Method for detecting intermediate III in salbutamol bulk drug

Abstract

The invention discloses a method for detecting an intermediate III in a salbutamol bulk drug. The method has high system applicability, has incomparable advantages in specificity, quantitation limit, detection limit, linear range, repeatability and durability, and has high precision.

Description

Technical Field

[0001] This invention belongs to the field of chemical drug analysis and detection, specifically relating to a method for detecting intermediate III in salbutamol raw material. Background Technology

[0002] Salbutamol is a potent and selective β2-receptor agonist. Clinically, it is widely used to treat respiratory diseases such as bronchospasm in patients with asthmatic bronchitis, bronchial asthma, and emphysema. It is listed in the Chinese Pharmacopoeia, the European Pharmacopoeia, and the United States Pharmacopeia. Currently, the main production process for salbutamol is the original research process, using p-hydroxyacetophenone as the starting material, which involves chloromethylation, esterification, bromination, substitution, hydrolysis, reduction, and debenzylation. The process route is as follows:

[0003]

[0004] To ensure the quality of salbutamol raw material, it is necessary to test the residual levels of each substance in the raw material and strictly control them below the limits. Intermediate III is prepared by chloromethylating p-hydroxyacetophenone with formaldehyde and concentrated hydrochloric acid to generate 3-chloromethyl-4-hydroxyacetophenone, then esterifying it with acetic anhydride to obtain 3-hydroxymethyl-4-hydroxyacetophenone diacetate, which is then brominated with bromine to obtain salbutamol intermediate.

[0005] Intermediate III is an important intermediate in the synthesis of salbutamol. However, existing technologies do not disclose relevant detection methods for intermediate III in salbutamol. Salbutamol is soluble in ethanol, slightly soluble in water, and insoluble in ether. Intermediate III has poor stability in ethanol or water solutions and is easily degraded. Furthermore, it cannot be directly measured by liquid chromatography-mass spectrometry (LC-MS). Therefore, developing a detection method that can both ensure the solubility of salbutamol and accurately detect intermediate III is of great practical significance. Summary of the Invention

[0006] Purpose of the invention: The purpose of this invention is to provide an HPLC detection method for intermediate III in salbutamol raw material, which fully meets the standards in terms of specificity, limit of quantitation, limit of detection, linear range, repeatability, and accuracy, and has high precision.

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for detecting intermediate III in salbutamol raw material, wherein the content of intermediate III in salbutamol raw material is detected by HPLC, and the solution conditions include:

[0008] (1) Solvent: water-ethanol-acetonitrile = 1:(7~9):(12~10);

[0009] (2) Concentration of the test solution: 29–31 mg / ml;

[0010] (3) Concentration of reference solution: 0.55~0.65μg / ml.

[0011] The intermediate III, chemically named 1-{4-(acetoxy)-3-[(acetoxy)methyl]phenyl}-2-bromoethylone, has the following structural formula:

[0012]

[0013] Preferably, the solution conditions include:

[0014] (1) Solvent: Water-ethanol-acetonitrile = 1:8:11;

[0015] (2) Concentration of the test solution: 30 mg / ml;

[0016] (3) Concentration of reference solution: 0.6 μg / ml.

[0017] Specifically, the present invention provides a method for detecting intermediate III in salbutamol raw material, comprising the following steps:

[0018] 1) Preparation of the test solution

[0019] Weigh approximately 300 mg of salbutamol accurately and place it in a 10 mL volumetric flask. Add 4 mL of ethanol and 0.5 mL of water, sonicate until the solution is clear, and dilute to the mark with acetonitrile. Shake well.

[0020] 2) Preparation of reference solution

[0021] Take an appropriate amount of intermediate III reference standard, dissolve it in acetonitrile, and dilute quantitatively to prepare a solution containing approximately 6 μg per ml, thus obtaining the reference standard stock solution. Accurately transfer 1 mL of the reference standard stock solution into a 10 mL volumetric flask, add 4 mL of ethanol and 0.5 mL of water, and dilute to the mark with acetonitrile to prepare a solution containing approximately 0.6 μg of intermediate III per ml, thus obtaining the reference standard solution.

[0022] 3) Accurately measure the test solution and the reference solution separately, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of intermediate III in the test solution by peak area using the external standard method.

[0023] Specifically, the calculation formula in step 3) is:

[0024]

[0025] in:

[0026] Where: f—response factor; m S —The sample weight of intermediate III in the reference solution; c S —Content of intermediate III; V S—Total dilution volume of intermediate III in the reference solution; A S —The peak area f of intermediate III in the chromatogram of the reference solution 平均 —The average value of the response factor; A i —Peak area of ​​intermediate III in the test sample; m i —Sample weight of the test sample; V i —Dilution volume of the test sample.

[0027] Specifically, the detection conditions for the liquid chromatography are as follows:

[0028] Chromatographic column: Kromasil 100-5-C18 column, 4.6×250mm, 5μm;

[0029] Mobile phase A: 2.87 g sodium heptanesulfonate, 2.5 g potassium dihydrogen phosphate, add 1000 mL of water, and adjust the pH to 3.65 with phosphoric acid;

[0030] Mobile phase B: Acetonitrile;

[0031] Elution method: isocratic elution;

[0032] Elution ratio: Mobile phase A: Mobile phase B = 48:52;

[0033] Flow rate: 1.0 ml / min;

[0034] Column temperature: 30℃;

[0035] Detection wavelength: 255nm;

[0036] Injection volume: 20 μl.

[0037] Preferably, this detection method can quantitatively detect intermediate III at a minimum limit of 6 ppm in the test sample.

[0038] Beneficial effects: (1) This invention proposes ethanol, acetonitrile, and water as a mixed solvent, which makes the solubility of salbutamol reach 30 mg / mL. The mixed solvent has the least impact on the response of intermediate III in the test solution to the UV detector, making intermediate III stable in the solution. It also meets the requirements of sensitivity and lower limit. The peak shape is good, the specificity is strong, it is not affected by other impurities, the method is reliable, and the specific content value of intermediate III can be calculated. (2) This invention shows unparalleled advantages in specificity, limit of quantitation, limit of detection, linear range, accuracy, and precision. (3) The use of HPLC to determine intermediate III in salbutamol solves the problem that intermediate III cannot be directly measured by liquid chromatography-mass spectrometry. Attached Figure Description

[0039] Figure 1 This refers to the examination of the blank solvent liquid chromatogram in the method of this invention;

[0040] Figure 2 This is a liquid chromatogram of the system suitability solution in the method of this invention;

[0041] Figure 3 This is the liquid chromatogram of the reference solution used in the method of this invention;

[0042] Figure 4 This refers to the liquid chromatogram of the test sample solution examined in the method of this invention;

[0043] Figure 5 This is the liquid chromatogram of a spiked test sample with methanol as the solvent;

[0044] Figure 6 This is the liquid chromatogram of a reference standard with ethanol as the solvent;

[0045] Figure 7 This is a blank solvent liquid chromatogram with acetonitrile as the solvent;

[0046] Figure 8 This is the liquid chromatogram of a reference standard with acetonitrile as the solvent;

[0047] Figure 9 This is the liquid chromatogram of the test sample with acetonitrile as the solvent;

[0048] Figure 10 This is the liquid chromatogram of a spiked test sample with acetonitrile as the solvent;

[0049] Figure 11 The chromatogram of the reference standard is obtained using ethanol-acetonitrile (7:3) as the solvent.

[0050] Figure 12 The HPLC chromatogram of the reference standard is prepared in a solvent of water-ethanol-acetonitrile (1:5:4).

[0051] Figure 13 The HPLC chromatogram of the reference standard is prepared in a solvent of water-ethanol-acetonitrile (1:8:11).

[0052] Figure 14 This is a blank solvent liquid chromatogram with water-ethanol-acetonitrile (1:8:11) as the solvent;

[0053] Figure 15 The HPLC chromatogram of the reference standard is prepared in a solvent of water-ethanol-acetonitrile (1:8:11).

[0054] Figure 16 The liquid-liquid chromatogram of the test sample is prepared in a solvent of water-ethanol-acetonitrile (1:8:11).

[0055] Figure 17 The chromatogram is of the spiked test sample in a solvent of water-ethanol-acetonitrile (1:8:11).

[0056] Figure 18 This is the standard curve diagram of intermediate III. Detailed Implementation

[0057] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0059] According to the detection method described in this invention, the detection of intermediate III is mainly performed using the external standard method.

[0060] The information on the instruments and reagents used in the implementation of the detection method of the present invention is shown in the table below:

[0061]

[0062] Example 1

[0063] This embodiment provides a method for detecting intermediate III in salbutamol raw material.

[0064] I. Chromatographic conditions

[0065] Instrument: Waters e2695;

[0066] Chromatographic column: Kromasil 100-5-C18 column, 4.6×250mm, 5μm;

[0067] Mobile phase A: 2.87 g sodium heptanesulfonate, 2.5 g potassium dihydrogen phosphate, add 1000 mL of water, and adjust the pH to 3.65 with phosphoric acid;

[0068] Mobile phase B: Acetonitrile;

[0069] Elution method: isocratic elution;

[0070] Elution ratio: Mobile phase A: Mobile phase B = 48:52;

[0071] Flow rate: 1.0 ml / min;

[0072] Column temperature: 30℃;

[0073] Detection wavelength: 255nm;

[0074] Injection volume: 20 μl.

[0075] II. Detection Methods

[0076] (1) Preparation of solvent

[0077] Transfer 4 mL of ethanol and 0.5 mL of water to a 10 mL volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0078] (2) Preparation of test solution

[0079] Weigh approximately 300 mg of salbutamol accurately and place it in a 10 mL volumetric flask. Add 4 mL of ethanol and 0.5 mL of water, sonicate until the solution is clear, and dilute to the mark with acetonitrile. Shake well.

[0080] (3) Preparation of reference solution

[0081] Take an appropriate amount of intermediate III reference standard, dissolve it in acetonitrile, and dilute quantitatively to prepare a solution containing approximately 6 μg per ml, which will serve as the reference standard stock solution. Accurately transfer 1 mL of the reference standard stock solution into a 10 mL volumetric flask, add 4 mL of ethanol and 0.5 mL of water, and dilute to the mark with acetonitrile to prepare a solution containing approximately 0.6 μg of intermediate III per ml.

[0082] (4) Prepare standard limit spiked test solution

[0083] Take an appropriate amount of intermediate III reference standard, dissolve it in acetonitrile, and quantitatively dilute it to prepare a solution containing approximately 6 μg per 1 ml, as the reference standard stock solution. Accurately weigh approximately 300 mg of salbutamol, place it in a 10 ml volumetric flask, add 4 mL of ethanol, 0.5 mL of water, and 1 mL of the reference standard stock solution. Dilute to the mark with acetonitrile to prepare a standard limit spiked test solution containing approximately 30 mg of salbutamol and approximately 0.6 μg of intermediate III per 1 ml.

[0084] (5) Accurately measure the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of intermediate III in the test solution by peak area using the external standard method.

[0085] III. Formula for Calculating the Content of Intermediate III

[0086]

[0087] Where: f—response factor;

[0088] ms — The amount of intermediate III in the reference solution;

[0089] The content of cs-intermediate III;

[0090] Vs—Total dilution volume of intermediate III in the reference solution;

[0091] As—the peak area of ​​intermediate III in the chromatogram of the reference solution.

[0092] 2.

[0093] Where: f_average — the average value of the response factors;

[0094] Ai—Peak area of ​​intermediate III in the test sample;

[0095] mi—sample weight;

[0096] Vi—Dilution volume of the test sample.

[0097] IV. Verification of the Method of the Invention

[0098] 1. Specificity test

[0099] (1) Blank solution: water-ethanol-acetonitrile = 1:8:11.

[0100] (2) System suitability solution: Take an appropriate amount of intermediate III reference standard, dissolve it in acetonitrile, and dilute quantitatively to prepare a solution containing approximately 6 μg per ml, which serves as the reference standard stock solution. Accurately weigh approximately 300 mg of salbutamol, place it in a 10 ml volumetric flask, add 4 mL of ethanol, 0.5 mL of water, and 1 mL of the reference standard stock solution. Dilute to the mark with acetonitrile to prepare a system suitability solution containing approximately 30 mg of salbutamol and approximately 0.6 μg of intermediate III per ml.

[0101] (3) Reference solution: Take an appropriate amount of intermediate III reference standard, dissolve it in acetonitrile and dilute it quantitatively to prepare a solution containing about 6 μg per ml. Accurately transfer 1 mL to a 10 mL volumetric flask, add 4 mL of ethanol and 0.5 mL of water, and dilute to the mark with acetonitrile to prepare a solution containing about 0.6 μg of intermediate III per ml.

[0102] (4) Test solution: Weigh about 300 mg of salbutamol accurately, place it in a 10 ml volumetric flask, add 4 mL of ethanol and 0.5 mL of water, sonicate until the solution is clear, dilute to the mark with acetonitrile, and shake well.

[0103] Inject blank solution, system suitability solution, reference solution, and test solution according to the chromatographic conditions of Example 1, and record the chromatograms (see attached instructions). Figures 1-4 See Table 1.

[0104] Table 1: Results of Specificity Tests

[0105] name Intermediate III retention time (min) Theoretical number of plates blank solution / / Reference solution 5.980 8558.1 Test solution / / System suitability solution 5.983 8547.4

[0106] Specificity tests showed that the blank solvent, the main component of the test sample, and adjacent peaks did not interfere with the detection of intermediate III.

[0107] 2. Limit of Quantitation and Limit of Detection Tests

[0108] The limits of detection (LOD) and quantitation (LOQ) were determined using the signal-to-noise ratio method. The reference solution was serially diluted, and the measured signal was compared with the baseline noise to calculate the lowest concentration that could be reliably detected. The results are shown in Table 2.

[0109] Table 2: Results of Limit of Quantitation and Limit of Detection

[0110]

[0111] Note: Sensitivity = Limit of Quantitation or Limit of Detection Concentration of Intermediate III / Concentration of Test Sample (30 mg / ml)

[0112] The experimental results show that the quality control limit of intermediate III in the sample is 20 ppm, and the sensitivity of the quantitation limit and detection limit is 30% or less of the quality control limit of intermediate III, proving that the present invention has good sensitivity.

[0113] 3. Linearity and range detection

[0114] Take an appropriate amount of intermediate III reference standard, dissolve it in acetonitrile, and quantitatively dilute it to prepare a solution containing approximately 6 μg per ml, as the reference standard stock solution. Take the reference standard stock solution and quantitatively dilute it to prepare solutions with concentrations of 6 ppm, 10 ppm, 16 ppm, 20 ppm, 30 ppm, and 40 ppm relative to the test sample concentration, as solutions for each linear gradient concentration. The linear relationship is plotted as a function of the measured peak area and the analyte concentration. Linear regression is performed using the least squares method, requiring the linear regression coefficient r to be no less than 0.990. The results are shown in Table 3. Figure 18 .

[0115] Table 3: Linearity Measurement Results

[0116]

[0117] Note: Intercept ratio = Linear equation intercept / 100% concentration peak area

[0118] As shown in the table above, the detection method described in this invention has a linear correlation coefficient r of 0.9963 for intermediate III in the range of 6 ppm to 40 ppm relative to the concentration of the test sample, which proves that it has a good linear relationship.

[0119] 4. Precision detection of reference solution injection

[0120] The reference solution from Example 1 was measured six times consecutively, and the relative standard deviation of the peak area was examined. The results are shown in Table 4.

[0121] Table 4: Results of Precision Test for Reference Solution Injection

[0122]

[0123] As shown in the table above, the detection method described in this invention has good precision in determining the injection precision of the reference solution, with a peak area RSD of less than 2%.

[0124] 5. Stability testing of reference and test solutions

[0125] Inject 20 μl of the reference solution and the test solution from Example 1 at 0 h, 0.5 h, 1.5 h, 2.5 h, 4.5 h and 8.5 h respectively, record the chromatograms, and calculate the relative standard deviation of the peak area of ​​intermediate III. The test results are shown in Table 5.

[0126] Table 5: Results of Solution Stability Test

[0127] time Reference Test sample 0h 26945 Not detected 0.5h 25878 Not detected 1.5h 25940 Not detected 2.5h 26053 Not detected 4.5h 25934 Not detected 8.5h 25891 Not detected average 26107 / RSD 1.6％ /

[0128] As shown in the table above, after standing at room temperature for 8.5 hours, the peak area RSD of the reference solution was <2.0%, and intermediate III was not detected in the test solution at any time point, indicating good solution stability.

[0129] 6. Repeatability testing

[0130] The standard limit spiked test solution was taken and the detection method of Example 1 of this invention was repeated 6 times to verify the good repeatability of the method. The results are shown in Table 6.

[0131] Table 6: Repeatability Test Results

[0132] serial number Recovery rate (%) of spiked test solution at standard limit A 1 90.7 A 2 89.4 A 3 91.2 A 4 92.5 A 5 92.4 A 6 89.9 average 91.0 RSD 1.5％

[0133] As shown in the table above, the recovery rate of intermediate III was <5.0% in 6 tests, proving that the method has good repeatability.

[0134] 7. Intermediate precision testing

[0135] The standard limit spiked test solution was taken and measured by different personnel at different times and on different instruments using the same method as in Example 7 for repeatability. The differences between the two measurement results were compared, and the results are shown in Table 7.

[0136] Table 7: Intermediate Precision Test Results

[0137] serial number Recovery rate (%) of spiked test solution at standard limit A 1 90.7 A 2 89.4 A 3 91.2 A 4 92.5 A 5 92.4 A 6 89.9 B 1 90.0 B 2 88.8 B 3 90.1 B 4 88.2 B 5 90.6 B 6 88.1 average 90.1 RSD 1.6％

[0138] As shown in the table above, the recovery rate (RSD) of intermediate III in twelve standard limit spiked test solutions measured by different personnel at different times and on different instruments was less than 10.0%, indicating that the intermediate precision of the detection method was good.

[0139] 8. Accuracy Testing

[0140] The recovery method was used to determine the ratio between the actual measured amount and the theoretical amount of intermediate III in the spiked sample (recovery rate), expressed as a percentage (%). The recovery rate was required to be between 85% and 110% to confirm that the method has good accuracy. The results are shown in Table 8.

[0141] Table 8: Accuracy Test Results

[0142]

[0143] As shown in the table above, the recovery rate of intermediate III ranged from 89.4% to 103.1%, which met the validation requirements (85% to 110%), confirming that the method has good accuracy; the RSD value of the recovery rate was less than 10%, indicating good accuracy.

[0144] In summary, in specific tests, blank solvent, main component of the test sample, and adjacent peaks do not interfere with the detection of intermediate III.

[0145] In the limit of quantitation test, the concentration of intermediate III at the limit of quantitation was 30% or less below the control limit (20 ppm); the concentration of intermediate III at the limit of detection was 15% or less below the control limit (20 ppm).

[0146] In the linearity test, intermediate III showed good linearity in the range of 6 ppm (limit of quantitation) to 40 ppm relative to the concentration of the test sample, with a correlation coefficient r of not less than 0.990 and the percentage of the peak area of ​​the y-axis intercept relative to the limit concentration reference standard was much less than 25%.

[0147] In the accuracy test, the recovery rate of intermediate III was between 85% and 110%, with RSD ≤ 10%.

[0148] In repeatability tests, the recoveries of intermediate III in all six spiked samples met the validation requirements (85%–110%).

[0149] In the intermediate precision test, the recovery rates of intermediate III in 12 spiked samples, measured by different personnel on different dates and on different instruments, all met the validation requirements (85%–110%), with RSD ≤ 10%.

[0150] Example 2

[0151] 1. Selection of solvent for dissolving the test sample: Water has low solubility for salbutamol, with a tested concentration of only about 2 mg / mL. When the limit for intermediate III is 20 ppm, the control concentration needs to be prepared to 0.04 ug / mL, which is not suitable as a solvent for this invention.

[0152] Second: Methanol and ethanol have high solubility as solvents, but they both produce a solvent effect when used in chromatography, which cannot be improved by adjusting the chromatographic conditions.

[0153] Specific steps:

[0154] Solution preparation: Take an appropriate amount of salbutamol sample and intermediate III reference standard, and dilute with methanol to prepare a spiked test solution containing approximately 30 mg of salbutamol and approximately 0.6 μg of intermediate III per ml; take intermediate III reference standard and dilute with ethanol to prepare a control solution containing approximately 0.6 μg of intermediate III per ml.

[0155] Detection: Take the solution prepared with methanol and ethanol solvents as described above, and detect it according to the chromatographic conditions of this invention. The test chromatogram is shown in [reference needed]. Figure 5 , 6 .

[0156] Conclusion: Based on Figure 5 It is evident that when methanol is used as the solvent, the peak shape of intermediate III is poor, and the peak is broadened; according to Figure 6 It is evident that using ethanol as a solvent results in high baseline noise, and intermediate III splits into two peaks. Both solvents produce varying degrees of solvent effect and are therefore unsuitable as solvents.

[0157] 3. Using acetonitrile as solvent, there was no interference in the blank, the peak shape of intermediate III was good, and the recovery rate of the spiked sample was acceptable. The test showed that it could dissolve up to 12 mg / mL of salbutamol, which corresponds to a lower response to the control concentration of 20 ppm.

[0158] Specific steps:

[0159] Solution preparation: Take an appropriate amount of intermediate III reference standard and dilute with acetonitrile to prepare a reference solution containing approximately 0.24 μg of intermediate III per ml; take an appropriate amount of salbutamol test sample and dilute with acetonitrile to prepare a test solution containing approximately 12 mg of salbutamol per ml; take appropriate amounts of salbutamol test sample and intermediate III reference standard and dilute with acetonitrile to prepare a spiked test solution containing approximately 12 mg of salbutamol and approximately 0.24 μg of intermediate III per ml.

[0160] Detection: Take the above acetonitrile, control solution, test sample, and spiked test sample solution and detect them according to the chromatographic conditions of this invention. The test chromatogram is shown in [reference needed]. Figure 7 , 8 9, 10.

[0161] Conclusion: Based on Figure 7 , 8 As shown in points 9 and 10, acetonitrile as a solvent results in low baseline noise and no interference with the elution of intermediate III. The main peak of the test sample solution does not interfere with the elution of intermediate III, and the recovery rate of the spiked test sample is satisfactory. However, the response of the control at certain concentrations is low, leading to a larger error in quantitative detection. Due to the limited selection of solvents and the inability to improve the response by adjusting the instrument method, ethanol can be introduced into the acetonitrile base to increase the solubility of the test sample, thereby improving the response.

[0162] 4. Improve the response of intermediate III

[0163] Intermediate III showed the best elution in acetonitrile and the highest solubility in ethanol. An attempt to dissolve it in a mixed solvent of acetonitrile and ethanol improved solubility, but the baseline was unstable, and the elution of the target peak in the spiked sample was interfered with, resulting in a lower response. Introducing a small amount of water significantly improved solubility, stabilized the baseline, and the elution of the target peak in the spiked sample was normal.

[0164] Specific steps:

[0165] Solution preparation: Weigh appropriate amounts of intermediate III reference standard and dilute them with ethanol-acetonitrile (7:3), water-ethanol-acetonitrile (1:5:4), and water-ethanol-acetonitrile (1:8:11) respectively to prepare a reference solution containing approximately 0.6 μg of intermediate III per ml.

[0166] Detection: Take solutions prepared with solvents in different proportions as described above, and detect them according to the chromatographic conditions of this invention. The test chromatograms are shown below. Figure 11 , 12 13.

[0167] Results: Solutions of the same concentration were prepared using different proportions of solvent for optimization. The results of the optimization are shown in Table 9 below.

[0168] Table 9: Solvent Comparison

[0169] Proportion response Signal-to-noise ratio Figure number Ethanol-acetonitrile (7:3) 22153 22.5 11 Water-ethanol-acetonitrile (1:5:4) 20952 12.5 12 Water-ethanol-acetonitrile (1:8:11) 22326 65.2 13

[0170] Conclusion: As the proportion of water and acetonitrile in the mixed solvent increases, the baseline becomes more stable and the peak shape is better. When the water-ethanol-acetonitrile mixture is 1:8:11, the peak shape is ideal and the peak response meets the detection requirements.

[0171] 5. Confirmation of the Invention Method

[0172] Confirm the solvent ratio; the specific steps are as follows:

[0173] (1) Blank solution: Transfer 4 mL of ethanol and 0.5 mL of water into a 10 mL volumetric flask, dilute to the mark with acetonitrile, and shake well.

[0174] (2) Reference solution: Take an appropriate amount of intermediate III reference standard, dissolve it in acetonitrile and dilute it quantitatively to prepare a solution containing about 6 μg per ml. Accurately transfer 1 mL to a 10 mL volumetric flask, add 4 mL of ethanol and 0.5 mL of water, and dilute to the mark with acetonitrile to prepare a solution containing about 0.6 μg of intermediate III per ml.

[0175] (3) Test solution: Weigh about 300 mg of salbutamol accurately, place it in a 10 ml volumetric flask, add 4 mL of ethanol and 0.5 mL of water, sonicate until the solution is clear, dilute to the mark with acetonitrile, and shake well.

[0176] (4) Spiked test solution: Take an appropriate amount of intermediate III reference standard, dissolve it in acetonitrile and dilute quantitatively to prepare a solution containing approximately 6 μg per ml, as the reference standard stock solution. Accurately weigh approximately 300 mg of salbutamol, place it in a 10 ml volumetric flask, add 4 mL of ethanol, 0.5 mL of water, and 1 mL of the reference standard stock solution. Dilute to the mark with acetonitrile to prepare a spiked test solution containing approximately 30 mg of salbutamol and approximately 0.6 μg of intermediate III per ml.

[0177] Detection: Take the above blank solution, control solution, test solution, and spiked test solution and detect them according to the chromatographic conditions of this invention. The test chromatograms are shown in the figure. Figure 14 , 15 16, 17, the results are shown in Table 10.

[0178] Table 10: Confirmation of the Invention Method

[0179]

[0180] Therefore, this invention uses ethanol, acetonitrile, and water as a mixed solvent, which maintains the stability of intermediate III in the test solution and exhibits good peak symmetry, facilitating the detection of intermediate III and demonstrating high system applicability. Furthermore, it exhibits unparalleled advantages in specificity, limit of quantitation, limit of detection, linear range, and repeatability, demonstrating high precision.

[0181] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the invention.

Claims

1. A high-performance liquid chromatography (HPLC) method for detecting intermediate III in salbutamol raw material, characterized in that, The content of intermediate III in salbutamol raw material was determined by HPLC. The solution conditions included: (1) Solvent: water-ethanol-acetonitrile = 1:(7~9):(12~10); (2) Concentration of the test solution: 29–31 mg / ml; (3) Concentration of reference solution: 0.55–0.65 μg / ml; The intermediate III has the following structural formula:

2. The method according to claim 1, characterized in that, Solution conditions include: (1) Solvent: Water-ethanol-acetonitrile = 1:8:11; (2) Concentration of the test solution: 30 mg / ml; (3) Concentration of reference solution: 0.6 μg / ml.

3. The method according to claim 2, characterized in that, The preparation method for the test solution is as follows: Weigh approximately 300 mg of salbutamol raw material accurately, place it in a 10 ml volumetric flask, add 4 mL of ethanol and 0.5 mL of water, sonicate until the solution is clear, dilute to the mark with acetonitrile, and shake well.

4. The method according to claim 2, characterized in that, The preparation method for the reference solution is as follows: Take an appropriate amount of intermediate III reference standard, dissolve it in acetonitrile and dilute it quantitatively to prepare a solution containing about 6 μg per 1 ml, which is used as the reference standard stock solution; accurately transfer 1 mL of the reference standard stock solution into a 10 mL volumetric flask, add 4 mL of ethanol and 0.5 mL of water, and dilute to the mark with acetonitrile to prepare a solution containing about 0.6 μg of intermediate III per 1 ml, which is the reference standard solution.

5. The method according to claim 2, characterized in that, Specifically, the steps include the following: 1) Prepare the test solution; 2) Prepare the reference solution; 3) Accurately measure the test solution and the reference solution separately, inject them into the liquid chromatograph, record the chromatograms, and calculate the content of intermediate III in the test solution by peak area using the external standard method.

6. The method according to claim 2, characterized in that, The detection conditions for the liquid chromatography are as follows: Chromatographic column: Kromasil 100-5-C18 column, 4.6×250mm, 5μm; Mobile phase A: 2.87 g sodium heptanesulfonate, 2.5 g potassium dihydrogen phosphate, add 1000 mL of water, and adjust the pH to 3.65 with phosphoric acid; Mobile phase B: Acetonitrile; Elution method: isocratic elution; Elution ratio: Mobile phase A: Mobile phase B = 48:52; Flow rate: 1.0 ml / min; Column temperature: 30℃; Detection wavelength: 255nm; Injection volume: 20 μl.

7. The method according to claim 2, characterized in that, The minimum quantitative detection limit for intermediate III in this detection method is 6 ppm.

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