28 mL / min, C 0.21 mL / min;

[0066] Temperature: 30℃

[0067] Injection volume: 5 μL

[0068] Detection: ELSD (Evap: 65℃; Neb: 55℃; Gas: 1.6SLM)

[0069] Sample: Azithromycin eye drops (diluted 10 times)

[0070] Peak name: 1. Polycarbophil, 2. Poloxamer 407

[0071] Table 1. Chromatographic results of Example 2

[0072]

[0073] The obtained chromatogram is shown in Figure 2, where A is 0.35 mL / min, B is 0.28 mL / min, and C is 0.21 mL / min.

[0074] The results showed that the separation of polycarbophil from other components in azithromycin eye drops was not affected by the flow rate within the range of 0.21-0.35 mL / min. Considering the analysis throughput, a flow rate of 0.35 mL / min was used for subsequent studies.

[0075] Example 4: Use in size exclusion mode Study on the effect of pore size of SEC chromatographic column on the concentration of mobile phase buffer salt

[0076] Column: BioCoreSEC-150, 5 μm

[0077] Specification: 4.6×300mm

[0078] Mobile Phase:

[0079] C: 10 mM ammonium acetate, pH 5.2

[0080] B: 30 mM ammonium acetate, pH 5.2

[0081] A: 50 mM ammonium acetate, pH 5.2

[0082] Flow rate: 0.35mL / min

[0083] Temperature: 30℃

[0084] Injection volume: 5 μL

[0085] Detection: ELSD (Evap: 65℃, Neb: 55℃, Gas: 1.6SLM)

[0086] Sample: Azithromycin eye drops (diluted 10 times)

[0087] Peak names: 1. Polycarbophil, 2. Poloxamer 407, 3. Edetate, 4. Citrate, 5. Chloride ion, 6. Mannitol, 7. Sodium ion, 8. Azithromycin.

[0088] The obtained chromatogram is shown in Figure 3 , wherein A is 50 mM ammonium acetate, pH 5.2, B is 30 mM ammonium acetate, pH 5.2, and C is 10 mM ammonium acetate, pH 5.2.

[0089] The results showed that polycarbophil and poloxamer 407 in azithromycin eye drops were basically not affected by the buffer salt concentration. However, the separation of other components was greatly affected by the buffer salt concentration: the separation effect improved with the decrease of buffer salt concentration.

[0090] Example 5 Analysis of chromatographic separation of components of azithromycin eye drops under recommended conditions Chromatographic column: BioCoreSEC-150, 5 μm

[0091] Specification: 4.6×300mm

[0092] Mobile phase: 10 mM ammonium acetate, pH 5.2 (0.77 g ammonium acetate dissolved in 1000 mL water, pH adjusted to 5.2 with glacial acetic acid)

[0093] Flow rate: 0.35mL / min

[0094] Temperature: 30℃

[0095] Injection volume: 5 μL

[0096] Detection: ELSD (Evap: 65℃, Neb: 55℃, Gas: 1.6SLM)

[0097] sample:

[0098] A: diluent (water)

[0099] B: Polycarbophil (1 mg / mL)

[0100] C: Poloxamer 407 (1 mg / mL)

[0101] D: Blank excipient (diluted 10 times)

[0102] E: Azithromycin eye drops (diluted 10 times)

[0103] Peak names: 1. Polycarbophil, 2. Poloxamer 407, 3. Edetate, 4. Citrate, 5. Chloride ion, 6. Mannitol, 7. Sodium ion, 8. Azithromycin.

[0104] Figure 4 This is a chromatographic separation diagram for analyzing the components of azithromycin eye drops under recommended conditions, where A is the diluent (water), B is polycarbophil (1 mg / mL), C is poloxamer 407 (1 mg / mL), D is the blank excipient (diluted 10 times), and E is azithromycin eye drops (diluted 10 times).

[0105] The results showed that under the recommended conditions, azithromycin eye drops components were analyzed, azithromycin and its excipients were well separated, and the polycarbophil peak shape was good and free from interference.

[0106] Example 6. Polycarbophil solution (0.1 mg / mL) (10 injections) was continuously analyzed under the recommended chromatographic method, and the sample reproducibility was good.

[0107] Column: BioCoreSEC-150, 5 μm

[0108] Specification: 4.6×300mm

[0109] Mobile phase: 10 mM ammonium acetate, pH 5.2

[0110] Flow rate: 0.35mL / min

[0111] Temperature: 30℃

[0112] Injection volume: 10 μL

[0113] Detection: ELSD (Evap: 65℃, Neb: 55℃, Gas: 1.6SLM)

[0114] Sample: Polycarbophil (0.1 mg / mL)

[0115] Table 2. Precision experiment

[0116]

[0117]

[0118] Under the chromatographic conditions, polycarbophil (0.1 mg / mL) solution was repeatedly injected, and the retention time RSD of polycarbophil was 0.087%, and the peak area RSD was 0.118%, with good reproducibility.

[0119] Example 7. Linear quantitative range, detection limit and quantification limit of polycarbophil under the recommended chromatographic method

[0120] Column: BioCoreSEC-150, 5 μm

[0121] Specification: 4.6×300mm

[0122] Mobile phase: 10 mM ammonium acetate, pH 5.2

[0123] Flow rate: 0.35mL / min

[0124] Temperature: 30℃

[0125] Injection volume: 10 μL

[0126] Detection: ELSD (Evap: 65℃, Neb: 55℃, Gas: 1.6SLM)

[0127] Sample: Polycarbophil

[0128] Table 3. Regression equation and linear range

[0129]

[0130] Detection limit and quantification limit: Take the polycarbophil control solution and dilute it stepwise with water. When the signal-to-noise ratio is 3, the detection limit (LOD) of polycarbophil is 9 μg / mL. When the signal-to-noise ratio is 10, the quantification limit (LOQ) of polycarbophil is 30 μg / mL.

[0131] Under the preferred chromatographic conditions, the linear equation of polycarbophil in the concentration range of 50-200 μg / mL is Log(y)=1.494Log(x)-2.3387, and the correlation coefficient R 2 The peak area had a good linear relationship with the concentration. The detection limit was 9 μg / mL and the quantification limit was 30 μg / mL.

[0132] Example 8. Study on the tolerance of chromatographic columns under the recommended chromatographic method

[0133] Column: BioCoreSEC-150, 5 μm

[0134] Specification: 4.6×300mm

[0135] Mobile phase: 10 mM ammonium acetate, pH 5.2

[0136] Flow rate: 0.35mL / min

[0137] Temperature: 30℃

[0138] Injection volume: 10 μL

[0139] Detection: ELSD (Evap: 65℃, Neb: 55℃, Gas: 1.6SLM)

[0140] Sample: Polycarbophil (0.1 mg / mL)

[0141] Figure 5 is a schematic diagram of the comparison of peak areas in the obtained chromatograms.

[0142] The chromatographic column tolerance study showed that when the chromatographic column continuously analyzes a higher concentration of polycarbophil (1 mg / mL), the chromatographic column performance will decrease, resulting in abnormal peak shape. When the chromatographic column continuously analyzes 0.1 mg / mL polycarbophil, the performance of the chromatographic column is relatively stable. Therefore, when analyzing samples, the concentration of polycarbophil needs to be controlled below 200 μg / mL or a guard column should be added in front of the column.

[0143] The column tolerance study under this recommended chromatographic method showed that this method can provide stable separation results when the polycarbophil concentration in the sample is about 0.1 mg / mL.

[0144] Example 9. UV detection

[0145] Since polycarbophil has ultraviolet absorption, its content can also be determined under ultraviolet light. The chromatographic method is: the mobile phase is 10 / 90v / v acetonitrile / 20mMKH 2 PO 4 , pH 6.5, UV detection wavelength is 210nm.

[0146] Column: BioCoreSEC-150, 5 μm

[0147] Specification: 4.6×300mm

[0148] Mobile phase: 10 / 90v / v acetonitrile / 20mM KH 2 PO 4 , pH 6.5

[0149] Flow rate: 0.35mL / min

[0150] Temperature: 30℃

[0151] Injection volume: 10 μL

[0152] Detection: UV@210nm

[0153] Sample: Polycarbophil (0.1 mg / mL)

[0154] Peak name: 1. Polycarbophil

[0155] UV test results are shown in Figure 6 .

[0156] Example 10. Linear quantitative range, detection limit and quantification limit of polycarbophil under the recommended UV method

[0157] Column: BioCoreSEC-150, 5 μm

[0158] Specification: 4.6×300mm

[0159] Mobile phase: 10 / 90v / v acetonitrile / 20mM KH 2 PO 4 , pH 6.5

[0160] Flow rate: 0.35mL / min

[0161] Temperature: 30℃

[0162] Injection volume: 10 μL

[0163] Detection: UV@210nm

[0164] Sample: Polycarbophil (0.1 mg / mL)

[0165] Table 4. Regression equation and linear range

[0166]

[0167] Detection limit and quantification limit: Take the polycarbophil control solution and dilute it stepwise with water. When the signal-to-noise ratio is 3, the detection limit of polycarbophil is 6 μg / mL. When the signal-to-noise ratio is 10, the quantification limit of polycarbophil is 20 μg / mL.

[0168] The invention provides a method for determining the content of polycarbophil in azithromycin eye drops, which specifically involves the selection of a chromatographic column, the optimization of a mobile phase and the verification of a chromatographic method. The method can accurately determine the content of polycarbophil in azithromycin eye drops, eliminate the interference of other auxiliary materials, and has good separation effect, good repeatability, small interference and good quantitative linearity, thus filling the gap in the prior art.

[0169] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for determining the content of polycarbophil in azithromycin eye drops, characterized in that: The determination method is to use liquid chromatography to determine the content of polycarbophil in azithromycin eye drops in size exclusion mode; The determination method adopts size exclusion conditions, and the chromatographic column filler used has a neutral hydrophilic surface functional group; the surface functional group is ethylene glycol, polyethylene glycol or polyol; The particle size of the chromatographic column filler is 1-50 μm; The pore size of the chromatographic column packing is 100-200Å; The detection method for determining the content of polycarbophil adopts evaporative light detection, electrospray detection, and ultraviolet detection; When evaporative light detection or electrospray detection is used, the mobile phase of the chromatography is: 10-50 mM ammonium formate or ammonium acetate aqueous solution, or 0.01-2% formic acid or acetic acid aqueous solution; When UV detection was used, the mobile phase of the chromatography was: 10 / 90 v / v acetonitrile / 5-200 mM phosphate aqueous solution; the UV detection wavelength was 210 nm.

2. The method for determining the content of polycarbophil in azithromycin eye drops according to claim 1, characterized in that: When evaporative light detection was used, the chromatographic conditions were as follows: column: BioCore SEC-150, 4.6 × 300 mm, 5 μm; Mobile phase: 10 mM ammonium acetate, pH 5.2, flow rate: 0.35 mL / min, temperature: 30°C, injection volume: 5 μL, detector: ELSD, detector conditions: Evap: 65°C, Neb: 55°C, Gas: 1.6 SLM.

3. The method for determining the content of polycarbophil in azithromycin eye drops according to claim 1, characterized in that: When using UV detection, the chromatographic conditions were: column: BioCore SEC-150, mobile phase: 10 / 90 v / v acetonitrile / 20 mMKH2PO4, pH 6.5, flow rate: 0.35 mL / min, temperature: 30 °C, and UV detection wavelength was 210 nm.

4. The method for determining the content of polycarbophil in azithromycin eye drops according to any one of claims 1 to 3, characterized in that: When analyzing samples, the concentration of polycarbophil must be controlled below 200 μg / mL or a guard column must be added before the column.

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