Method for determining content of p-hydroxybenzoic acid in procaterol hydrochloride oral solution

Abstract

The invention discloses a method for determining the content of p-hydroxybenzoic acid in a procaterol hydrochloride oral solution. The method comprises the following steps: (1) preparing chromatographic conditions of a high performance liquid chromatography; (2) taking a procaterol hydrochloride oral solution to be detected as a test solution; (3) dissolving a p-hydroxybenzoic acid reference substance in a solvent, and quantitatively diluting to prepare a reference substance solution for later use, wherein about 0.22 mu g of p-hydroxybenzoic acid is contained in each 1ml of reference substance solution; (4) measuring the test solution and the reference solution, respectively injecting the test solution and the reference solution into a liquid chromatograph, recording chromatograms, and calculating the concentration by peak area according to an external standard method, the detection wavelength being 254-300nm; the column temperature of the chromatographic column is 35-45 DEG C; the flow rate is 0.9 to 1.1 ml per minute; the sample injection volume is 20-100 [mu] l. According to the method, the separation degree of a p-hydroxybenzoic acid peak and an adjacent peak is good, interference generated by blank auxiliary materials and solvents can be effectively eliminated, and the method can be used for quality control of a final product of the procaterol hydrochloride oral solution.

Description

Technical Field

[0001] The invention relates to a method for determining the content of p-hydroxybenzoic acid in Procaterol hydrochloride oral solution. Background Art

[0002] Procaterol hydrochloride is a third-generation highly selective bronchial β2 receptor agonist, which has a high selectivity for the β2 adrenergic receptors of bronchial smooth muscle, thereby relaxing bronchial smooth muscle. Its bronchial dilation effect is strong and lasting. For details, see Chinese patent CN114028330A. It also has a certain anti-allergic effect, which can not only inhibit the rapid increase in airway resistance, but also inhibit the delayed increase in airway responsiveness. It can also promote the movement of respiratory cilia. It is clinically used to treat various types of bronchial asthma, asthmatic bronchitis, chronic obstructive pulmonary disease and acute bronchitis. Its oral solution was first developed by Otsuka Pharmaceutical Co., Ltd. of Japan and was launched in Japan in March 1984 under the trade name Meptin.

[0003] Compared with other dosage forms, oral solution has the advantages of flexible dosage and good patient compliance. It is especially suitable for elderly patients with dysphagia and children, and is a commonly used dosage form for children. Multi-dose oral solutions are prone to microbial contamination due to the need to open multiple times, so one or more preservatives are usually required. Parabens are a class of highly efficient, low-toxic bactericidal preservatives, commonly used for the preservation of liquid oral preparations. Sodium benzoate, ethylparaben, and butylparaben are added as preservatives to Procaterol Hydrochloride Oral Solution. Ethylparaben and butylparaben will hydrolyze into parahydroxybenzoic acid in the liquid state. The antibacterial efficacy of ethylparaben and butylparaben will be affected after hydrolysis, and their hydrolysis product, parahydroxybenzoic acid, will have a certain irritation to the mucosa. Therefore, monitoring the content of parahydroxybenzoic acid in oral solutions is of great significance in the quality control of oral solutions. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for determining the content of p-hydroxybenzoic acid in a Procaterol hydrochloride oral solution. The method has good separation between a p-hydroxybenzoic acid peak and adjacent peaks, can effectively eliminate the interference caused by blank excipients and solvents, and can be used for quality control of the final product of the Procaterol hydrochloride oral solution.

[0005] In order to solve the above technical problems, the present invention provides a method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution, comprising the following steps:

[0006] (1) Preparation of chromatographic conditions for high performance liquid chromatography:

[0007] a. Add octadecylsilane bonded silica gel as filler in the chromatographic column;

[0008] b. Preparation of mobile phase A: Take 0.25 g of sodium heptane sulfonate, add 1000 ml of water to dissolve it, add 40 ml of glacial acetic acid, shake well to obtain a sodium heptane sulfonate solution, and then mix it with methanol in a volume ratio of 80:20 to obtain a mixed solution, which is mobile phase A;

[0009] c. Mobile phase B uses methanol;

[0010] d. Elution: Gradient elution was used, and the elution procedure was as shown in Table 1.

[0011] Table 1:

[0012] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 26 100 0 45 20 80 46 100 0 65 100 0

[0013] (2) Take the Procaterol hydrochloride oral solution to be tested and use it as the test solution for later use;

[0014] (3) Take an appropriate amount of p-hydroxybenzoic acid reference substance, accurately weigh it, dissolve it in a solvent, and quantitatively dilute it to prepare a reference solution for use. The solvent is one of methanol, mobile phase A, and water. Each 1 ml of the reference solution contains about 0.22 μg of p-hydroxybenzoic acid.

[0015] (4) Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the concentration by the peak area according to the external standard method. The detection wavelength is 254-300 nm; the column temperature is 35-45°C; the flow rate is 0.9-1.1 ml per minute; and the injection volume is 20-100 μl.

[0016] Preferably, the specifications of the chromatographic column are an inner diameter of 4.6 mm, a length of 250 mm, and a filler particle size of 5 μm.

[0017] Preferably, the detection wavelength is 254 nm.

[0018] Preferably, the column temperature of the chromatographic column is 40°C.

[0019] Preferably, the flow rate of the mobile phase is 1.0 ml.

[0020] Preferably, the injection volume is 50 μl.

[0021] For the purpose of simple explanation, the method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution described in the present invention is referred to as this method below.

[0022] Advantages of this method: This method has good separation between the p-hydroxybenzoic acid peak and the adjacent peaks, can effectively eliminate the interference caused by blank excipients and solvents, and can be used for quality control of the final product of Procaterol Hydrochloride Oral Solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the chromatogram of verification experiment 1 of the present invention.

[0024] Figure 2 It is the linear regression diagram of the third verification experiment of the present invention.

[0025] Figure 3 It is a diagram showing the content of p-hydroxybenzoic acid determined by HPLC in the comparative example of the present invention. DETAILED DESCRIPTION

[0026] Embodiment 1:

[0027] The method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution comprises the following steps:

[0028] (1) Preparation of chromatographic conditions for high performance liquid chromatography:

[0029] a. Add octadecylsilane bonded silica gel as filler in the chromatographic column;

[0030] b. Preparation of mobile phase A: Take 0.25 g of sodium heptane sulfonate, add 1000 ml of water to dissolve it, add 40 ml of glacial acetic acid, shake well to obtain a sodium heptane sulfonate solution, and then mix it with methanol in a volume ratio of 80:20 to obtain a mixed solution, which is mobile phase A;

[0031] c. Mobile phase B uses methanol;

[0032] d. Elution: Gradient elution was used, and the elution procedure was as shown in Table 1.

[0033] Table 1:

[0034] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 26 100 0 45 20 80 46 100 0 65 100 0

[0035] (2) Take the Procaterol hydrochloride oral solution to be tested and use it as the test solution for later use;

[0036] (3) Take an appropriate amount of p-hydroxybenzoic acid reference substance, accurately weigh it, dissolve it in a solvent, and quantitatively dilute it to prepare a reference solution for later use. The solvent is methanol, and each 1 ml of the reference solution contains about 0.22 μg of p-hydroxybenzoic acid;

[0037] (4) Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the concentration by the peak area according to the external standard method. The detection wavelength is 254 nm; the column temperature is 35°C; the flow rate is 0.9 ml per minute; and the injection volume is 20 μl.

[0038] The specifications of the chromatographic column are an inner diameter of 4.6 mm, a length of 250 mm, and a filler particle size of 5 μm.

[0039] Embodiment 2:

[0040] The method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution comprises the following steps:

[0041] (1) Preparation of chromatographic conditions for high performance liquid chromatography:

[0042] a. Add octadecylsilane bonded silica gel as filler in the chromatographic column;

[0043] b. Preparation of mobile phase A: Take 0.25 g of sodium heptane sulfonate, add 1000 ml of water to dissolve it, add 40 ml of glacial acetic acid, shake well to obtain a sodium heptane sulfonate solution, and then mix it with methanol in a volume ratio of 80:20 to obtain a mixed solution, which is mobile phase A;

[0044] c. Mobile phase B uses methanol;

[0045] d. Elution: Gradient elution was used, and the elution procedure was as shown in Table 1.

[0046] Table 1:

[0047] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 26 100 0 45 20 80 46 100 0 65 100 0

[0048] (2) Take the Procaterol hydrochloride oral solution to be tested and use it as the test solution for later use;

[0049] (3) Take an appropriate amount of p-hydroxybenzoic acid reference substance, accurately weigh it, dissolve it in a solvent, and quantitatively dilute it to prepare a reference solution for later use. The solvent is mobile phase A, and each 1 ml of the reference solution contains about 0.22 μg of p-hydroxybenzoic acid;

[0050] (4) Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the concentration by the peak area according to the external standard method. The detection wavelength is 280 nm; the column temperature is 40°C; the flow rate is 1 ml per minute; and the injection volume is 50 μl.

[0051] The specifications of the chromatographic column are an inner diameter of 4.6 mm, a length of 250 mm, and a filler particle size of 5 μm.

[0052] Embodiment three:

[0053] The method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution comprises the following steps:

[0054] (1) Preparation of chromatographic conditions for high performance liquid chromatography:

[0055] a. Add octadecylsilane bonded silica gel as filler in the chromatographic column;

[0056] b. Preparation of mobile phase A: Take 0.25 g of sodium heptane sulfonate, add 1000 ml of water to dissolve it, add 40 ml of glacial acetic acid, shake well to obtain a sodium heptane sulfonate solution, and then mix it with methanol in a volume ratio of 80:20 to obtain a mixed solution, which is mobile phase A;

[0057] c. Mobile phase B uses methanol;

[0058] d. Elution: Gradient elution was used, and the elution procedure was as shown in Table 1.

[0059] Table 1:

[0060] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 26 100 0 45 20 80 46 100 0 65 100 0

[0061] (2) Take the Procaterol hydrochloride oral solution to be tested and use it as the test solution for later use;

[0062] (3) Take an appropriate amount of p-hydroxybenzoic acid reference substance, accurately weigh it, dissolve it in a solvent, and quantitatively dilute it to prepare a reference solution for later use. The solvent is water, and each 1 ml of the reference solution contains about 0.22 μg of p-hydroxybenzoic acid;

[0063] (4) Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the concentration by the peak area according to the external standard method. The detection wavelength is 300 nm; the column temperature is 45°C; the flow rate is 1.1 ml per minute; and the injection volume is 100 μl.

[0064] The specifications of the chromatographic column are an inner diameter of 4.6 mm, a length of 250 mm, and a filler particle size of 5 μm.

[0065] In order to demonstrate the advantages of this method, the following verification experiments are performed:

[0066] Verification experiment 1: Specificity

[0067] Take 50 μl of blank auxiliary material solution, reference substance solution and test substance solution respectively and test them according to the law. The results are shown in Table 2 and the chromatogram is shown in Figure 1 .

[0068] Table 2 Methodology validation - specificity

[0069] name Retention time (min) Peak attribution Blank Accessories There is no interfering peak at the position of p-hydroxybenzoic acid peak / Test solution 9.603 Parabens Reference solution 9.558 Parabens

[0070] Conclusion: The blank excipients did not interfere with the determination of p-hydroxybenzoic acid in the test solution and had good specificity.

[0071] Verification experiment 2: limit of quantification and limit of detection

[0072] Take an appropriate amount of p-hydroxybenzoic acid reference substance, weigh it accurately, add an appropriate amount of methanol to dissolve it, and dilute it with mobile phase A to make a solution with a peak height of about 10 times the noise, as the quantitative limit solution. Dilute it with mobile phase A to make a solution with a peak height of about 3 times the noise, as the detection limit solution. Accurately measure 50μl, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 3.

[0073] Table 3 Para-hydroxybenzoic acid method validation - detection limit results

[0074] name Concentration (μg / ml) Equivalent to test sample concentration (%) Peak area S / N Limit of Quantitation 0.0075 0.01 2.100 10.23 Detection limit 0.0023 0.003 / 3.21

[0075] Conclusion: The quantitative limit of p-hydroxybenzoic acid is 0.0075 μg / ml, equivalent to 0.01% of the test sample concentration, and the S / N value is 10.23. The detection limit is 0.0023 μg / ml, equivalent to 0.003% of the limit concentration, and the S / N value is 3.21, which meets the acceptable standard.

[0076] Verification Experiment 3: Linearity and Range

[0077] According to the method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution, the linearity of p-hydroxybenzoic acid will be investigated within the range of 30% to 160% of the test sample concentration. The linear correlation coefficient r is required to be ≥ 0.999.

[0078] Solution preparation

[0079] Linear stock solution: Take about 7.5 mg of p-hydroxybenzoic acid reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add mobile phase A to dissolve and dilute to the scale, shake well, accurately measure 2 ml, place it in a 100 ml volumetric flask, dilute to the scale with mobile phase A, and shake well as the linear stock solution.

[0080] Prepare the linear solution according to Table 4 and dilute to the mark with solvent.

[0081] Table 4 Para-hydroxybenzoic acid method validation - preparation of linear solution

[0082]

[0083]

[0084] 50 μl of each linear series solution was taken and tested according to the method. The peak area was used to perform linear regression on the main component concentration. The results are shown in Table 5.

[0085] Table 5 Para-hydroxybenzoic acid method validation - linearity results

[0086]

[0087] Conclusion: When the concentration of p-hydroxybenzoic acid is in the range of 0.0755 μg / ml to 0.3775 μg / ml, the linear equation is y=2.880×10 2 x+4.297, linear correlation coefficient r=0.9999; residual sum of squares is 7.41×10 -2 ; The linear relationship is good.

[0088] Verification experiment 4: precision

[0089] ① Injection precision

[0090] Prepare reference solution and measure six times continuously according to the method. The RSD of the peak area in the reference solution for six times is required to be ≤2%.

[0091] Solution preparation

[0092] Reference solution: Take an appropriate amount of p-hydroxybenzoic acid reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing approximately 0.22 μg of p-hydroxybenzoic acid per 1 ml.

[0093] The reference solution was sampled and tested according to the law. The results are shown in Table 6.

[0094] Table 6 Para-hydroxybenzoic acid method validation - injection precision study

[0095]

[0096] Conclusion: The RSD of the peak area in the reference solution was 0.9%, which met the acceptance criteria and the injection precision was good.

[0097] ②Repeatability

[0098] In order to examine the precision of the results obtained by the same analyst under the same conditions, a repeatability test was performed to evaluate the accuracy. Two reference solutions and six test solutions were prepared. The results were determined according to the law and the content of p-hydroxybenzoic acid was calculated by the peak area using the external standard method. The repeatability was required to be ≤2% for the six solutions.

[0099] Solution preparation

[0100] Test solution: Take an appropriate amount of this product and prepare 6 portions in parallel.

[0101] Reference solution: Take an appropriate amount of p-hydroxybenzoic acid reference substance, dissolve it in methanol and quantitatively dilute it to make a solution containing about 0.22 μg of p-hydroxybenzoic acid per 1 ml. Prepare 2 portions in parallel.

[0102] The above-mentioned reference solution and test solution were taken separately and tested according to the law. The results are shown in Table 7.

[0103] Table 7 Para-hydroxybenzoic acid method validation - precision study

[0104]

[0105]

[0106] Conclusion: The RSD of 6 repeatability results was 1.8%, indicating that the method had good precision.

[0107] Verification experiment 5: solution stability

[0108] Investigate the change of sample solution over time, prepare test solution and reference solution. Leave at room temperature for a certain period of time, and take samples for testing at different time points. The peak area of ​​p-hydroxybenzoic acid in reference solution and reference solution measured at each time point is required to be compared with that at 0 hour, and the |change rate| is ≤2%.

[0109] Solution preparation

[0110] Test solution: Take an appropriate amount of this product.

[0111] Reference solution: Take an appropriate amount of para-hydroxybenzoic acid reference substance, dissolve it in the mobile phase and quantitatively dilute it to make a solution containing approximately 0.22 μg per 1 ml.

[0112] The test solution and the reference solution were taken separately and tested according to the method. The results are shown in Table 8.

[0113] Table 8 Para-hydroxybenzoic acid method validation - solution stability results

[0114]

[0115]

[0116] Conclusion: When the p-hydroxybenzoic acid reference solution and the test solution were placed at room temperature for 72 hours, the maximum value of the peak area | change rate | was 1.92%, indicating that the reference solution and the test solution were stable within 72 hours at room temperature.

[0117] Verification experiment 6: Accuracy

[0118] The recovery rate will be investigated at three concentrations: 50%, 100%, and 150% of the limit concentration. Three test solutions are prepared in parallel at each concentration. The recovery rate is required to be between 92% and 105%; the RSD of the recovery rate of the 9 samples is ≤6%.

[0119] Solution preparation

[0120] Reference substance stock solution: Take an appropriate amount of p-hydroxybenzoic acid reference substance, accurately weigh it, dissolve it in methanol and dilute it to make a solution containing about 2.25 μg per 1 ml, which is used as the p-hydroxybenzoic acid reference substance stock solution.

[0121] Reference solution: Take an appropriate amount of p-hydroxybenzoic acid, weigh it accurately, dissolve it in methanol and dilute it to make a solution containing about 0.22 μg per 1 ml, which serves as the reference solution.

[0122] 50% test solution: accurately measure 1 ml of reference stock solution, place in a 20 ml volumetric flask, add blank excipient solution (without ethylparaben and butylparaben) to dilute to scale, shake well, and prepare in 3 parallel portions.

[0123] 100% test solution: accurately measure 2 ml of reference stock solution, place in a 20 ml volumetric flask, add blank excipient solution (without ethylparaben and butylparaben) to dilute to scale, shake well, and prepare in 3 parallel portions.

[0124] 150% test solution: accurately measure 3 ml of reference stock solution, place in a 20 ml volumetric flask, add blank excipient solution (without ethylparaben and butylparaben) to dilute to scale, shake well, and prepare in 3 parallel portions.

[0125] Take the reference solution and the recovery test solution separately, and test them according to the law, as shown in Table 9.

[0126] Table 9 Para-Hydroxybenzoic Acid Methodology Validation - Recovery

[0127]

[0128] Conclusion: The recovery of the test solution was between 100.1% and 104.2%, with a mean of 101.9% and a RSD of 1.7%, which met the acceptable standard.

[0129] Verification experiment seven: durability

[0130] By slightly changing the chromatographic parameters (flow rate, column temperature, wavelength, replacement of chromatographic column), the tolerance of the determination results is evaluated. It is required that the RSD of the p-hydroxybenzoic acid content is ≤ 2% when the parameters of each condition are slightly changed.

[0131] Solution preparation

[0132] Test solution: Take an appropriate amount of this product.

[0133] Reference solution: Take an appropriate amount of para-hydroxybenzoic acid reference substance, dissolve it in water and quantitatively dilute it to make a solution containing approximately 0.22 μg per 1 ml.

[0134] The test solution and the reference solution were taken separately and tested according to the method. The results are shown in Table 10.

[0135] Table 10 Para-Hydroxybenzoic Acid Methodology Validation - Durability Results

[0136]

[0137]

[0138] Conclusion: When the flow rate was adjusted to ±0.1min / ml, column temperature to ±5℃, wavelength to ±2nm, and different chromatographic columns were used, the RSD of the content of the same batch of test samples under each condition was 1.3%, indicating that the method has good durability.

[0139] Comparative Example

[0140] The content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution was tested by the method for determining the content of p-hydroxybenzoic acid in the quality standard of ethyl parabens included in the 2020 edition (Part IV) of the Chinese Pharmacopoeia. The content of p-hydroxybenzoic acid in this product was determined by HPLC. The test results are as follows: Figure 3 It can be seen that under the chromatographic conditions recorded in the Chinese Pharmacopoeia, the blank excipient peak overlaps with the p-hydroxybenzoic acid peak, interfering with the determination of p-hydroxybenzoic acid.

Claims

1. A method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution, characterized in that: The following steps are involved: (1) Preparation of chromatographic conditions for high performance liquid chromatography: a. Add octadecylsilane bonded silica gel as filler in the chromatographic column; b. Preparation of mobile phase A: Take 0.25 g of sodium heptane sulfonate, add 1000 ml of water to dissolve it, add 40 ml of glacial acetic acid, shake well to obtain a sodium heptane sulfonate solution, and then mix it with methanol in a volume ratio of 80:20 to obtain a mixed solution, which is mobile phase A; c. Mobile phase B uses methanol; d. Elution: Gradient elution was used, and the elution procedure was as shown in Table 1. Table 1: (2) Take the Procaterol hydrochloride oral solution to be tested and use it as the test solution for later use; (3) Take an appropriate amount of p-hydroxybenzoic acid reference substance, accurately weigh it, dissolve it in a solvent, and quantitatively dilute it to prepare a reference solution for use. The solvent is one of methanol, mobile phase A, and water. Each 1 ml of the reference solution contains about 0.22 μg of p-hydroxybenzoic acid. (4) Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the concentration by the peak area according to the external standard method. The detection wavelength is 254-300 nm; the column temperature is 35-45°C; the flow rate is 0.9-1.1 ml per minute; and the injection volume is 20-100 μl.

2. The method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution according to claim 1, characterized in that: The specifications of the chromatographic column are an inner diameter of 4.6 mm, a length of 250 mm, and a filler particle size of 5 μm.

3. The method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution according to claim 1, characterized in that: The detection wavelength is 254 nm.

4. The method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution according to claim 1, characterized in that: The column temperature of the chromatographic column is 40°C.

5. The method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution according to claim 1, characterized in that: The flow rate of the mobile phase is 1.0 ml.

6. The method for determining the content of p-hydroxybenzoic acid in Procaterol Hydrochloride Oral Solution according to claim 1, characterized in that: The injection volume is 50 μl.

Images