A method for simultaneously detecting multiple genotoxic impurities in drugs

Abstract

The present invention provides a method for simultaneously detecting multiple genotoxic impurities in a drug, wherein the genotoxic impurities are selected from one or more of budesonide triene impurity 5 (IM-5) of structural formula I and budesonide triene impurity 6 (IM-6) of structural formula II: wherein the method is liquid chromatography, and the liquid chromatography comprises the following chromatographic conditions: chromatographic column: a liquid chromatography column with core-shell octylsilane as a stationary phase; detection wavelength: 238-242nm; mobile phase A: 0.1v / v% phosphoric acid solution; mobile phase B: acetonitrile. The method of the present invention has good separation; no interference; strong specificity; high sensitivity; good linear relationship, can realize effective control of impurities in budesonide triene, and further ensures the product quality of budesonide and the drug safety of patients.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a method for simultaneously detecting multiple genotoxic impurities in a drug, such as budesonide triene. Background Art

[0002] Budesonide triene, chemical name is 11β-hydroxy-pregnane-1,4,16-triene-3,20-dione-21-acetate, CAS number is 3044-42-6, and the structural formula is shown in Formula 1 below.

[0003]

[0004] Budesonide is a glucocorticoid drug that induces the expression of angiotensin I converting enzyme and has a good therapeutic effect on both exogenous and intrinsic asthma. Compared with other asthma treatment drugs, budesonide can also relieve airway inflammatory remodeling lesions after treatment. When inhaled in trace amounts, even if some is absorbed or swallowed through the airways, it does not produce obvious systemic glucocorticoid adverse reactions. It is clinically used for patients with glucocorticoid-dependent or non-dependent bronchial asthma and asthmatic chronic bronchitis. The products that have been launched on the market include budesonide dry powder inhaler (trade name: Pulmicort Dubao), budesonide inhalation aerosol (trade name: Jishu), budesonide suspension for inhalation (trade name: Pulmicort Respules), etc.

[0005] The starting material budesonide triene in the synthesis route of budesonide raw materials is a key material, which provides the basic skeleton and key active groups for the synthesis of budesonide. The starting material may produce a variety of impurities during the production process, such as budesonide triene impurity 1 (represented by material code 52002-0-1IM-1 in this specification, hereinafter referred to as IM-1, chemical name: pregnane-1,4,16-triene-3,11,20-trione-21-acetic acid), budesonide triene impurity 2 (represented by material code 52002-0-1IM-2 in this specification, hereinafter referred to as IM-2, chemical name: 11β-hydroxy-pregnane-4,16-diene-11,20-dione-21-acetate), budesonide triene impurity 3 (represented by material code 52002-0-1IM-3 in this specification, hereinafter referred to as IM-3, chemical name: 11β-hydroxy-pregnane-1,4,16-triene-3,20 -dione-21-acetate), budesonide triene impurity 4 (represented by material code 52002-0-1IM-4 in this specification, hereinafter referred to as IM-4, chemical name: pregnane-1,4,9,16-tetraene-3,20-dione-21-acetate), budesonide triene impurity 5 containing bromocyclohexyl (structural formula as shown in I, represented by material code 52002-0-1IM-5 in this specification, hereinafter referred to as IM-5, chemical name: 9α-bromo-11β-hydroxy-pregnane-1,4,16-triene-3,20-dione-21-acetate), budesonide triene impurity 6 containing epoxy group (structural formula as shown in II, represented by material code 52002-0-1IM-6 in this specification, hereinafter referred to as IM-6, CAS No.:103466-44-0; chemical name: 9,11-epoxy-pregnane-1,4,16-triene-3,20-dione-21-acetate), budesonide triene impurity 7 (represented by material code 52002-0-1IM-7 in this specification, hereinafter referred to as IM-7, chemical name: 11β,17α-dihydroxy-pregnane-1,4-diene-3,20-dione-21-acetate), among which impurity 5 and impurity 6 are two genotoxic impurities.

[0006]

[0007] Although the reaction steps of impurities 5 and 6 are far from the final product and the content is very low, such impurities can induce gene mutations at very low levels and have the risk of carcinogenesis, so they need to be detected and controlled in budesonide raw materials. It is precisely because of its low content limit that the sensitivity of the detection method is high and it is difficult to achieve quantitative detection. There are many impurities in budesonide triene, and the impurities have similar structures. It is difficult to separate impurities 5 and 6 from many impurities, and the polarity of impurities 5 and 6 is close, which makes it more difficult to further separate the two. There is no literature report on the method of separating these potential genotoxic impurities and qualitatively and quantitatively detecting them.

[0008] Therefore, it is of great significance to develop a method for simultaneously detecting multiple genotoxic impurities in budesonide trienes with the advantages of good separation, strong specificity, high sensitivity, and good linear relationship, so as to achieve effective control of budesonide impurities and further ensure the product quality of budesonide and the safety of patients' medication. Summary of the invention

[0009] In order to overcome the shortcomings of the prior art, the present invention provides a method for simultaneously detecting genotoxic impurities containing bromocyclohexyl and epoxy groups in budesonide triene, thereby being able to effectively control the genotoxic impurities in budesonide triene, thereby reducing the occurrence of adverse reactions to medication in patients and ensuring medication safety.

[0010] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions:

[0011] A method for detecting genotoxic impurities containing bromocyclohexyl and epoxy groups in budesonide triene, wherein the genotoxic impurities are selected from one or more of budesonide triene impurity 5 (IM-5) of structural formula I and budesonide triene impurity 6 (IM-6) of structural formula II:

[0012]

[0013] Wherein, the method is liquid chromatography, and the liquid chromatography includes the following chromatographic conditions:

[0014] Chromatographic column: Liquid chromatography column with core-shell octylsilane as stationary phase;

[0015] Detection wavelength: 238-242nm;

[0016] Mobile phase A: 0.1 v / v% phosphoric acid solution;

[0017] Mobile phase B: acetonitrile.

[0018] Preferably, the genotoxic impurities include both the budesonide triene impurity 5 of structural formula I and the budesonide triene impurity 6 of structural formula II.

[0019] Preferably, the chromatographic column is Phenomenex Kinetex C8, with a specification of 2.6 μm and 150×4.6 mm.

[0020] Preferably, the detection wavelength is 238 nm, 240 nm or 242 nm.

[0021] Preferably, the liquid chromatography adopts gradient elution, and the conditions of the gradient elution are: the initial proportion of mobile phase B is 28-32 v / v%, increased to 30-34 v / v% after 4 minutes, maintained for 1 minute, then increased to 33-37 v / v% within 4 minutes, and then increased to 68-72 v / v% within 4 minutes, maintained for 3 minutes, and finally returned to the initial gradient within 1 minute and balanced for 4 minutes.

[0022] More preferably, the gradient elution is selected from one of the following:

[0023] Gradient 1:

[0024]

[0025] Gradient 2:

[0026]

[0027] Gradient Three:

[0028]

[0029] Preferably, the flow rate of the mobile phase is 0.8-1.2 ml / min, preferably 0.8 ml / min, 1.0 ml / min or 1.2 ml / min.

[0030] Preferably, the chromatographic conditions further include:

[0031] Column temperature: 18-22°C;

[0032] Sample pan temperature: 4°C;

[0033] Injection volume: 10 μl;

[0034] More preferably, the chromatographic conditions further include:

[0035] Column temperature: 18, 20 or 22°C;

[0036] Sample pan temperature: 4°C;

[0037] Injection volume: 10 μl;

[0038] Preferably, the method further comprises the preparation of a reference solution, the specific operations being:

[0039] Take 12 mg of budesonide triene working standard in a 200 ml volumetric flask, dissolve and dilute to the scale with 50 v / v% acetonitrile, and shake well; accurately measure 1 ml and place it in a 500 ml volumetric flask, dilute to the scale with 50 v / v% acetonitrile, and shake well.

[0040] Preferably, the method further comprises the preparation of a sensitivity solution, and the specific operations are:

[0041] Accurately measure 5 ml of the reference solution and place it in a 10 ml volumetric flask. Dilute to the mark with 50 v / v% acetonitrile and shake well.

[0042] Preferably, the method further comprises the preparation of a test solution, the specific operations being:

[0043] Weigh 25 mg of the sample to be tested, accurately weigh it, place it in a 25 ml volumetric flask, dissolve it in 50 v / v% acetonitrile and dilute it to the scale, shake well, and obtain it.

[0044] Preferably, the method further comprises the step of performing a determination, the specific operation being:

[0045] Accurately pipette 10 μl of the reference solution and the test solution, inject into the liquid chromatograph, and record the chromatogram.

[0046] As a preferred embodiment, the present invention provides a method for detecting genotoxic impurities containing bromocyclohexyl and epoxy groups in budesonide triene, wherein the genotoxic impurities are selected from one or more of budesonide triene impurity 5 (IM-5) of structural formula I and budesonide triene impurity 6 (IM-6) of structural formula II.

[0047]

[0048] The method is liquid chromatography, which includes the following chromatographic conditions:

[0049] Column: Phenomenex Kinetex C8, 2.6 μm, 150 × 4.6 mm;

[0050] Column temperature: 20°C;

[0051] Sample pan temperature: 4°C;

[0052] Flow rate: 1.0ml / min;

[0053] Detection wavelength: 240nm;

[0054] Injection volume: 10 μl;

[0055] Mobile phase A: 0.1 v / v% phosphoric acid solution;

[0056] Mobile phase B: acetonitrile;

[0057] Gradient elution, the elution program is as follows:

[0058]

[0059] Preparation of reference solution: Take 12 mg of budesonide triene working standard in a 200 ml volumetric flask, dissolve and dilute to scale with 50 v / v% acetonitrile, shake well; accurately measure 1 ml and place in a 500 ml volumetric flask, dilute to scale with 50 v / v% acetonitrile, shake well, and obtain;

[0060] Preparation of sensitivity solution: accurately measure 5 ml of reference solution into a 10 ml volumetric flask, dilute to scale with 50 v / v% acetonitrile, shake well, and obtain;

[0061] Preparation of test solution: Weigh 25 mg of the sample to be tested, accurately weigh it, place it in a 25 ml volumetric flask, dissolve it in 50 v / v% acetonitrile and dilute it to the scale, shake well, and obtain;

[0062] Determination: Accurately pipette 10 μl of reference solution and test solution, inject into liquid chromatograph, and record the chromatogram;

[0063] Quantitative detection: The content of budesonide triene genotoxic impurities in the test solution was calculated using the principal component external standard method; the content of IM-5 and IM-6 in the test solution was calculated using formula 1:

[0064]

[0065] in,

[0066] A X : refers to the peak area of ​​impurities in the test solution;

[0067] A S : refers to the average peak area of ​​the main peak in the reference solution;

[0068] V: refers to the dilution volume (ml);

[0069] C s : refers to the concentration of the reference solution (μg / ml);

[0070] M: refers to the sample weight of the test product (mg);

[0071] F: refers to the correction factor for impurities, where: IM-5 is 1.30 and IM-6 is 1.40.

[0072] In the above preferred embodiment, it is preferred to simultaneously detect two genotoxic impurities IM-5 and IM-6 in budesonide triene.

[0073] Using the detection method of the present invention, the impurity IM-5 has a good linear relationship in the concentration range of 0.0599 to 0.2396 μg / ml (equivalent to 0.006% to 0.024% of the main component of the test sample), the linear equation is Y=28137.9659X-12.1982, and the correlation coefficient is R=0.9997; the RSD of the 6 spiked recoveries is 0.7%, less than 5.0%, and the precision is good; the quantitative limit concentration is 0.0197 μg / ml (equivalent to about The S / N of the six injections was greater than 10, and the RSD of the impurity peak area in the six solutions was 1.3%, which was less than 10.0%; the detection limit concentration was 0.0098μg / ml (approximately equivalent to 0.001% of the main component of the test sample); at concentrations of 50%, 100% and 200%, the recoveries of the nine test samples were between 80% and 120%; the RSD of the nine recoveries was 1.5%, which was less than 10.0%, and the method had good accuracy.

[0074] By using the detection method of the present invention, the impurity IM-6 has a good linear relationship in the concentration range of 0.0601-0.2405 μg / ml (equivalent to 0.006%-0.024% of the main component of the test sample); the linear equation is Y=34131.7557X+5.5252, R=0.9998; the RSD of the 6 spiked recoveries is 0.5%, less than 5.0%, and the precision is good; the quantitative limit concentration is 0.0200 μg / ml (approximately equivalent to the main component of the test sample The S / N of the six injections was greater than 10, and the RSD of the impurity peak area in the six solutions was 2.2%, which was less than 10.0%; the detection limit concentration was 0.0100 μg / ml (approximately equivalent to 0.001% of the main component of the test sample); at concentrations of 50%, 100% and 200%, the recoveries of the nine test samples were between 80% and 120%; the RSD of the nine recoveries was 1.8%, which was less than 10.0%, and the method had good accuracy.

[0075] The beneficial effects of the present invention are as follows:

[0076] The present invention adopts a liquid chromatography analysis method to separate two genotoxic impurities (IM-5 and IM-6) from numerous impurities of budesonide triene, and further separates and detects the two genotoxic impurities (IM-5 and IM-6). In the detection method for simultaneously detecting budesonide triene established by the present invention, the separation between the two genotoxic impurities and the adjacent chromatographic peaks is greater than 1.5, and the separation is good; the blank has no interference at the retention time of the two genotoxic impurities, the specificity is strong, the sensitivity solution S / N is greater than 10, and the sensitivity is high; the linear correlation coefficient is not less than 0.995, there is a good linear relationship, and the effective control of impurities in budesonide triene can be achieved, and the product quality of budesonide and the drug safety of patients are further guaranteed. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] The present invention will be further described below in conjunction with the accompanying drawings.

[0078] Figure 1 The chromatogram of the test solution I obtained by the chromatographic condition 1 in the research example 1;

[0079] Figure 2 The chromatogram of the test solution obtained after the gradient optimization of chromatographic condition 1 in Research Example 1;

[0080] Figure 3 The chromatogram of the test solution obtained after chromatographic condition 2 in Study Example 1;

[0081] Figure 4 is the chromatogram of the blank solution in Example 1;

[0082] Figure 5 is the chromatogram of the system suitability solution in Example 1;

[0083] Figure 6 It is the chromatogram of the test solution in Example 1;

[0084] Figure 7 is the chromatogram of the IM-5 positioning solution in Example 1;

[0085] Figure 8 is the chromatogram of the IM-6 positioning solution in Example 1;

[0086] Fig. 9 Shown is the linear regression curve of budesonide triene in Example 1;

[0087] Fig.10 Shown is the linear regression curve of IM-5 in Example 1;

[0088] Fig.11 Shown is the linear regression curve of IM-6 in Example 1;

[0089] Fig.12 The chromatogram recorded in the durability experiment in Example 1 is shown; wherein: the chromatographic peaks of impurities IM-5 and IM-6 are marked in the front and back circles respectively;

[0090] Fig.13 It is the chromatogram of the test solution of batch ST019-19082-79A in Example 1;

[0091] Fig.14 This is the chromatogram of the test solution of batch ST019-19082-79B in Example 1. DETAILED DESCRIPTION

[0092] The present invention is described below with reference to specific embodiments. It will be appreciated by those skilled in the art that these embodiments are only intended to illustrate the present invention and do not limit the scope of the present invention in any way. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art may make various changes or modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the present application.

[0093] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0094] For budesonide triene, material code 52002-0-1 is used instead in some spectra.

[0095] The specific information of impurities IM-1, IM-2, IM-3, IM-4, IM-5, IM-6 and IM-7 are as follows:

[0096]

[0097] In the following examples, % involved in the mobile phase represents volume percentage.

[0098] Study Example 1: Establishment of Chromatographic Conditions for the Detection Method of the Present Invention

[0099] In order to detect the content of two genotoxic impurities (IM-5 and IM-6) in budesonide triene, according to the structure of impurities IM-5 and IM-6, a new type of bonded and double-end-capped octadecylsilane bonded chromatographic column suitable for the analysis of steroidal compounds was initially selected. The brand is Welch Ultimate XB-C18, the specifications are 3.0μm, 150×4.6mm. Because there are multiple steroidal impurities with similar polarity and similar structure in the process route of budesonide triene, in order to improve the separation efficiency and avoid the degradation of the main component interfering with the detection of genotoxic impurities, the column temperature is selected at 40℃ and the sample is controlled at 4℃. The initial mobile phase ratio is 5% acetonitrile, slowly eluted to 95% within 15 minutes, and then rinsed at this ratio for 5 minutes to avoid residues, and then returned to the starting ratio within 0.1 minutes, and balanced for 7.9 minutes. The detection wavelength is selected as 240nm, and the injection volume is 10μl. The above chromatographic conditions are named chromatographic conditions 1, and the gradient is as follows:

[0100]

[0101] Preparation of test solution I: Weigh 2.52 mg of budesonide triene, place in a 10 ml volumetric flask, add budesonide triene impurity 1 (represented by material code 52002-0-1IM-1 in this specification, hereinafter referred to as IM-1, chemical name: pregnane-1,4,16-triene-3,11,20-trione-21-acetic acid), budesonide triene impurity 2 (represented by material code 52002-0-1IM-2 in this specification, hereinafter referred to as IM-2, chemical name: 11β-hydroxy-pregnane-4,16-diene-11,20-dione-21-acetate) , budesonide triene impurity 3 (represented by material code 52002-0-1IM-3 in this specification, hereinafter referred to as IM-3, chemical name: 11β-hydroxy-pregnane-1,4,16-triene-3,20-dione-21-acetate), budesonide triene impurity 4 (represented by material code 52002-0-1IM-4 in this specification, hereinafter referred to as IM-4, chemical name: pregnane-1,4,9,16-tetraene-3,20-dione-21-acetate), IM-5 and IM-6 positioning solution 50μl, add 50% acetonitrile to dilute to the scale, shake well, and obtain. Preparation of positioning solution: weigh about 5mg of each impurity, place it in a 100ml volumetric flask, dissolve it with 50% acetonitrile solution and dilute to the scale, shake well, and obtain.

[0102] Measure the test solution under the chromatographic condition 1 and record the chromatogram. Figure 1 . Figure 1 As shown, the chromatographic peaks of IM-2, IM-5 and IM-6 cannot be baseline separated.

[0103] The mobile phase gradient was optimized based on chromatographic condition 1. The initial proportion of mobile phase B was adjusted to 25% and maintained for 30 minutes, then increased to 75% within 15 minutes, flushed for 5 minutes, returned to the initial proportion within 1 minute and balanced for 9 minutes. The specific gradient is as follows:

[0104]

[0105] The remaining chromatographic conditions are the same as those in chromatographic condition 1. The test solution is measured under this chromatographic condition and the chromatogram is recorded. Figure 2 . Figure 2 As shown, the chromatographic peaks of IM-2 and IM-6 cannot be baseline separated.

[0106] On this basis, the relevant parameters in chromatographic condition 1 were optimized as follows: the chromatographic column was changed from Welch Ultimate XB-C18 (3.0μm, 150×4.6mm) to a high-performance core-shell octylsilane column Phenomenex Kinetex C8, with specifications of 2.6μm, 150×4.6mm; to improve the peak shape and avoid hydrolysis of various impurities, 0.1% phosphoric acid was added to mobile phase A. The mobile phase gradient was changed to: the initial proportion of mobile phase B was 30%, increased to 32% within 4 minutes, maintained for 1 minute, then increased to 35% within 4 minutes, and then increased to 70% within 4 minutes, maintained for 3 minutes, and then returned to the initial proportion within 1 minute, and balanced for 4 minutes. The specific gradient is as follows:

[0107]

[0108] The flow rate was maintained at 1 ml / min, the column temperature was adjusted to 20°C to reduce the degradation of the main component in the column, and the detection wavelength was adjusted to 240 nm, the strong absorption band K band of conjugated double bonds, to increase the response intensity. The above chromatographic conditions were named chromatographic conditions 2.

[0109] Considering the low limit level of genotoxic impurities, IM-5 and IM-6 at the limit level were used for spike experiments to examine the separation.

[0110] Preparation of test solution II: Weigh 25.08 mg of budesonide triene, place in a 25 ml volumetric flask, accurately add 1 ml of resolution impurity stock solution and 5 ml of genotoxic impurity stock solution, dilute to scale with diluent, shake well, and obtain (resolution impurity stock solution preparation: weigh IM-1, IM-2, IM-3, IM-4 and budesonide triene impurity 7 (represented by material code 52002-0-1IM-7 in this specification, hereinafter referred to as IM-7, chemical name: 11β,17α-dihydroxy-pregnant-1,4-diene-3, 20-diketo-21-acetate) reference substance 3.758745mg, 2.5735mg, 2.5215mg, 2.5425mg and 2.5105mg are placed in a 50ml volumetric flask, dissolved and diluted to the scale with a diluent, and shaken to obtain; preparation of genotoxic impurity stock solution: 3.2095mg and 3.1075mg IM-5 and IM-6 are weighed respectively and placed in a 50ml volumetric flask, dissolved and diluted to the scale with a diluent, shaken to obtain 1ml each, placed in a 200ml volumetric flask, diluted to the scale with a diluent, shaken to obtain).

[0111] Measure the test solution II under the chromatographic condition 2 and record the chromatogram. Figure 3 . Figure 3 It is shown that the separation between the chromatographic peaks of impurities IM-1, IM-2, IM-3, IM-4, IM-5, IM-6, IM-7 and budesonide triene in the test solution II is greater than 1.5.

[0112] Through the above research, the preferred chromatographic conditions of the present invention were preliminarily established, namely:

[0113] Chromatographic column: high-performance core-shell octylsilane column, specification 2.6μm, 150×4.6mm;

[0114] Column temperature: 20°C;

[0115] Sample plate temperature: 4°C

[0116] Flow rate: 1.0ml / min;

[0117] Detection wavelength: 240nm;

[0118] Injection volume: 10 μl;

[0119] Mobile phase A: 0.1% phosphoric acid solution;

[0120] Mobile phase B: acetonitrile;

[0121] gradient:

[0122]

[0123] Example 1 Methodological verification of the detection method established by the present invention

[0124] 1.1 Specificity and system adaptability

[0125] Solution preparation:

[0126] (1) Blank solution: Take 50% acetonitrile solution.

[0127] (2) Test solution: Accurately weigh 25.20 mg of budesonide triene (batch number: 615D052007001, source: Shandong Sirui Pharmaceutical Co., Ltd.) and place it in a 25 ml volumetric flask. Dissolve and dilute to the mark with 50% acetonitrile solution and shake well to obtain the solution.

[0128] (3) Impurity reference mixed stock solution: Weigh the impurity reference substances IM-1, IM-2, IM-3, IM-4 and IM-7 according to the table below, place them in 20 ml volumetric bottles, dissolve and dilute to the mark with 50% acetonitrile solution, shake well, and record them as IM-1, IM-2, IM-3, IM-4 and IM-7 impurity stock solutions. Weigh the impurity reference substances IM-5 and IM-6 according to the table below, place them in 50 ml volumetric bottles, dissolve and dilute to the mark with 50% acetonitrile solution, shake well, and record them as IM-5 and IM-6 impurity stock solutions. Take 20 ml of IM-1, IM-2, IM-3, IM-4 and IM-7 impurity stock solutions and 1 ml of IM-5 and IM-6 impurity stock solutions respectively, place them in 200 ml volumetric bottles, dilute to the mark with 50% acetonitrile solution, shake well, and obtain. The weighed amount of each impurity reference substance is shown in Table 1.

[0129] Table 1 Sample weights of seven impurity reference substances

[0130] Impurity Reference IM-1 IM-2 IM-3 IM-4 IM-5 IM-6 IM-7 Sample weight (mg) 15.10 10.29 5.0505 10.49 3.2095 3.1075 10.58

[0131] (4) Positioning solution: Take 100ul of each impurity stock solution, add 900ul of diluent and shake well.

[0132] (5) System suitability solution: Weigh about 25.11 mg of budesonide triene into a 25 ml volumetric flask, transfer 1.0 ml of the mixed reference substance stock solution, dilute to the mark with 50% acetonitrile solution, and shake well to prepare the system suitability solution.

[0133] (6) Reference solution: Weigh about 12.33 mg of budesonide triene into a 200 ml volumetric flask, dissolve and dilute to the mark with 50% acetonitrile solution, shake well, accurately pipette 1 ml into a 500 ml volumetric flask, dilute to the mark with 50% acetonitrile solution, shake well, and use this as the working reference solution. Prepare two aliquots in parallel.

[0134] Determination: Take 10 μl of blank solution, each positioning solution, system suitability solution, reference solution, and test solution, and record the chromatogram. The chromatogram of the blank solution is shown in Figure 4 , the chromatogram of the system suitability solution is shown in Figure 5 The chromatogram of the test solution is shown in Figure 6 , the chromatogram of IM-5 positioning solution is shown in Figure 7 , the chromatogram of IM-6 positioning solution is shown in Figure 8 .

[0135] Results: There was no obvious interference in the blank solution at the retention time of the main peak and the known impurity peaks; the separation between the main peak and the adjacent impurity peaks and the known impurity peaks in the system suitability solution spectrum was not less than 1.2; the peak purity of each impurity peak in the system suitability solution met the requirements (purity angle was less than the purity threshold), and was consistent with the retention time of the corresponding peak in each positioning solution.

[0136] 2.1 Limit of quantification and limit of detection

[0137] Solution preparation:

[0138] (1) Limit of quantitation (LQQ) solution: Accurately weigh 6.1464 mg and 6.0618 mg of the impurity IM-5 and IM-6 reference substances, place in a 100 ml volumetric flask, add 50% acetonitrile solution to dissolve and dilute to the mark, shake well. Accurately pipette 1 ml, place in a 200 ml volumetric flask, add 50% acetonitrile solution to dilute to the mark, shake well. Accurately measure 2 ml of the above solution, place in a 25 ml volumetric flask, dilute to the mark with 50% acetonitrile solution, shake well, and the solution is obtained.

[0139] (2) Detection limit solution: Accurately pipette 1 ml of limit of quantitation (LOQ) solution, add 1 ml of diluent, and shake well.

[0140] Determination: Accurately pipette 10 μl of the quantitative limit solution and the detection limit solution for injection and determination. Repeat the quantitative limit determination 6 times. The results are shown in Tables 2 to 4.

[0141] Table 2 IM-5 limit of quantification (LOQ) determination results

[0142]

[0143] Table 3 IM-6 limit of quantification (LOQ) determination results

[0144]

[0145]

[0146] Table 4 Detection limit (LOD) determination results

[0147] name Concentration (μg / ml) S / N IM-5 0.0098 9.3 IM-6 0.0100 9.5

[0148] 2.3 Linear investigation

[0149] Preparation of series mixed reference solution:

[0150] (1) Preparation of linear stock solution: Accurately weigh 4.8535 mg, 4.9920 mg and 4.8635 mg of budesonide triene, IM-5 and IM-6 reference substances, respectively, into a 100-ml volumetric flask, add 50% acetonitrile solution to dissolve and dilute to the scale, shake well, accurately pipette 1 ml, place into a 100-ml volumetric flask, add 50% acetonitrile solution to dilute to the scale, shake well, and obtain.

[0151] (2) L-0.024%: Accurately measure 5 ml of the linear stock solution and place it in a 10 ml volumetric flask. Add 50% acetonitrile solution to dilute to the mark and shake well.

[0152] (3) L-0.019%: Accurately measure 4 ml of the linear stock solution and place it in a 10 ml volumetric flask. Add 50% acetonitrile solution to dilute to the mark and shake well.

[0153] (4) L-0.014%: Accurately measure 3 ml of the linear stock solution and place it in a 10 ml volumetric flask. Add 50% acetonitrile solution to dilute to the mark and shake well.

[0154] (5) L-0.012%: Accurately measure 5 ml of the linear stock solution and place it in a 20 ml volumetric flask. Add 50% acetonitrile solution to dilute to the mark and shake well.

[0155] (6) L-0.0096%: Accurately measure 4 ml of the linear stock solution and place it in a 20 ml volumetric flask. Add 50% acetonitrile solution to dilute to the mark and shake well.

[0156] (7) L-0.006%: Accurately measure 5 ml of L-0.012% solution and place it in a 10 ml volumetric flask. Add 50% acetonitrile solution to dilute to the mark and shake well.

[0157] Determination: 10 μl of each of L-0.006%, L-0.096%, L-0.012%, L-0.014%, L-0.019% and 0.024% solutions were respectively injected for determination, and a linear regression curve was drawn with concentration as the X-axis and peak area as the Y-axis.

[0158] Results: The test results are shown in Tables 5 to 7, and the linear regression curve Figures 9-11 .

[0159] Table 5 Linear determination results of desonide triene (52002-0-1)

[0160]

[0161]

[0162] Table 6 Desonide triene impurity IM-5 linear determination results

[0163]

[0164] Table 7 Desonide triene impurity IM-6 linear determination results

[0165]

[0166] 2.4 Accuracy and precision

[0167] Solution preparation:

[0168] (1) Mixed reference stock solution: Accurately weigh 6.1464 mg of the impurity IM-5 reference substance and 6.0618 mg of the IM-6 reference substance into a 100 ml volumetric flask, dissolve and dilute to the mark with 50% acetonitrile solution, and shake well. Then, pipette 1 ml of each of the above solutions into a 200 ml volumetric flask, dilute to the mark with 50% acetonitrile solution, and shake well.

[0169] (2) Reference substance solution: Pipette 10 ml of the mixed reference substance stock solution into a 25 ml volumetric flask, dilute to the mark with 50% acetonitrile solution, shake well, and prepare 2 portions in parallel.

[0170] (3) Recovery R-50% solution: Accurately weigh 25.04 mg of budesonide triene and place it in a 25 ml volumetric flask. Add an appropriate amount of 50% acetonitrile solution to dissolve it. Accurately measure 5 ml of the mixed reference stock solution into the same volumetric flask and dilute it to the mark with 50% acetonitrile solution. Shake well and prepare 3 portions in parallel. The other two portions have sample weights of 25.34 mg and 25.25 mg, respectively.

[0171] (4) Recovery R-100% solution: Accurately weigh 25.08 mg of budesonide triene and place it in a 25 ml volumetric flask. Add an appropriate amount of 50% acetonitrile solution to dissolve it. Accurately measure 10 ml of the mixed reference stock solution into the same volumetric flask and dilute it to the mark with 50% acetonitrile solution. Shake well and prepare 3 portions in parallel. The other two portions have sample weights of 25.25 mg and 25.14 mg, respectively.

[0172] (5) Recovery R-200% solution: Accurately weigh 25.16 mg of budesonide triene and place it in a 25 ml volumetric flask. Add an appropriate amount of 50% acetonitrile solution to dissolve it. Accurately measure 20 ml of the mixed reference stock solution into the same volumetric flask and dilute it to the mark with 50% acetonitrile solution. Shake well and prepare 3 portions in parallel. The other two portions have sample weights of 25.06 mg and 25.13 mg, respectively.

[0173] (6) Test solution: Accurately weigh 25.01 mg of budesonide triene and place it in a 25 ml volumetric flask. Add 50% acetonitrile solution to dissolve and dilute to the mark. Shake well to obtain the solution.

[0174] Determination: Take 10μl of each reference solution, recovery solution and test solution for injection, record the peak area of ​​each chromatographic peak, calculate the content by peak area according to the external standard method, and calculate the recovery rate with the impurity content in the test solution as the "background". The results are shown in Tables 8 to 9.

[0175] Table 8 Impurity IM-5 accuracy and recovery determination results

[0176]

[0177] Table 9 Accuracy and recovery results of impurity IM-6

[0178]

[0179]

[0180] The results showed that the recoveries of impurities IM-5 and IM-6 were between 80.0% and 120.0% at 50%, 100% and 200% limit concentrations, indicating that the method had good accuracy. The RSDs of the three recoveries at the same level and the nine recoveries at three concentration levels were all less than 10.0%, which met the requirements and indicated that the method had good precision.

[0181] 2.5 Durability

[0182] Solution preparation: Take the first portion of the recovery rate R-100% solution under 2.4(3).

[0183] Reference solution: Take the reference solution under 1.1(6).

[0184] System suitability solution: Take the system suitability solution under 1.1(5).

[0185] Determination: At different flow rates (0.8 ml / min, 1.2 ml / min); different column temperatures (18°C, 22°C); different detection wavelengths (238 nm, 242 nm); different mobile phase ratios (Table 10, Table 11, Table 12); under different chromatographic column conditions, 10 μl of the recovery rate R-100% solution was taken for injection and determination, and the chromatogram was recorded.

[0186] Table 10 Different mobile phase ratios - original conditions

[0187]

[0188] Table 11 Different mobile phase ratios - Variation 1

[0189]

[0190] Table 12 Different mobile phase ratios - variation 2

[0191]

[0192]

[0193] result: Fig.12 As shown in Table 13, under the above chromatographic conditions, the separation degree between each impurity and the adjacent peak is greater than 1.5, and the system applicability meets the requirements; the absolute difference between the impurity content measured under each condition and the result measured by the current method is no more than 0.004%, which meets the requirements, and the method has good durability. Changing the chromatographic conditions such as flow rate, column temperature, mobile phase gradient, chromatographic column and detection wavelength within a small range will not affect the detection of genotoxic impurities IM-5 and IM-6.

[0194] Table 13 Durability test results

[0195]

[0196] Example 2 Determination of genotoxic impurities in different batches of budesonide triene

[0197] Chromatographic conditions:

[0198] Column: Phenomenex Kinetex C8, 2.6 μm, 150 × 4.6 mm;

[0199] Column temperature: 20°C;

[0200] Sample pan temperature: 4°C;

[0201] Flow rate: 1.0ml / min;

[0202] Detection wavelength: 240nm;

[0203] Injection volume: 10 μl;

[0204] Mobile phase A: 0.1% phosphoric acid solution;

[0205] Mobile phase B: acetonitrile;

[0206] gradient:

[0207]

[0208]

[0209] Working reference solution: Weigh about 12.22 mg of budesonide triene into a 200 ml volumetric flask, dissolve and dilute to the mark with 50% acetonitrile solution, shake well, accurately transfer 1 ml into a 500 ml volumetric flask, dilute to the mark with 50% acetonitrile solution, shake well, and use this as the working reference solution. Prepare two portions in parallel, with the other portion weighing 12.23 mg.

[0210] Preparation of the test solution: Weigh 25 mg of budesonide triene (see Table 14 for specific sample weight), place in a 25 ml volumetric flask, dissolve and dilute to the scale with 50% acetonitrile solution, shake well, and obtain.

[0211] Table 14 Budesonide triene sample weighing record

[0212] batch number ST019-19082-79A ST019-19082-79B Sample weight (mg) 25.03 25.14

[0213] Determination: Accurately pipette 10 μl of reference solution and test solution, inject into liquid chromatograph, and record the chromatogram. Figures 13-14 .

[0214] Determination results: The content of budesonide triene (52002-0-1) genotoxic impurities in the test solution was calculated using the principal component external standard method; the content of IM-5 and IM-6 in the test solution was calculated using formula 1:

[0215]

[0216] in,

[0217] A X : refers to the peak area of ​​impurities in the test solution;

[0218] A S : refers to the average peak area of ​​the main peak in the reference solution;

[0219] V: refers to the dilution volume (ml);

[0220] C s : refers to the concentration of the reference solution (μg / ml);

[0221] M: refers to the sample weight of the test product (mg);

[0222] F: refers to the correction factor for impurities, where: IM-5 is 1.30 and IM-6 is 1.40.

[0223] In the above preferred embodiment, it is preferred to simultaneously detect two genotoxic impurities IM-5 and IM-6 in budesonide triene.

[0224] The measurement results are shown in Table 15.

[0225] Table 15 Determination results of genotoxic impurities in two batches of budesonide triene

[0226] Related substances IM-5 IM-6 ST019-19082-79A Not detected 0.003％ ST019-19082-79A Not detected 0.002％

[0227] In summary, the present invention provides a method for simultaneously determining multiple genotoxic impurities (selected from one or more of IM-5 and IM-6) that may be present in budesonide triene. The method has good separation, strong specificity and high sensitivity, so that the product quality of budesonide triene can be further detected, and the drug safety of patients can be better guaranteed.

Claims

1. A method for detecting genotoxic impurities containing bromocyclohexyl and epoxy groups in budesonide triene, wherein the genotoxic impurities are budesonide triene impurity 5 of structural formula I and budesonide triene impurity 6 of structural formula II: in, The method is liquid chromatography, which includes the following chromatographic conditions: Chromatographic column: Liquid chromatography column with core-shell octylsilane as stationary phase; Detection wavelength: 238-242nm; Mobile phase A: 0.1 v / v% phosphoric acid solution; Mobile phase B: acetonitrile; Liquid chromatography adopted gradient elution, and the conditions of the gradient elution were as follows: the initial proportion of mobile phase B was 28-32 v / v%, increased to 30-34 v / v% after 4 minutes, maintained for 1 minute, then increased to 33-37 v / v% within 4 minutes, then increased to 68-72 v / v% within another 4 minutes, maintained for 3 min, and finally returned to the initial gradient within 1 minute and equilibrated for 4 minutes.

2. The method according to claim 1, characterized in that The genotoxic impurities include both budesonide triene impurity 5 of structural formula I and budesonide triene impurity 6 of structural formula II.

3. The method according to claim 1 or 2, characterized in that: The chromatographic column is Phenomenex Kinetex C8, with a specification of 2.6 μm and 150×4.6 mm.

4. The method according to claim 1 or 2, characterized in that: The detection wavelength is 238nm, 240nm or 242nm.

5. The method according to claim 1 or 2, characterized in that: The gradient elution is selected from one of the following: Gradient 1: Gradient 2: Gradient Three:

6. The method according to claim 1 or 2, characterized in that: The flow rate of the mobile phase is 0.8-1.2 ml / min.

7. The method according to claim 1 or 2, characterized in that: The flow rate of the mobile phase is 0.8 ml / min, 1.0 ml / min or 1.2 ml / min.

8. The method according to claim 1 or 2, characterized in that: The chromatographic conditions also include: Column temperature: 18-22°C; Sample pan temperature: 4°C; Injection volume: 10 μl.

9. The method according to claim 1 or 2, characterized in that: The chromatographic conditions also include: Column temperature: 18, 20 or 22°C; Sample pan temperature: 4°C; Injection volume: 10 μl.

10. The method according to claim 1 or 2, characterized in that: The method also includes the preparation of a reference solution, which is specifically performed as follows: Take 12 mg of budesonide triene working standard in a 200 ml volumetric flask, dissolve it with 50 v / v% acetonitrile and dilute it to the mark, shake well; accurately measure 1 ml and place it in a 500 ml volumetric flask, dilute it with 50 v / v% acetonitrile to the mark, shake well; The method also includes the preparation of a sensitivity solution, which is specifically performed as follows: Accurately measure 5 ml of the reference solution and place it in a 10 ml volumetric flask, dilute to the mark with 50 v / v% acetonitrile, and shake well to obtain; The method also includes the preparation of a test solution, which is specifically performed as follows: Weigh 25 mg of the sample to be tested, accurately weigh it, place it in a 25 ml volumetric flask, dissolve it in 50 v / v% acetonitrile and dilute it to the mark, shake well, and you have it; The method further comprises the step of performing a determination, which is specifically performed as follows: Accurately pipette 10 μl of the reference solution and the test solution, inject into the liquid chromatograph, and record the chromatogram.

11. A method for detecting genotoxic impurities containing bromocyclohexyl and epoxy groups in budesonide triene, wherein the genotoxic impurities are budesonide triene impurity 5 of structural formula I and budesonide triene impurity 6 of structural formula II; The method is liquid chromatography, which includes the following chromatographic conditions: Column: Phenomenex Kinetex C8, 2.6 μm, 150 × 4.6 mm; Column temperature: 20°C; Sample pan temperature: 4°C; Flow rate: 1.0ml / min; Detection wavelength: 240nm; Injection volume: 10 μl; Mobile phase A: 0.1 v / v% phosphoric acid solution; Mobile phase B: acetonitrile; gradient: Preparation of reference solution: Take 12 mg of budesonide triene working standard in a 200 ml volumetric flask, dissolve and dilute to scale with 50 v / v% acetonitrile, and shake well; accurately measure 1 ml and place it in a 500 ml volumetric flask, dilute to scale with 50 v / v% acetonitrile, and shake well; Preparation of sensitivity solution: accurately measure 5 ml of reference solution into a 10 ml volumetric flask, dilute to scale with 50 v / v% acetonitrile, shake well, and obtain; Preparation of test solution: Weigh 25 mg of the sample to be tested, accurately weigh it, place it in a 25 ml volumetric flask, dissolve it in 50 v / v% acetonitrile and dilute it to the scale, shake well, and obtain; Determination: Accurately pipette 10 μl of each of the sensitivity solution, reference solution and test solution, inject into the liquid chromatograph, and record the chromatogram; Quantitative detection: The content of budesonide triene genotoxic impurities in the test solution was calculated using the principal component external standard method; the content of budesonide triene impurity 5 and budesonide triene impurity 6 in the test solution was calculated using formula 1: in, A X : refers to the peak area of ​​impurities in the test solution; A S : refers to the average peak area of ​​the main peak in the reference solution; V: refers to the dilution volume (ml); C s : refers to the concentration of the reference solution (μg / ml); M: refers to the sample weight of the test product (mg); F: refers to the correction factor for impurities; among them: budesonide triene impurity 5 is 1.30, impurity 6 is 1.

40.

12. The method according to claim 11, characterized in that The method simultaneously detects two genotoxic impurities, budesonide triene impurity 5 and impurity 6, in budesonide triene.

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