Method for determining related substances in procaterol hydrochloride or oral solution of procaterol hydrochloride

Abstract

The invention relates to a method for determining related substances in procaterol hydrochloride or an oral solution of procaterol hydrochloride. Specifically, the invention provides the method for determining the related substances in the procaterol hydrochloride-containing sample, and the method comprises the step of determining the related substances in the procaterol hydrochloride-containing sample by high performance liquid chromatography. The high performance liquid chromatography provided by the invention has excellent specificity, detection limit, quantitation limit, linear range, repeatability, precision, accuracy, stability and durability, so that related substances in a procaterol hydrochloride-containing sample can be qualitatively and quantitatively analyzed, and the method is used for quality control of the procaterol hydrochloride-containing sample.

Description

Technical Field

[0001] The present invention relates to the field of drug analysis, and in particular to a method for determining related substances in Procaterol hydrochloride or its oral solution. Background Art

[0002] Procaterol Hydrochloride (CAS No. 81262-93-3) is a β2-adrenergic receptor agonist used to treat bronchial asthma, asthmatic bronchitis, acute bronchitis with increased bronchial reactivity, and chronic obstructive pulmonary disease. Among clinical formulations of Procaterol Hydrochloride, Procaterol Hydrochloride Oral Solution offers advantages such as rapid onset of action and convenient administration. Children and the elderly in particular have a high oral compliance with Procaterol Hydrochloride Oral Solution, resulting in its widespread clinical application.

[0003] Related substances in Procaterol hydrochloride or its oral solution affect drug purity and safety, are one of the key items in drug quality research, and are also a direct indicator of drug purity. Therefore, effective detection of related substances in Procaterol hydrochloride or its oral solution, such as qualitative or quantitative detection, can achieve quality control of Procaterol hydrochloride or its oral solution. However, existing detection methods for related substances in Procaterol hydrochloride or its oral solution have many shortcomings, such as specificity, accuracy, detection limit, and quantitative limit, making it difficult to conduct effective qualitative and quantitative detection of related substances in Procaterol hydrochloride or its oral solution, thereby affecting its quality control.

[0004] Therefore, there is a need in the art to develop a method for effectively detecting related substances in Procaterol hydrochloride or its oral solution. Summary of the Invention

[0005] The object of the present invention is to provide a method for determining related substances in a sample containing Procaterol hydrochloride.

[0006] In a first aspect, the present invention provides a method for determining related substances in a sample containing Procaterol hydrochloride, the method comprising the steps of:

[0007] (1) Determination of related substances in samples containing Procaterol hydrochloride by high performance liquid chromatography;

[0008] The chromatographic conditions of the high performance liquid chromatography are:

[0009] Chromatographic column: reverse phase column;

[0010] Mobile phase: mobile phase A and mobile phase B, wherein the mobile phase A comprises phosphate buffer; the mobile phase B comprises methanol; and

[0011] Mobile phase elution: The mobile phase elution is gradient elution, and the order of the gradient elution is:

[0012]

[0013] Preferably, the sample containing Procaterol hydrochloride contains related substances.

[0014] Preferably, the related substances include impurity I and / or impurity II, and the structures of impurity I and / or impurity II are as follows:

[0015] and enantiomers;

[0016]

[0017] Preferably, the pH of the phosphate buffer is 2.5-4.0, more preferably 2.7-3.5, more preferably 3.0-3.2, and most preferably 3.1.

[0018] Preferably, the phosphate buffer is prepared by the following method:

[0019] Take 7.4-8.2 ml of sodium dihydrogen phosphate dihydrate, add 950-1050 ml of water to dissolve it, and adjust the pH to 2.7-3.5 with phosphoric acid to obtain phosphate buffer.

[0020] Preferably, the phosphate buffer is prepared by the following method:

[0021] Take 7.6-8.0 ml of sodium dihydrogen phosphate dihydrate, add 980-1020 ml of water to dissolve it, and adjust the pH to 3.0-3.2 with phosphoric acid to obtain phosphate buffer.

[0022] Preferably, the phosphate buffer is prepared by the following method:

[0023] Take 7.8 g of sodium dihydrogen phosphate dihydrate, add 1000 ml of water to dissolve it, and adjust the pH to 3.1 with phosphoric acid.

[0024] Preferably, the high performance liquid chromatography method further comprises one or more chromatographic conditions selected from the group consisting of:

[0025] Chromatographic column temperature: 35-45°C, preferably 38-42°C, more preferably 40°C;

[0026] Mobile phase flow rate: 0.2-3 mL / min, preferably 0.5-1.5 mL / min, more preferably 0.8-1.2 mL / min, most preferably 1.0 mL / min; and / or

[0027] The detection wavelength is 230-270 nm, preferably 240-260 nm, more preferably 245-255 nm, and most preferably 259 nm.

[0028] Preferably, the filler of the reverse phase chromatography column comprises octadecylsilane bonded silica gel.

[0029] Preferably, the reverse phase chromatography column comprises a Waters XSelect HSS T3 reverse phase chromatography column.

[0030] Preferably, the specifications of the reverse phase chromatography column are 4.6 mm×150 mm, 3.5 μm.

[0031] Preferably, the injection volume of the HPLC method is 5-200 μl, more preferably 5-150 μl, more preferably 90-100 μl, and most preferably 95 μl.

[0032] Preferably, the sample containing Procaterol hydrochloride includes Procaterol hydrochloride or Procaterol hydrochloride oral solution.

[0033] Preferably, the Procaterol hydrochloride comprises Procaterol hydrochloride bulk drug.

[0034] Preferably, the sample containing Procaterol hydrochloride includes Procaterol hydrochloride bulk drug or Procaterol hydrochloride oral solution.

[0035] Preferably, the Procaterol hydrochloride oral solution comprises Procaterol hydrochloride, ethylparaben, butylparaben, sodium benzoate, citric acid, sodium citrate, ethanol and water.

[0036] Preferably, the water comprises purified water.

[0037] Preferably, the Procaterol hydrochloride oral solution comprises:

[0038]

[0039]

[0040] Preferably, the Procaterol hydrochloride oral solution comprises:

[0041] Components Dosage Procaterol Hydrochloride 0.004-0.006 parts by weight Ethylparaben 0.04-0.06 parts by weight Butylparaben 0.02-0.03 parts by weight Sodium benzoate 1.8-2.2 parts by weight Citric acid 2.3-2.7 parts by weight Sodium citrate 0.8-1.2 parts by weight ethanol 24-28 parts by weight; and water 980-1020 parts by weight.

[0042] Preferably, the Procaterol hydrochloride oral solution comprises:

[0043] Components Dosage Procaterol Hydrochloride 0.005 parts by weight Ethylparaben 0.05 parts by weight Butylparaben 0.025 parts by weight Sodium benzoate 2.0 parts by weight Citric acid 2.5 parts by weight Sodium citrate 1.0 parts by weight ethanol 26.0 parts by weight; and water 1000 parts by weight.

[0044] Preferably, the method is a quantitative and / or qualitative method.

[0045] Preferably, the determination is a quantitative determination and / or a qualitative determination.

[0046] Preferably, the method is an in vitro method.

[0047] Preferably, the method is a non-diagnostic and non-therapeutic method.

[0048] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 HPLC spectrum of blank solvent water.

[0050] Figure 2 HPLC spectrum of blank excipient test solution.

[0051] Figure 3 HPLC chromatogram of the spiked test solution.

[0052] Figure 4 This is the HPLC spectrum of the undamaged self-developed Procaterol Hydrochloride Oral Solution.

[0053] Figure 5 This is the HPLC spectrum of the undamaged original reference formulation of Procaterol Hydrochloride Oral Solution.

[0054] Figure 6 This is the HPLC spectrum of the self-developed Procaterol Hydrochloride Oral Solution destroyed by high temperature.

[0055] Figure 7 This is the HPLC spectrum of the original reference preparation of Procaterol Hydrochloride Oral Solution destroyed by high temperature.

[0056] Figure 8 This is the HPLC spectrum of the self-developed Procaterol Hydrochloride oral solution damaged by light.

[0057] Figure 9 This is the HPLC spectrum of the original reference formulation of Procaterol Hydrochloride Oral Solution damaged by light.

[0058] Figure 10 This is the HPLC spectrum of acid-destroyed self-developed Procaterol Hydrochloride oral solution.

[0059] Figure 11 This is the HPLC profile of the original reference formulation of acid-destroyed Procaterol Hydrochloride Oral Solution.

[0060] Figure 12 This is the HPLC spectrum of the alkali-destroyed self-developed Procaterol Hydrochloride oral solution.

[0061] Figure 13 This is the HPLC profile of the original reference formulation of alkali-destroyed Procaterol Hydrochloride Oral Solution.

[0062] Figure 14 This is the HPLC spectrum of the self-developed Procaterol Hydrochloride Oral Solution damaged by oxidation. DETAILED DESCRIPTION

[0063] The present invention develops a method for determining related substances in a sample containing Procaterol hydrochloride. The method comprises determining the related substances in the sample containing Procaterol hydrochloride by high performance liquid chromatography. The high performance liquid chromatography has excellent specificity, detection limit, quantification limit, linear range, repeatability, precision, accuracy, stability and durability, thereby being capable of performing qualitative and quantitative analysis of the related substances in the sample containing Procaterol hydrochloride, and being used for quality control of the sample containing Procaterol hydrochloride.

[0064] the term

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0066] As used herein, the terms "include," "comprise," and "contain" are used interchangeably to encompass not only open definitions but also semi-closed and closed definitions. In other words, the terms encompass "consisting of," "consisting essentially of."

[0067] As used herein, "HPLC" refers to high performance liquid chromatography.

[0068] As used herein, "ethylparaben" is also known as ethyl parahydroxybenzoate.

[0069] As used herein, "butylparaben" is also known as butylparaben.

[0070] In the present invention, it should be understood that the flow rate of the mobile phase is the sum of the flow rates of mobile phase A and mobile phase B.

[0071] method

[0072] The present invention provides a method for determining related substances in a sample containing Procaterol hydrochloride, the method comprising the steps of:

[0073] (1) Determination of related substances in samples containing Procaterol hydrochloride by high performance liquid chromatography;

[0074] The chromatographic conditions of the high performance liquid chromatography are:

[0075] Chromatographic column: reverse phase column;

[0076] Mobile phase: mobile phase A and mobile phase B, wherein the mobile phase A comprises phosphate buffer; the mobile phase B comprises methanol; and

[0077] Mobile phase elution: The mobile phase elution is gradient elution, and the order of the gradient elution is:

[0078]

[0079] Specifically, the method for determining related substances in a sample containing Procaterol hydrochloride according to the present invention is as described in the first aspect of the present invention.

[0080] The main technical effects of the present invention include:

[0081] The present invention develops a method for determining related substances in a sample containing Procaterol hydrochloride (such as a Procaterol hydrochloride bulk drug or a Procaterol hydrochloride oral solution) by high performance liquid chromatography (HPLC). The high performance liquid chromatography method has excellent specificity, detection limit, quantification limit, linear range, repeatability, precision, accuracy, stability, and durability, meeting the requirements. Thus, the method can perform qualitative and quantitative analysis of related substances in a sample containing Procaterol hydrochloride, and is used for quality control of samples containing Procaterol hydrochloride.

[0082] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0083] Example 1

[0084] This Example 1 provides a method for determining related substances in Procaterol Hydrochloride Oral Solution by high performance liquid chromatography (HPLC).

[0085] 1. Self-developed Procaterol Hydrochloride Oral Solution and its related substances (impurities), and its original reference preparation

[0086] The formula of the self-developed Procaterol Hydrochloride Oral Solution of Example 1 and its related substances (impurities), as well as its original research reference preparation are as follows:

[0087] 1.1 The prescription of the self-developed Procaterol Hydrochloride Oral Solution is shown in Table 1 below:

[0088] Table 1 Prescription composition of self-developed Procaterol Hydrochloride Oral Solution

[0089] Components Dosage Procaterol Hydrochloride 0.005g Ethylparaben 0.05g Butylparaben 0.025g Sodium benzoate 2.0g Citric acid 2.5g Sodium citrate 1.0g ethanol 26.0g Add purified water to To 1000ml.

[0090] Preparation method:

[0091] The Procaterol hydrochloride oral solution is obtained by mixing Procaterol hydrochloride, ethylparaben, butylparaben, sodium benzoate, citric acid, sodium citrate, ethanol and purified water.

[0092] 1.2 The structures of impurities I and II in Procaterol Hydrochloride Oral Solution are as follows:

[0093] and enantiomers;

[0094]

[0095] 1.3 Original reference preparation of Procaterol Hydrochloride Oral Solution:

[0096] The original reference preparation of Procaterol Hydrochloride Oral Solution is produced by the original manufacturer, Otsuka Pharmaceutical Co., Ltd. of Japan, with the trade name "Meptin", specification of 5μg / ml, batch number OB94C, and the prescription contains Procaterol Hydrochloride, ethylparaben, butylparaben, sodium benzoate, ethanol, citric acid, sodium citrate and purified water.

[0097] 2. Chromatographic conditions of HPLC:

[0098] Chromatographic column: octadecylsilane bonded silica gel as filler, Waters XSelect HSS T3, 4.6 mm × 150 mm, 3.5 μm;

[0099] Mobile phase: Mobile phase A + mobile phase B, wherein phosphate buffer (7.8 g of sodium dihydrogen phosphate dihydrate, dissolved in 1000 ml of water, and adjusted to pH 3.1 with phosphoric acid) is used as mobile phase A, and methanol is used as mobile phase B;

[0100] Mobile phase elution: Gradient elution according to Table 2:

[0101] Table 2 Mobile phase gradient elution

[0102]

[0103] The mobile phase flow rate was 1.0 ml / min;

[0104] The column temperature was 40°C;

[0105] The detection wavelength is 259 nm.

[0106] 3. Validation of the HPLC Method

[0107] 3.1 Specificity Investigation

[0108] 3.1.1 Interference experiments with blank solvents and blank excipients

[0109] According to the prescription of the self-developed Procaterol Hydrochloride Oral Solution, take the blank excipient (i.e., the solution obtained by removing Procaterol Hydrochloride from the self-developed Procaterol Hydrochloride Oral Solution), filter it, and use it as the blank excipient test solution. Accurately measure 95 μl of each of the blank solvent water and the blank excipient test solution and inject them into the high performance liquid chromatograph to record the chromatogram. The spectrum is shown in Figure 1 and Figure 2 shown.

[0110] from Figure 1 and Figure 2 It can be seen that the blank solvent and blank excipients have no absorption at the positions corresponding to the main peak of Procaterol hydrochloride and its related substances and known impurities, and have no interference with the detection. Among them, the peak with a retention time of 12.618 min is p-hydroxybenzoic acid, which is the degradation product of ethylparaben and butylparaben. Therefore, the specificity of the HPLC method is good.

[0111] 3.1.2 Separation of known impurities

[0112] Preparation of impurity reference stock solution:

[0113] (a) Stock solution of impurity I reference substance: accurately weigh 6.25 mg of impurity I reference substance and place it in a 50 ml volumetric flask. Soluble it in water and dilute it to the mark by ultrasonication. Shake well to obtain the stock solution.

[0114] (b) Stock solution of impurity II reference substance: Take 5 mg of impurity II reference substance, accurately weigh it, and place it in a 50 ml volumetric flask. Add solvent water and ultrasonically dissolve it and dilute it to the scale. Shake well to obtain the solution.

[0115] (c) 8-Hydroxyquinolone stock solution: Accurately weigh 5 mg of 8-hydroxyquinolone and place it in a 100 ml volumetric flask. Add solvent water and sonicate to dissolve and dilute to the mark. Shake well to obtain the stock solution.

[0116] (d) Stock solution of p-hydroxybenzoic acid: accurately weigh 3.75 mg of p-hydroxybenzoic acid reference substance and place it in a 100 ml volumetric flask. Add solvent water and sonicate to dissolve and dilute to the mark. Shake well to obtain the stock solution.

[0117] (e) Mixed reference stock solution: Accurately measure 2 ml each of the impurity I reference stock solution, the impurity II reference stock solution, and the 8-hydroxyquinolone stock solution into a 20 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain the mixed reference stock solution.

[0118] (f) Spiked test solution: Take 20 ml of the self-ground Procaterol hydrochloride oral solution, add 0.2 ml each of the above mixed reference stock solution and p-hydroxybenzoic acid stock solution, shake well, and obtain the product.

[0119] Accurately measure 95 μl of the above-mentioned spiked test solution, inject it into the high performance liquid chromatograph, and record the chromatogram (such as Figure 3 The results are shown in Table 3 below.

[0120] Table 3 Separation of known impurities

[0121]

[0122] From Table 3 and Figure 3 It can be seen that the separation between Procaterol hydrochloride and the adjacent impurity peaks is greater than 4.0, the separation between each impurity peak is greater than 1.2, and the purity of the main peak is greater than 99.9%, which meets the requirements, indicating that the HPLC method has good specificity.

[0123] 3.1.3 Forced degradation test

[0124] Preparation of test solution:

[0125] Undamaged sample: Take an appropriate amount of self-developed Procaterol Hydrochloride Oral Solution, filter it through a microporous filter membrane, and collect the filtrate. Simultaneously, perform the same operation on the original reference preparation.

[0126] High-temperature destruction sample: Take an appropriate amount of self-developed Procaterol Hydrochloride Oral Solution and place it in a glass bottle. Place it at 60°C for 7 days. Filter it through a microporous filter membrane and collect the filtrate. Simultaneously, perform the same operation on the original reference preparation.

[0127] Photodestruction sample: Take an appropriate amount of self-developed Procaterol hydrochloride oral solution and place it in a transparent glass bottle. 2 ) for 7 days, filter with a microporous membrane, and take the filtrate to obtain the product. At the same time, take the original reference preparation and perform the same operation in parallel.

[0128] Acid-damaged sample: Take 10 ml of the self-developed Procaterol Hydrochloride Oral Solution, add 0.1 ml of 5 mol / L HCl solution, and place in a 40°C oven for 45 hours. Remove, cool, and then add 0.1 ml of 5 mol / L NaOH solution to neutralize. Filter through a microporous filter membrane and collect the filtrate. Simultaneously, perform the same operation on the original reference preparation.

[0129] Alkali-damaged sample: Take 10 ml of the self-developed Procaterol Hydrochloride Oral Solution, add 0.1 ml of 5 mol / L NaOH solution, and place in a 40°C oven for 19 hours. Remove, cool, and then add 0.1 ml of 5 mol / L HCl solution to neutralize. Filter through a microporous filter membrane and collect the filtrate. Simultaneously, perform the same operation on the original reference preparation.

[0130] Oxidative damage sample: Take 10 ml of the self-developed Procaterol hydrochloride oral solution, add 0.1 ml of 30% H2O2 solution, and place in a 40°C oven for 48 hours. Remove, cool, and filter through a microporous membrane. The filtrate is obtained. Simultaneously, perform the same procedure on the original reference preparation.

[0131] Accurately measure 95 μl of each of the above test solution and inject them into the high performance liquid chromatograph, and record the chromatogram (such as Figure 4 、 Figure 5 、 Figure 6 、 Figure 7 、 Figure 8 、 Figure 9 、 Figure 10 、 Figure 11 、 Figure 12 、 Figure 13 and Figure 14 The results are shown in Table 4.

[0132] Table 4 Forced degradation test

[0133]

[0134]

[0135]

[0136] Note: RRT refers to relative retention time; equilibrium index = total peak area of ​​the destroyed solution / total peak area of ​​the original solution.

[0137] from Figure 4-14 As can be seen from Table 4, under conditions of high temperature, acid, alkali, and oxidation, the main degradation products in Procaterol Hydrochloride Oral Solution are impurities I and II. Under light exposure, the main degradation impurity in Procaterol Hydrochloride Oral Solution is an unknown single impurity. The resolution between the main Procaterol Hydrochloride peak and adjacent impurities in both Procaterol Hydrochloride Oral Solution and the original reference formulation was greater than 1.5, and the resolution between impurity peaks was also greater than 1.5, meeting the separation requirements. Peak purities were greater than 99.0%. No interfering peaks were detected at the Procaterol Hydrochloride peak or at the known impurity peaks in the blank excipient solution. This demonstrates that the HPLC method has good specificity for the determination of related substances in Procaterol Hydrochloride Oral Solution and that degradation products do not affect the determination of related substances in Procaterol Hydrochloride Oral Solution.

[0138] 3.2 Limit of detection and limit of quantification

[0139] 3.2.1 Limit of Detection and Limit of Quantification of Procaterol Hydrochloride

[0140] Accurately weigh 5.0 mg of Procaterol hydrochloride reference substance and place it in a 100 ml volumetric flask. Add an appropriate amount of solvent water, sonicate to dissolve and dilute to the scale, shake well, then accurately measure 5 ml and place it in a 50 ml volumetric flask, dilute to the scale with solvent water, shake well, and use as the reference substance stock solution.

[0141] Accurately measure 1 ml of the reference stock solution and place it in a 200 ml volumetric flask, dilute it to the mark with solvent water, shake well, then accurately measure 5 ml and place it in a 200 ml volumetric flask, dilute it to the mark with solvent water, shake well, accurately measure 95 μl and inject it into the high performance liquid chromatograph, calculate the quantitative limit concentration of Procaterol hydrochloride, and after meeting the quantitative limit requirement, make 5 consecutive injections and calculate the relative standard deviation (RSD) (%).

[0142] Accurately measure 3 ml of the above-mentioned quantitative limit solution and place it in a 10 ml volumetric flask, dilute it to the scale with solvent water, shake it well, accurately measure 95 μl and inject it into the high performance liquid chromatograph to calculate the detection limit concentration of Procaterol hydrochloride.

[0143] The limit of quantification and limit of detection of Procaterol hydrochloride are shown in Table 5 below.

[0144] Table 5 Limit of quantification and detection limit of Procaterol hydrochloride

[0145]

[0146] As can be seen from Table 5, at the detection limit concentration, the concentration of Procaterol hydrochloride is equivalent to 0.004% of the concentration of the related substance test sample, which meets the detection limit requirement; at the quantification limit concentration, the concentration of Procaterol hydrochloride is equivalent to 0.013% of the concentration of the related substance test sample, which meets the quantitative detection requirement, and after 5 consecutive injections, the RSD of the peak area is less than 10%, which meets the quantification limit acceptance standard.

[0147] 3.2.2 Limits of detection and quantification of impurity I

[0148] Accurately weigh 5.0 mg of impurity I reference substance and place it in a 100 ml volumetric flask. Add an appropriate amount of solvent water, sonicate to dissolve and dilute to the scale, shake well, then accurately measure 5 ml and place it in a 50 ml volumetric flask, dilute to the scale with solvent water, shake well, and use as the reference substance stock solution.

[0149] Accurately measure 1 ml of the reference stock solution and place it in a 200 ml volumetric flask, dilute it to the scale with solvent water, shake well, then accurately measure 5 ml and place it in a 200 ml volumetric flask, dilute it to the scale with solvent water, shake well, accurately measure 95 μl and inject it into the high performance liquid chromatograph, calculate the quantitative limit concentration of impurity I, and after meeting the quantitative limit requirement, make 5 consecutive injections and calculate the relative standard deviation RSD (%).

[0150] Accurately measure 5 ml of the above quantitative limit solution and place it in a 10 ml volumetric flask, dilute it to the scale with solvent water, shake it well, accurately measure 95 μl and inject it into the high performance liquid chromatograph to calculate the detection limit concentration of hydrochloric acid impurity I.

[0151] The quantitative limit concentration and detection limit concentration of impurity I are shown in Table 6 below.

[0152] Table 6 Quantitation limit and detection limit concentration of impurity Ⅰ

[0153]

[0154] It can be seen from Table 6 that at the detection limit concentration, the concentration of impurity I is equivalent to 0.006% of the concentration of the related substance test sample, which meets the detection limit requirement; at the quantification limit concentration, the concentration of impurity I is equivalent to 0.012% of the concentration of the related substance test sample, which meets the quantitative detection requirement, and after 5 consecutive injections, the RSD of the peak area is less than 10%, which meets the quantitative limit acceptance standard.

[0155] 3.2.3 Detection Limit and Quantification Limit of Impurity II

[0156] Accurately weigh 5.0 mg of impurity II reference substance and place it in a 100 ml volumetric flask. Add an appropriate amount of solvent water and sonicate to dissolve and dilute to the scale. Shake well. Then accurately measure 5 ml and place it in a 50 ml volumetric flask. Dilute to the scale with solvent water and shake well. This is used as the reference substance stock solution.

[0157] Accurately measure 1 ml of the reference stock solution and place it in a 200 ml volumetric flask, dilute it to the scale with solvent water, shake well, then accurately measure 5 ml and place it in a 200 ml volumetric flask, dilute it to the scale with solvent water, shake well, accurately measure 95 μl and inject it into the high performance liquid chromatograph, calculate the quantitative limit concentration of impurity II, and after meeting the quantitative limit requirement, make 5 consecutive injections and calculate the relative standard deviation (RSD) (%).

[0158] Accurately measure 5 ml of the above quantitative limit solution and place it in a 10 ml volumetric flask, dilute it to the scale with solvent water, shake it well, accurately measure 95 μl and inject it into the high performance liquid chromatograph to calculate the detection limit concentration of hydrochloric acid impurity II.

[0159] The quantitative limit concentration and detection limit concentration of impurity II are shown in Table 7 below.

[0160] Table 7 Quantitation limit and detection limit concentration of impurity Ⅱ

[0161]

[0162]

[0163] It can be seen from Table 7 that at the detection limit concentration, the concentration of impurity II is equivalent to 0.006% of the concentration of the related substance test sample, which meets the detection limit requirement; at the quantification limit concentration, the concentration of impurity II is equivalent to 0.012% of the concentration of the related substance test sample, which meets the quantitative detection requirement, and after 5 consecutive injections, the RSD of the peak area is less than 10%, which meets the quantitative limit acceptance standard.

[0164] 3.3 Linearity and correction factor determination

[0165] Impurity I reference substance stock solution: accurately weigh 5 mg of impurity I reference substance and place it in a 100 ml volumetric flask. Add an appropriate amount of solvent water and sonicate to dissolve and dilute to the scale. Shake well. Accurately measure 5 ml and place it in a 50 ml volumetric flask. Dilute to the scale with solvent water and shake well.

[0166] Impurity II reference substance stock solution: accurately weigh 5 mg of impurity II reference substance and place it in a 100 ml volumetric flask. Add an appropriate amount of solvent water and ultrasonically dissolve it and dilute it to the scale. Shake well. Accurately measure 5 ml and place it in a 50 ml volumetric flask. Dilute it to the scale with solvent water and shake well.

[0167] Linear solution I: Accurately weigh 5 mg of Procaterol hydrochloride reference substance and place it in a 100 ml volumetric flask. Add an appropriate amount of solvent water and sonicate to dissolve and dilute to the scale. Shake well. Accurately measure 5 ml and place it in a 50 ml volumetric flask. Dilute to the scale with solvent water and shake well.

[0168] Linear solution II: Accurately measure 5 ml of linear solution I, place it in a 10 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0169] Linear solution III: Accurately measure 5 ml of linear solution I, place it in a 25 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0170] Linear solution IV-1: Accurately measure 3 ml each of linear solution I and impurity II reference stock solution, place them in the same 100 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0171] Linear solution IV-2: Accurately measure 3 ml of the stock solution of impurity I reference substance, place it in a 100 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0172] Linear solution V-1: Accurately measure 2 ml each of linear solution I and impurity II reference stock solution, place them in the same 100 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0173] Linear solution V-2: Accurately measure 2 ml of the stock solution of impurity I reference substance, place it in a 100 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0174] Linear solution VI-1: Accurately measure 1 ml each of linear solution I and impurity II reference stock solution, place them in the same 100 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0175] Linear solution VI-2: Accurately measure 1 ml of the stock solution of impurity I reference substance, place it in a 100 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0176] Linear solution VII-1: Accurately measure 5 ml of linear solution VI-1, place it in a 10 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0177] Linear solution VII-2: Accurately measure 5 ml of linear solution VI-2, place it in a 10 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0178] Linear solution VIII-1: Accurately measure 2 ml of linear solution VI-1, place it in a 10 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0179] Linear solution VIII-2: Accurately measure 2 ml of linear solution VI-2, place it in a 10 ml volumetric flask, dilute to the mark with solvent water, and shake well to obtain;

[0180] Linear solution IX-1 (Procaterol hydrochloride and impurity II limit of quantitation solution): Accurately measure 15 ml of linear solution VI-1, place it in a 200 ml volumetric flask, dilute to the mark with solvent water, shake well, then accurately measure 5 ml, place it in a 10 ml volumetric flask, dilute to the mark with solvent water, shake well, and the solution is obtained;

[0181] Linear solution IX-2 (limit of quantitation solution of impurity I): Accurately measure 5 ml of linear solution VI-2, place it in a 200 ml volumetric flask, dilute to the mark with solvent water, shake well, then accurately measure 5 ml, place it in a 10 ml volumetric flask, dilute to the mark with solvent water, shake well, and the solution is obtained;

[0182] Determination

[0183] Accurately measure 95 μl of each of the linear solutions I to IX and inject them into a high performance liquid chromatograph. Plot the graph with the peak area (A) as the ordinate and the concentrations of Procaterol hydrochloride, impurity I, and impurity II (C) as the abscissa. Calculate the regression equation and correlation coefficient. Calculate the correction factors for impurities I and II based on the slopes of the linear equations for impurities I and II and the slope of the linear equation for Procaterol hydrochloride. The results are shown in Tables 8, 9, 10, and 11 below.

[0184]

[0185] Table 8 Procaterol hydrochloride linear results

[0186]

[0187] Table 9 Procaterol hydrochloride correction factor linear determination results

[0188]

[0189]

[0190] Table 10 Impurity I linearity and correction factor determination results

[0191]

[0192] Table 11 Impurity II linearity and correction factor determination results

[0193]

[0194]

[0195] As can be seen from Tables 8-11, the linear relationships of Procaterol Hydrochloride are good in the range of 0.0006μg / ml to 5.0375μg / ml (equivalent to the test sample concentration of the related substance of 0.01% to 100%), impurity I is in the range of 0.0006μg / ml to 0.1446μg / ml (equivalent to the test sample concentration of the related substance of 0.01% to 3%), and impurity II is in the range of 0.0006μg / ml to 0.1535μg / ml (equivalent to the test sample concentration of the related substance of 0.01% to 3%). The average value was calculated based on the relative correction factor results measured by two test personnel, and the relative correction factor of impurity II was 0.72, and the relative correction factor of impurity I was 1.03. Since the relative correction factor of impurity I is between 0.9 and 1.1, the correction factor of impurity I can be calculated as 1.0 in the quality standard.

[0196] 3.4 Repeatability and precision test

[0197] 3.4.1 Preparation of solution:

[0198] Stock solution of impurity I reference substance: Take 6.25 mg of impurity I reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add solvent water, ultrasonically dissolve it and dilute it to the scale, shake well, and obtain it.

[0199] Impurity II reference substance stock solution: Take 5 mg of impurity II reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add solvent water, ultrasonically dissolve it and dilute it to the scale, shake well, and obtain it.

[0200] Mixed reference substance stock solution: Accurately measure 2 ml of impurity I reference substance stock solution and impurity II reference substance stock solution respectively and place them in a 20 ml volumetric flask, dilute to the scale with solvent water, shake well, and obtain the mixed reference substance stock solution.

[0201] Spiked test solution: Take 50 ml of self-ground Procaterol hydrochloride oral solution, add 0.5 ml of mixed reference stock solution, shake well, and prepare 6 parallel portions.

[0202] 3.4.2 Repeatability determination

[0203] 95 μl of each of the six spiked test solutions was measured and injected into a high performance liquid chromatograph. The contents of impurity I, impurity II, and total impurities were calculated using the external standard method with a correction factor. The results are shown in Table 12 below.

[0204] Table 12 Repeatability test results

[0205]

[0206] As can be seen from Table 12, in the six test solutions, the RSDs of the measured impurity I, impurity II and total impurity contents were all less than 5.0%, indicating that the HPLC method had good repeatability in the determination of impurity I and impurity II.

[0207] 3.4.3 Precision determination

[0208] Separately, six portions of the spiked test solution were measured by different individuals, using different equipment, and on different dates using the repeatability method. The contents of Impurity I, Impurity II, and total impurities were calculated as the intermediate precision test results. The results of the repeatability and intermediate precision tests were combined to calculate the RSDs for the 12 samples. The results are shown in Table 13 below.

[0209] Table 13 Intermediate precision test results

[0210]

[0211] As can be seen from Table 13, in the 12 test solutions operated by two people, A and B, the RSDs of the measured impurity I, impurity II and total impurities were all less than 5.0%, indicating that the HPLC method has good precision.

[0212] 3.5 Accuracy test

[0213] Solution preparation:

[0214] Stock solution of impurity I reference substance: Take 6.25 mg of impurity I reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add solvent water, ultrasonically dissolve it and dilute it to the scale, shake well, and obtain it.

[0215] Impurity II reference substance stock solution: Take 5 mg of impurity II reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add solvent water, ultrasonically dissolve it and dilute it to the scale, shake well, and obtain it.

[0216] Mixed reference substance stock solution: Accurately measure 2 ml of impurity I reference substance stock solution and impurity II reference substance stock solution respectively and place them in a 20 ml volumetric flask, dilute to the scale with solvent water, shake well, and obtain the mixed reference substance stock solution.

[0217] Mixed reference substance stock solution 1: Accurately measure 1 ml of impurity I reference substance stock solution and impurity II reference substance stock solution respectively and place them in a 20 ml volumetric flask, dilute to the scale with solvent water, and shake well to obtain the mixed reference substance stock solution.

[0218] Mixed reference substance stock solution 2: Accurately measure 3 ml of impurity I reference substance stock solution and impurity II reference substance stock solution respectively and place them in a 20 ml volumetric flask, dilute to the scale with solvent water, and shake well to obtain the mixed reference substance stock solution.

[0219] 50% test solution: Take 50 ml of self-ground Procaterol hydrochloride oral solution, add 0.5 ml of mixed reference substance stock solution 1, shake well, and prepare three replicates.

[0220] 100% test solution: Take 50 ml of Procaterol Hydrochloride Oral Solution and add 0.5 ml of the mixed reference stock solution. Shake well. Prepare three replicates.

[0221] 150% test solution: Take 50 ml of Procaterol Hydrochloride Oral Solution and add 0.5 ml of Mixed Reference Stock Solution 2. Shake well. Prepare three replicates.

[0222] Determination:

[0223] 95 μl of each of the above solutions were measured and injected into the high performance liquid chromatograph. The measured amounts of impurities I and II were calculated using the external standard method with the addition of correction factors, and their recoveries were calculated. The results are shown in Tables 14 and 15.

[0224] Table 14 Impurity I recovery test results

[0225]

[0226] Table 15 Impurity II recovery test results

[0227]

[0228] It can be seen from Table 14 and Table 15 that the recovery rate of impurity I is 95.12%, and the recovery rate of impurity II is 104.68%, both within the range of 90% to 110%; the RSD value of the recovery rate of impurity I in the test samples of each concentration is 0.61%, and the RSD value of the recovery rate of impurity II is 4.01%, both less than 5.0%, indicating that the accuracy of the HPLC determination of impurities I and impurity II is good.

[0229] 3.6 Stability test

[0230] Take the spiked test solution in "3.4 Repeatability and Precision Test" and inject 95 μl into the HPLC at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 h. Calculate the change in peak area of ​​each impurity peak in the spiked test solution. The results are shown in Table 16 below.

[0231] Table 16 Stability test results of spiked test solution

[0232]

[0233] As can be seen from Table 16, compared with the stability study at 0h, the changes in the peak areas of impurities I and II and the total peak area of ​​impurities in the spiked test solution within 24 hours were all less than 5%, and no new impurities were added, indicating that the HPLC method for the determination of impurities I and II had good stability.

[0234] 3.7 Durability test

[0235] With other chromatographic conditions unchanged, the column temperature was varied by ±5°C, the flow rate by ±0.1ml / min, the buffer salt concentration in the mobile phase by ±50%, the mobile phase pH by ±0.2, and different serial numbers of the columns were used for comparison. The method and results are as follows:

[0236] Solution preparation:

[0237] System suitability solution: Take an appropriate amount of self-ground Procaterol hydrochloride oral solution, heat it in a 90℃ water bath for 90 minutes, and let it cool to prepare the system suitability solution.

[0238] Reference solution: Take an appropriate amount of Procaterol hydrochloride reference substance, accurately weigh it, add an appropriate amount of solvent water to dissolve it and quantitatively dilute it to make a solution containing approximately 50ng of Procaterol hydrochloride per 1ml.

[0239] Sensitivity solution: Accurately measure 1 ml of the reference solution and place it in a 20 ml volumetric flask, dilute to the mark with solvent water, and shake well.

[0240] Determination:

[0241] 95 μl of each of the system suitability solution and the sensitivity solution were injected into the high performance liquid chromatograph. The resolution between the Procaterol hydrochloride peak and impurity I and the signal-to-noise ratio of the Procaterol hydrochloride peak in the chromatogram of the sensitivity solution were compared. The results are shown in Table 17 below.

[0242] Table 17 Durability test results

[0243]

[0244] As can be seen from Table 17, when the column temperature changes by ±5°C, the flow rate changes by ±0.1 ml / min, the buffer salt concentration in the mobile phase by ±50%, the pH value of the mobile phase by ±0.2, and when chromatographic columns with different serial numbers are selected, the system suitability test meets the requirements, indicating that the HPLC method has good durability.

[0245] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A method for determining related substances in a sample containing Procaterol hydrochloride, characterized in that: The method comprises the steps of: (1) Determination of related substances in samples containing Procaterol hydrochloride by high performance liquid chromatography; The chromatographic conditions of the high performance liquid chromatography are: Chromatographic column: reverse phase column; Mobile phase: mobile phase A and mobile phase B, wherein the mobile phase A comprises phosphate buffer; the mobile phase B comprises methanol; and Mobile phase elution: The mobile phase elution is gradient elution, and the order of the gradient elution is:

2. The method according to claim 1, wherein The related substances include impurity I and / or impurity II, and the structures of impurity I and / or impurity II are as follows: and enantiomers; 3. The method according to claim 1, wherein The pH of the phosphate buffer is 2.5-4.0, preferably 2.7-3.5, more preferably 3.0-3.2, and most preferably 3.

1.

4. The method according to claim 1, wherein The phosphate buffer was prepared by the following method: Take 7.6-8.0 ml of sodium dihydrogen phosphate dihydrate, add 980-1020 ml of water to dissolve it, and adjust the pH to 3.0-3.2 with phosphoric acid to obtain phosphate buffer.

5. The method according to claim 1, wherein The phosphate buffer was prepared by the following method: Take 7.8 g of sodium dihydrogen phosphate dihydrate, add 1000 ml of water to dissolve it, and adjust the pH to 3.1 with phosphoric acid.

6. The method according to claim 1, wherein The high performance liquid chromatography method further comprises one or more chromatographic conditions selected from the group consisting of: Chromatographic column temperature: 35-45°C, preferably 38-42°C, more preferably 40°C; Mobile phase flow rate: 0.2-3 mL / min, preferably 0.5-1.5 mL / min, more preferably 0.8-1.2 mL / min, most preferably 1.0 mL / min; and / or The detection wavelength is 230-270 nm, preferably 240-260 nm, more preferably 245-255 nm, and most preferably 259 nm.

7. The method according to claim 1, wherein The filler of the reverse phase chromatography column includes octadecylsilane bonded silica gel.

8. The method according to claim 1, wherein The specifications of the reverse phase chromatography column are 4.6 mm×150 mm and 3.5 μm.

9. The method according to claim 1, wherein The sample containing Procaterol hydrochloride includes Procaterol hydrochloride bulk drug or Procaterol hydrochloride oral solution.

10. The method according to claim 9, wherein The Procaterol hydrochloride oral solution comprises Procaterol hydrochloride, ethylparaben, butylparaben, sodium benzoate, citric acid, sodium citrate, ethanol and water.

Images