Detection method of residues in homopiperazine
阅读说明：本技术 一种高哌嗪中残留物的检测方法 (Detection method of residues in homopiperazine ) 是由 尤梅 陈云建 彭劭 杨建玲 于 2020-05-13 设计创作，主要内容包括：本发明公开了一种高哌嗪中残留物的检测方法,包括以下步骤：(1)称量高哌嗪产品,加入稀释剂溶解,定容,混匀,作为供试品溶液；(2)分别称取对照品N,N′-二对甲苯磺酰基乙二胺、N,N′-1,4-二对甲苯磺酰基高哌嗪、苯酚,置于同一容器内,加入稀释剂溶解,定容,混匀,稀释,作为对照品溶液；(3)采用高效液相色谱法进行测定,测定条件包括：十八烷基硅烷键合硅胶为填充剂的色谱柱,以乙酸铵缓冲液为流动相A,乙腈为流动相B,进行梯度洗脱,检测波长为225nm,取对照品溶液和供试品溶液注入液相色谱仪,记录色谱图。本发明的残留物中苯酚和两个中间体的检测方法,能够避免溶剂峰的干扰,基线稳定,分离度良好。(The invention discloses a detection method of residues in homopiperazine, which comprises the following steps: (1) weighing a high piperazine product, adding a diluent to dissolve, fixing the volume, and uniformly mixing to obtain a test solution; (2) respectively weighing reference substances of N, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine and phenol, placing in the same container, adding a diluent for dissolving, fixing the volume, uniformly mixing, and diluting to obtain a reference substance solution; (3) the determination is carried out by adopting high performance liquid chromatography, and the determination conditions comprise: performing gradient elution by using an ammonium acetate buffer solution as a mobile phase A and acetonitrile as a mobile phase B through a chromatographic column using octadecylsilane chemically bonded silica as a filler, wherein the detection wavelength is 225nm, injecting a reference solution and a test solution into a liquid chromatograph, and recording a chromatogram. The method for detecting phenol and two intermediates in residues can avoid the interference of solvent peaks, and has stable baseline and good separation degree.)
1. A method for detecting residues in homopiperazine is characterized by comprising the following steps:
(1) weighing a high piperazine product, adding a diluent to dissolve, fixing the volume, and uniformly mixing to obtain a test solution;
(2) respectively weighing reference substances of N, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine and phenol, placing in the same container, adding a diluent for dissolving, fixing the volume, uniformly mixing, and diluting to obtain a reference substance solution;
(3) the determination is carried out by adopting high performance liquid chromatography, and the determination conditions comprise: performing gradient elution by using an ammonium acetate buffer solution as a mobile phase A and acetonitrile as a mobile phase B through a chromatographic column using octadecylsilane chemically bonded silica as a filler, wherein the detection wavelength is 225nm, injecting a reference solution and a test solution into a liquid chromatograph, and recording a chromatogram.
2. The method for detecting residues in homopiperazine as claimed in claim 1, wherein in step (3), the time and mobile phase ratio of gradient elution are as follows: and (3) 0-8min, reducing the volume percentage of the mobile phase A from 95% to 25%, increasing the volume percentage of the mobile phase B from 5% to 75%, and 8-13min, wherein the volume percentage of the mobile phase A is kept at 25%, the volume percentage of the mobile phase B is kept at 75%, and 13-13.1min, the volume percentage of the mobile phase A is increased from 25% to 95%, the volume percentage of the mobile phase B is reduced from 75% to 5%, and 13.1-15min, the volume percentage of the mobile phase A is kept at 95%, and the volume percentage of the mobile phase B is kept at 5%.
3. The method for detecting residues in homopiperazine according to claim 1 or 2, wherein in step (3), the concentration of the ammonium acetate buffer solution is 5mmol/L, and the pH is adjusted to 5.0 with acetic acid.
4. The method for detecting residues in homopiperazine of any one of claims 1-3, characterized in that, the concentration of N, N '-di-p-toluenesulfonyl ethylenediamine, N, N' -1, 4-di-p-toluenesulfonyl homopiperazine and phenol in the control solution are all 0.01 mg/mL; the concentration of high piperazine in the test solution was 10 mg/mL.
5. The method for detecting residues in homopiperazine of any one of claims 1-4, wherein the injection volume of the control solution and the test solution is 5 μ l.
6. The method for detecting residues in homopiperazine according to any one of claims 1 to 5, characterized in that the diluent in steps (1) and (2) is acetonitrile.
7. The method for detecting residues in homopiperazine according to any one of claims 1-6, characterized in that the chromatographic column in step (3) is a Waters XbridgeTMC18, 4.6mm by 150mm, 3.5 μm, or equivalent columns.
8. The method for detecting residues in homopiperazine of any one of claims 1 to 7, characterized in that, in the determination conditions of step (3), the column temperature is 30 ℃ and the flow rate is 1.0ml per minute.
9. The method for detecting residues in homopiperazine according to any one of claims 1 to 8, characterized in that, in the determination conditions of step (3), the collection time is 15min and the equilibration time is 5 min.
10. The method for detecting residues in homopiperazine of any one of claims 1 to 9, characterized in that, when the chromatogram of the test solution has chromatographic peaks of phenol, N '-di-p-toluenesulfonylethylenediamine, N' -1, 4-di-p-toluenesulfonylpiperazine, the content of phenol, N '-di-p-toluenesulfonylethylenediamine, N' -1, 4-di-p-toluenesulfonylpiperazine is not more than 0.10% as calculated by peak area according to the external standard method; the other single impurities are not more than 0.10 percent, and the total amount of the impurities is not more than 1.0 percent.
Technical Field
The invention belongs to the field of medicine quality control, and particularly relates to a detection method of residues in homopiperazine, and more particularly relates to a detection method of residues in homopiperazine, such as phenol, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine and other unknown impurities.
Background
The homopiperazine is synthesized by using ethylenediamine as a starting material through three steps of reactions including sulfonylation, cyclization and desulfonylation, reagents of phenol, N '-di-p-toluenesulfonyl ethylenediamine (intermediate 1), N' -1, 4-di-p-toluenesulfonyl homopiperazine (intermediate 2) and other unknown impurities may remain, and the homopiperazine is used as a starting material for drug synthesis, so that the residues of phenol, N '-di-p-toluenesulfonyl ethylenediamine (intermediate 1), N' -1, 4-di-p-toluenesulfonyl homopiperazine (intermediate 2) and other unknown impurities need to be detected and controlled in quality control.
The application number 2015100770097 discloses a method for preparing homopiperazine, which comprises the steps of using ethylenediamine as a raw material, carrying out acylation reaction with p-toluenesulfonyl chloride in an N-butanol solvent to obtain N, N '-di-p-toluenesulfonyl ethylenediamine, directly carrying out cyclization reaction with bromochloropropane under the action of sodium hydroxide without separating a product from a reaction solution to obtain N, N' -di-p-toluenesulfonyl homopiperazine, then carrying out desulfonylation under the action of hydrobromic acid and phenol to obtain homopiperazine hydrobromide, and finally carrying out sodium hydroxide dissociation and toluene carrying water to obtain high-purity homopiperazine. The technical scheme does not disclose a method for detecting residues in homopiperazine.
The present invention has been made in view of this situation.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a method for detecting residues in homopiperazine, in particular to a method for detecting the residues of phenol, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine and other unknown impurities in homopiperazine.
In order to solve the technical problems, the invention adopts the technical scheme that:
the application provides a detection method of residues in homopiperazine, which comprises the following steps:
(1) weighing a high piperazine product, adding a diluent to dissolve, fixing the volume, and uniformly mixing to obtain a test solution;
(2) respectively weighing reference substances of N, N '-di-p-toluenesulfonyl ethylenediamine, N' -1, 4-di-p-toluenesulfonyl homopiperazine and phenol, placing in the same container, adding a diluent for dissolving, fixing the volume, uniformly mixing, and diluting to obtain a reference substance solution;
(3) the determination is carried out by adopting high performance liquid chromatography, and the determination conditions comprise: performing gradient elution by using an ammonium acetate buffer solution as a mobile phase A and acetonitrile as a mobile phase B through a chromatographic column using octadecylsilane chemically bonded silica as a filler, wherein the detection wavelength is 225nm, injecting a reference solution and a test solution into a liquid chromatograph, and recording a chromatogram.
In a further scheme, in the step (3), the time and the mobile phase ratio of gradient elution are as follows: and (3) 0-8min, reducing the volume percentage of the mobile phase A from 95% to 25%, increasing the volume percentage of the mobile phase B from 5% to 75%, and 8-13min, wherein the volume percentage of the mobile phase A is kept at 25%, the volume percentage of the mobile phase B is kept at 75%, and 13-13.1min, the volume percentage of the mobile phase A is increased from 25% to 95%, the volume percentage of the mobile phase B is reduced from 75% to 5%, and 13.1-15min, the volume percentage of the mobile phase A is kept at 95%, and the volume percentage of the mobile phase B is kept at 5%.
In the step (3), the concentration of the ammonium acetate buffer solution is 5mmol/L, and the pH value is adjusted to 5.0 by adopting acetic acid.
In a further scheme, in the reference solution, the concentrations of N, N '-di-p-toluenesulfonyl ethylenediamine, N, N' -1, 4-di-p-toluenesulfonyl homopiperazine and phenol are all 0.01 mg/mL; the concentration of high piperazine in the test solution was 10 mg/mL.
In a further embodiment, the injection volumes of the control solution and the test solution are 5. mu.l.
In a further embodiment, the diluent in steps (1) and (2) is acetonitrile.
In a further scheme, the chromatographic column in the step (3) is Waters XbridgeTMC18, 4.6mm by 150mm, 3.5 μm, or equivalent columns.
In a further embodiment, in the determination conditions of step (3), the column temperature is 30 ℃ and the flow rate is 1.0ml per minute.
In a further scheme, in the determination conditions of the step (3), the acquisition time is 15min, and the equilibrium time is 5 min.
In a further scheme, when chromatographic peaks of phenol, N '-di-p-toluenesulfonyl ethylenediamine and N, N' -1, 4-di-p-toluenesulfonyl homopiperazine exist in a chromatogram of a test solution, the contents of the phenol, the N, N '-di-p-toluenesulfonyl ethylenediamine and the N, N' -1, 4-di-p-toluenesulfonyl homopiperazine are not more than 0.10% by calculating peak areas according to an external standard method; the other single impurities are not more than 0.10 percent, and the total amount of the impurities is not more than 1.0 percent.
As a specific scheme, a method for detecting residual phenol, N '-di-p-toluenesulfonylethylenediamine (intermediate 1), N' -1, 4-di-p-toluenesulfonyl homopiperazine (intermediate 2) and other unknown impurities in homopiperazine comprises the following steps:
(1) taking 0.5g of homopiperazine product, precisely weighing, placing in a 50ml measuring flask, adding acetonitrile about 30ml, carrying out ultrasonic treatment (if necessary) until the product is dissolved, carrying out constant volume to scale with the acetonitrile, and uniformly mixing to obtain a test solution.
(2) Precisely weighing 20mg of each of the intermediate 1, the intermediate 2 and the phenol reference substance, placing the intermediate 1, the intermediate 2 and the phenol reference substance into the same 100ml measuring flask, adding about 60ml of acetonitrile, carrying out ultrasonic treatment (if necessary) until the acetonitrile is dissolved, fixing the volume to scale by using the acetonitrile, and uniformly mixing the solution to be used as a reference substance stock solution; precisely measuring 5ml, placing in a 100ml measuring flask, adding acetonitrile to constant volume to scale, and mixing to obtain reference solution.
(3) Measuring by high performance liquid chromatography, using chromatographic column with octadecylsilane chemically bonded silica as filler, and buffering with 5mmol/L ammonium acetateThe solution (acetic acid to adjust pH to 5.0) is mobile phase A, acetonitrile is mobile phase B, and gradient elution is carried out according to the following table 1; the detection wavelength was 225nm, the column temperature was 30 ℃ and the flow rate was 1.0ml per minute. Precisely measuring 5 μ l of each of the reference solution and the sample solution, respectively injecting into a liquid chromatograph, and recording chromatogram. If the chromatogram of the test solution contains peaks of phenol, intermediate 1 and intermediate 2, the peak area is calculated according to an external standard method, and the peak area is not more than 0.10%. The content of other single impurities is not more than 0.10%, and the total content of impurities is not more than 1.0%. Preferably, the chromatographic column is a Waters XbridgeTMC18, 4.6mm × 150mm, 3.5 μm or its equivalent.
TABLE 1
After adopting the technical scheme, compared with the prior art, the invention has the following beneficial effects:
the detection method for the residual phenol, N '-di-p-toluenesulfonyl ethylenediamine (intermediate 1), N' -1, 4-di-p-toluenesulfonyl homopiperazine (intermediate 2) and other unknown impurities in homopiperazine provided by the invention can avoid the interference of a solvent peak, has stable base line, good separation degree of the intermediate I, II and phenol, good peak type of a chromatographic peak, short time and high efficiency, and can provide guarantee for the quality control of homopiperazine.
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention, are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention without limiting the invention to the right. It is obvious that the drawings in the following description are only some embodiments, and that for a person skilled in the art, other drawings can be derived from them without inventive effort. In the drawings:
FIG. 1 is a chromatogram of a mixed control of 2.1 of example 2 of the present invention;
FIG. 2 is a chromatogram of a 2.1 intermediate I localization solution of example 2 of the present invention;
FIG. 3 is a chromatogram of a 2.1 intermediate II localization solution of example 2 of the present invention;
FIG. 4 is a chromatogram of a 2.1 phenol positioning solution of example 2 of the present invention;
FIG. 5 is a chromatogram of a mixed control of 2.2 of example 2 of the present invention;
FIG. 6 is a chromatogram of a mixed control of 2.3 of example 2 of the present invention;
FIG. 7 is a chromatogram of the mixed control of 3.1 of example 3 of the present invention;
FIG. 8 is a chromatogram of the mixed control of 3.2 of example 3 of the present invention;
FIG. 9 is a chromatogram of an actual sample in 3.3 of example 3 of the present invention;
FIG. 10 is a chromatogram of the mixed control of 3.4 of example 3 of the present invention;
FIG. 11 is a chromatogram of a 0.002% level recovery test solution from 3.5 of example 3 of the present invention;
FIG. 12 is a chromatogram of the mixed control of 4.1 of example 4 of the present invention;
FIG. 13 is a chromatogram of the mixed control of 4.2 of example 4 of the present invention;
FIG. 14 is a chromatogram of the mixed control of 4.3 of example 4 of the present invention;
FIG. 15 is a chromatogram of the mixed control of 4.4 of example 4 of the present invention;
FIG. 16 is a chromatogram of the mixed control of 4.5 of example 4 of the present invention;
FIG. 17 is a chromatogram of the mixed control of 4.6 of example 4 of the present invention;
FIG. 18 is a chromatogram of the mixed control of 4.7 of example 4 of the present invention;
FIG. 19 is a chromatogram of the mixed control of 5.1 of example 5 of the present invention;
FIG. 20 is a chromatogram of the mixed control of 5.2 of example 5 of the present invention;
FIG. 21 is a chromatogram of the mixed control of 5.3 of example 5 of the present invention;
FIG. 22 is a 0.001%, 0.002%, 0.1%, 0.2% level recovery solution chromatogram of example 6 of the present invention;
FIG. 23 is a chromatogram of the mixed control of 1.1 of comparative example 1 of the present invention;
FIG. 24 is a chromatogram of a mixed control of 1.2 of comparative example 1 of the present invention;
FIG. 25 is a chromatogram of the mixed control of 1.3 of comparative example 1 of the present invention;
FIG. 26 is a chromatogram of an air-white solution, a mixed control, and a limiting solution in test example 1 of the present invention;
FIG. 27 is a chromatogram of a localization solution of intermediate I in Experimental example 1 of the present invention;
FIG. 28 is a chromatogram of a localization solution of intermediate II in Experimental example 1 of the present invention;
FIG. 29 is a chromatogram of a phenol positioning solution of test example 1 of the present invention;
FIG. 30 is a chromatogram of a 0.1% level normalized recovery solution in test example 1 of the present invention.
It should be noted that the drawings and the description are not intended to limit the scope of the inventive concept in any way, but to illustrate it by a person skilled in the art with reference to specific embodiments.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and the following embodiments are used for illustrating the present invention and are not intended to limit the scope of the present invention.
Sample information
Intermediate I (N, N' -di-p-toluenesulfonylethylenediamine) run No. 201710Z 07; intermediate II (N, N' -1, 4-di-p-toluenesulfonyl homopiperazine) with a batch number of XS 180329-15; phenol, lot number G171215-06; homopiperazine product, lot 160201.
Taking 0.5g of homopiperazine product, precisely weighing, placing in a 50ml measuring flask, adding acetonitrile about 30ml, carrying out ultrasonic treatment (if necessary) until the product is dissolved, carrying out constant volume to scale with the acetonitrile, and uniformly mixing to obtain a test solution.
Precisely weighing 20mg of each of the intermediate I, the intermediate II and the phenol reference substance, placing the intermediate I, the intermediate II and the phenol reference substance into the same 100ml measuring flask, adding about 60ml of acetonitrile, carrying out ultrasonic treatment (if necessary) until the mixture is dissolved, fixing the volume to the scale by using the acetonitrile, and uniformly mixing the solution to be used as a reference substance stock solution; precisely measuring 5ml, placing in a 100ml measuring flask, adding acetonitrile to constant volume to scale, and mixing to obtain reference solution.
Example 1 initial detection method
1.1 initial liquid phase parameters
TABLE 2
Example 2 preliminary optimization of liquid phase parameters and chromatograms of positioning solution and mixed control solution
2.1 Mobile phase A "0.5% ammonium acetate buffer (pH adjusted to 5.0 with aqueous ammonia)" was changed to "5 mM ammonium acetate buffer (pH adjusted to 5.0 with acetic acid)". The preparation process comprises the following steps: accurately weighing 385.4mg of anhydrous ammonium acetate in 1000mL of pure water, and dissolving; the pH was adjusted to 5.0. + -. 0.05 with acetic acid. A Waters Xbridge TM 18,150 x 4.6mm,3.5 μm column was selected for detection. The liquid phase parameters after optimization are shown in the following table:
TABLE 3
Mobile phase
A5 mM ammonium acetate buffer (pH adjusted to 5.0 with acetic acid) B acetonitrile A: B40: 60
Chromatographic column
Waters XBridgeTM C18,150×4.6mm,3.5μm
Column temperature
30℃
Flow rate of flow
1.0mL/min
Wavelength of light
225nm
Sample volume
10μL
Time of acquisition
10min
Diluent and needle washing solution
Acetonitrile
The concentrations of the intermediate I, the intermediate II and phenol in the prepared mixed reference solution are all 0.01 mg/mL. The mixed control solution was assayed using the above assay conditions, and the resulting chromatogram is shown in FIG. 1. The retention time for each sample was: the phenol content was 2.151min, intermediate I2.991 min, and intermediate II 5.444 min.
The chromatogram of the intermediate I localization solution (concentration: 1.0mg/mL) is shown in FIG. 2, the chromatogram of the intermediate II localization solution (concentration: 1.0mg/mL) is shown in FIG. 3, and the chromatogram of the phenol localization solution (concentration: 1.0mg/mL) is shown in FIG. 4.
2.2 detection limit solution chromatogram and System adaptability results
The liquid phase parameters were determined as in table 3.
0.02% level mixed control solution chromatogram and signal-to-noise ratio, separation degree, tailing factor results
The chromatogram of the mixed control solution at a level of 0.02% (all concentrations of intermediate I, intermediate II and phenol were 0.002mg/mL) is shown in FIG. 5.
The signal to noise ratio, resolution, and tailing factor results for the mixed control solution at a level of 0.02% (all intermediate I, intermediate II, and phenol concentrations were 0.002mg/mL) are analyzed as shown in Table 4.
TABLE 4
Sample name
Signal to noise ratio
Degree of separation
Tailing factor
Phenol and its preparation
253
N/A
1.1
Intermediate I
374
8.3
1.1
Intermediate II
183
17.4
1.0
And (4) conclusion:
the liquid phase parameters listed in table 3 can satisfy the requirements of intermediate I, intermediate II and phenol detection on the separation degree and the tailing factor. The separation degree is more than 1.5, and the tailing factors are all between 0.8 and 2.0. However, the peak time of phenol is about 2.1min, and in order to thoroughly avoid the interference of the solvent peak on the phenol detection, the liquid phase parameters need to be further optimized, and the peak time of phenol is prolonged.
The method has strong detection capability on three compounds, the signal-to-noise ratio of 0.02% level (the concentrations of the intermediate I, the intermediate II and phenol are all 0.002mg/mL) is more than 180, the concentration loses the significance of detection capability evaluation, the sample injection amount needs to be reduced or the sample concentration needs to be reduced, and the actual detection limit level of the analysis method on the three compounds is tested.
Determination of 2.30.05% level to 0.3% level
The liquid phase parameters were determined as in table 3.
The chromatogram of the solution from the 0.05% level (all concentrations of intermediate I, intermediate II and phenol are 0.005mg/mL) to the 0.3% level (all concentrations of intermediate I, intermediate II and phenol are 0.03mg/mL) is shown in FIG. 6.
From fig. 6, it can be seen that from 0.05%, 0.2% to 0.3%, the peak patterns of intermediates I and II gradually worsen with increasing injection concentration, and especially the peak pattern of intermediate II significantly widens. It is necessary to reduce the feed concentration or to optimize the liquid phase parameters.
Example 3 optimization of liquid phase parameters and corresponding chromatograms
3.1 liquid phase parameters were carried out according to Table 5. In the method, the sample injection amount is reduced from 10 mu L to 5 mu L. The proportion of the mobile phase B is reduced from 60% to 55%, so that the peak time of the phenol can be delayed, and the phenol and the solvent peak are completely separated.
TABLE 5
Mobile phase
A5 mM ammonium acetate buffer (pH adjusted to 5.0 with acetic acid) B acetonitrile A: B45: 55
Chromatographic column
Waters XBridgeTM C18,150×4.6mm,3.5μm
Column temperature
30℃
Flow rate of flow
1.0mL/min
Wavelength of light
225nm
Sample volume
5μL
Time of acquisition
15min
Diluent and needle washing solution
Acetonitrile
The chromatogram of the mixed control solution measured as described above is shown in FIG. 7.
3.2 liquid phase parameters were performed according to Table 6. In the method, the proportion of the mobile phase B is reduced from 55% to 50%.
TABLE 6
Mobile phase
A5 mM ammonium acetate buffer (pH adjusted to 5.0 with acetic acid) B acetonitrile A50: 50
Chromatographic column
Waters XBridgeTM C18,150×4.6mm,3.5μm
Column temperature
30℃
Flow rate of flow
1.0mL/min
Wavelength of light
225nm
Sample volume
5μL
Time of acquisition
15min
Diluent and needle washing solution
Acetonitrile
The chromatogram of the mixed control solution measured as described above is shown in FIG. 8.
And (4) conclusion: when the proportion of mobile phase B was reduced from 55% to 50%, the phenol front time was increased from 2.1min to 2.5min, interference of the solvent peak was avoided, and baseline separation was achieved for both intermediate I, II and phenol. The liquid phase conditions in table 6 were tentatively set as optimum parameters.
3.3 actual sample testing
The liquid phase parameters of the actual samples were examined according to the conditions in Table 6, and the resulting chromatograms are shown in FIG. 9. As can be seen from the chromatogram, neither intermediate I, II nor phenol was detected in the actual sample solution (the sample solution concentration was 10 mg/mL).
3.4 practical detection Limit level test
The concentration of the mixed control solution was reduced to obtain the actual detection capability of the assay for intermediate I, II and phenol, i.e., the detection limit level of the method. The 0.05% level mixed control solution was diluted 50-fold and tested for signal-to-noise ratio.
The detection limit solution concentrations were as follows:
TABLE 7
The liquid phase parameters were measured according to the conditions in table 6, and the chromatogram of the mixed control solution is shown in fig. 10. And (4) conclusion: the signal-to-noise ratio of the intermediate I and the phenol is larger than 10, while the signal-to-noise ratio of the intermediate II cannot reach 10 due to the broadening of chromatographic peaks. When the intermediate II is diluted to a 0.002% level (the concentration is 0.0002mg/mL), the signal-to-noise ratio can meet the requirement of more than 10.
3.5 recovery test
3.5.1 recovery test solution was prepared with a main compound high piperazine concentration of 10 mg/mL. The preparation process comprises the following steps:
TABLE 8
The liquid phase parameters were measured according to the conditions in table 6, and the 0.002% horizontal recovery test solution chromatogram is shown in fig. 11.
And (4) conclusion: the solvent peak at 2.0min in the recovery rate test solution is obvious, and the interference on phenol is generated, so that the phenol quantification is inaccurate, and particularly the low-concentration recovery rate solution is obtained. The liquid phase parameters need to be further optimized.
Example 4 further optimization of liquid phase parameters and corresponding chromatograms
The following liquid phase conditions are adopted, and a better scheme is selected, and the sample configuration mode is the same as the above.
4.1 measurements were made using the liquid phase parameters as shown in Table 9 below
TABLE 9
The chromatogram of the mixed control solution measured as described above is shown in FIG. 12. It can be seen that: intermediate I, II separated well from the phenol and continued optimization of liquid phase parameters. The inventors found that the peak appearance of intermediate I, II can be accelerated and the analysis efficiency can be improved by increasing the proportion of mobile phase B.
4.2 measurements were made using the liquid phase parameters as shown in Table 10 below
Watch 10
The chromatogram of the mixed control solution measured as described above is shown in FIG. 13.
4.3 measurements were made using the liquid phase parameters as shown in Table 11 below
TABLE 11
The chromatogram of the mixed control solution measured as described above is shown in FIG. 14. It can be seen that: intermediate I, II separated well from phenol, but had significant baseline fluctuations at 8min, requiring continued optimization of liquid phase parameters, decreasing the time to peak of intermediate I, II, and optimizing the baseline.
4.4 liquid phase parameters as shown in Table 12 below were used for the measurements
TABLE 12
The chromatogram of the mixed control solution measured as described above is shown in FIG. 15. It can be seen that: intermediate I, II separated well from phenol, but the intermediate I chromatographic peak was broad.
4.5 liquid phase parameters as shown in Table 13 below were used for the measurements
Watch 13
The chromatogram of the mixed control solution measured as described above is shown in FIG. 16. It can be seen that: intermediate I, II separated well from phenol, but the intermediate I chromatographic peak was broad.
4.6 Using the liquid phase parameters as shown in Table 14 below
TABLE 14
The chromatogram of the mixed control solution measured as described above is shown in FIG. 17.
4.7 Using the liquid phase parameters as shown in Table 15 below
Watch 15
The chromatogram of the mixed control solution measured as described above is shown in FIG. 18.
Of the above results, the chromatograms (fig. 17 and 18) under the liquid phase parameters (table 14 and table 15) for the 4.6 and 4.7 protocols were close, and both the baseline and the analysis time were superior to the chromatograms obtained under the 4.5 protocol. Comparing the liquid phase parameters under the above 4.1-4.8 schemes with the corresponding chromatograms, it can be known that the gradient change has obvious improvement on the chromatographic peak profile of phenol, but causes baseline fluctuation, and needs to be continuously optimized to obtain the optimal chromatographic peak profile and baseline.
Example 5 yet further optimization of liquid phase parameters and chromatograms
5.1 Using the liquid phase parameters as shown in Table 16 below
TABLE 16
The chromatogram of the mixed control solution measured as described above is shown in FIG. 19.
5.2 measurements were made using the liquid phase parameters as shown in Table 17 below
TABLE 17
The chromatogram of the mixed control solution measured as described above is shown in FIG. 20.
5.3 Using the liquid phase parameters as shown in Table 18 below
Watch 18
The chromatogram of the mixed control solution measured as described above is shown in FIG. 21.
As can be seen from the above schemes 5.1-5.3, the chromatograms (fig. 20 and 21) under the liquid phase parameters (table 17 and table 18) in the schemes 5.2 and 5.3 are close. And 5.3, the whole analysis time of the liquid phase parameters in the scheme is faster than 5.2, and the requirement of the resolution can be met. In order to reduce unnecessary chromatogram collection time and ensure that the system is in a fully balanced state, the collection time is set to be 15min, and the balance time is set to be 5 min.
Example 6 optimum liquid phase parameters
6.1 optimization by screening, the optimum liquid phase parameters are shown in Table 19 below.
Watch 19
6.2 recovery and linearity testing Using the Final liquid phase method
6.2.1 recovery test solutions were prepared with a main compound high piperazine concentration of 10 mg/mL. The preparation process comprises the following steps:
watch 20
6.2.2 control solutions
Control solutions (each of intermediate I, intermediate II and phenol concentration 0.005mg/mL) were mixed at a level of 0.05% as control solutions for external standard quantitation. The preparation process comprises the following steps:
TABLE 21
6.2.3 measurements were made using the liquid phase parameters in Table 19.
6.2.4 results
(1) The 0.001%, 0.002%, 0.1%, 0.2% level recovery solution chromatograms are shown in fig. 22.
(2) The system suitability results are shown in table 22 below.
TABLE 22
(3) The signal to noise ratio results are shown in table 23 below.
TABLE 23
(4) The recovery results are shown in table 24 below.
Watch 24
(5) The linearity results are shown in table 25 below.
TABLE 25
6.3 conclusion
(1) No interference exists at a target peak in a blank solution chromatogram; the separation degrees of three compounds in a 0.05% horizontal mixed reference solution (the concentrations of the intermediate I, the intermediate II and phenol are all 0.005mg/mL) meet the requirement of more than 1.5, and the tailing factors meet the requirement of 0.8-2.0.
(2) Determination of actual detection limit concentration: the 0.001% level mixed reference solution (the concentrations of the intermediate I, the intermediate II and the phenol are all 0.0001mg/mL), the signal-to-noise ratios of the three compounds are all more than 3, and the requirement that the signal-to-noise ratio of the detection limit solution is more than or equal to 3 is met. The limit of detection solution (LOD) was determined by mixing a control solution at a level of 0.001% (all concentrations of intermediate I, intermediate II and phenol were 0.0001 mg/mL).
(3) Determination of actual quantitative limiting concentration: the 0.002% level mixed reference solution (the concentrations of the intermediate I, the intermediate II and the phenol are all 0.0002mg/mL), the signal-to-noise ratios of the three compounds are all more than 10, and the requirement that the signal-to-noise ratio of the quantitative limit solution is more than or equal to 10 is met. A0.002% level of the mixed control solution (each of intermediate I, intermediate II and phenol concentrations was 0.0002mg/mL) was determined as a limiting quantitation solution (LOQ).
(4) Determination of the linear acceptance criteria: in the concentration range of 0.001 percent (the concentrations of the intermediate I, the intermediate II and the phenol are all 0.0001mg/mL) to 0.2 percent (the concentrations of the intermediate I, the intermediate II and the phenol are all 0.02mg/mL) in the pre-verification experiment, the linear correlation coefficients of the three compounds all meet the requirement of being more than 0.999. According to our SOP AD-VAL-001 requirements, the impurity level linear correlation coefficient should be greater than 0.995, so the acceptance criteria in the formal validation scheme are suggested to be: the linear correlation coefficient was greater than 0.995 at a concentration range from a level of 0.02% of the quantitation limit (0.0002 mg/mL for each of intermediate I, intermediate II and phenol) to a level of 0.2% (0.02 mg/mL for each of intermediate I, intermediate II and phenol). Specific linear concentrations and corresponding acceptance criteria are as follows:
watch 26
(5) Recovery was determined by standard: in a quantitative limit concentration level, namely a 0.02% level (the concentrations of the intermediate I, the intermediate II and the phenol are all 0.0002mg/mL), the recovery rate result in a pre-verification experiment is 102-118%, because the peak areas of the three compounds at the 0.02% level are small, and the difference of the integral parameters has a large influence on the recovery rate result. According to the requirements of SOP AD-VAL-001, when the impurity content is less than 0.15%, the difference between the theoretical content and the actually measured content is within +/-0.04%, and when the impurity content is more than or equal to 0.15%, the ratio of the theoretical content to the actually measured content is within +/-30%. In combination with the recovery results of the pre-validation, it is suggested that the acceptance criteria in the formal validation protocol are: the recovery rate of the level from the 0.02% level (concentration of the intermediate I, the intermediate II and the phenol is 0.0002mg/mL) to the 0.1% level (concentration of the intermediate I, the intermediate II and the phenol is 0.01mg/mL) of the limit of quantitation is between 60% and 140%. The recovery was accepted at 80% -120% from the 0.1% level to the 0.2% level. Specific recovery concentrations and corresponding acceptance criteria were as follows:
watch 27
Comparative example 1 different columns tested 0.1% horizontal recovery solutions
1.1 testing of 0.1% horizontal recovery solution Using ACE 5C 18,25 cm column
Referring to the liquid phase parameters of table 3 in example 2, the difference is: detecting with a chromatographic column ACE 5C 18,250 × 4.6mm,5 μm, sample size of 5ul, and collection time of 15 min.
The resulting chromatogram is shown in FIG. 23. It can be seen that using an ACE 5C 18,250 x 4.6mm,5 μm column, none of intermediate I, intermediate ii and phenol could be quantified and detected well and the overall baseline was poor, indicating that the column is not suitable for determining the content of intermediate I, intermediate ii and phenol in homopiperazine.
1.2 testing of 0.1% horizontal recovery solutions using an Agilent ZORBAX Eclipse XDB-C8,25 cm column
Referring to the liquid phase parameters of table 3 in example 2, the difference is: the mixed control solution was assayed using a chromatography column Agilent ZORBAX Eclipse XDB-C8, 250X 4.6mm,5 μm, 5ul sample size, 15min collection time.
The resulting chromatogram is shown in FIG. 24. It can be seen that using an Agilent ZORBAX Eclipse XDB-C8, 250X 4.6mm,5 μm column, the main compound produced a chromatographic peak around 2.5min that interfered with phenol and the overall baseline was poor between 0min and 4.5 min.
1.3 testing of 0.1% horizontal recovery solutions using a Waters Symmetry C18,25 cm column
Referring to the liquid phase parameters of table 3 in example 2, the difference is: the mixed control solution was assayed using a chromatographic column Waters Symmetry C18, 250X 4.6mm,5 μm, a sample size of 5ul, a collection time of 15 min.
The resulting chromatogram is shown in FIG. 25. It can be seen that using a Waters Symmetry C18,250 x 4.6mm,5 μm column, the main compound produced a chromatographic peak around 2.5min that interferes with phenol, with a changing baseline, presenting a risk for subsequent phenol quantification, a condition where the durability of the method does not meet the target requirements.
Conclusion, the chromatographic column Waters Xbridge was usedTMC18,150X 4.6mm,3.5 μm is the best solution.
Test example 1 specificity
Sample preparation: blank solution (diluent acetonitrile), 0.002% limiting solution (LOQ), control solution. Blank solution (diluent acetonitrile), 0.002% limiting solution (LOQ) and control solution were each prepared according to the analytical method.
An intermediate I positioning solution, an intermediate II positioning solution and a phenol positioning solution.
Preparing 0.05mg/mL of each of an intermediate I positioning solution, an intermediate II positioning solution and a phenol positioning solution, wherein the preparation process comprises the following steps:
for example: accurately weighing 10mg of the intermediate I reference substance into a 200mL volumetric flask, adding 150mL of diluent, performing ultrasonic treatment (if necessary) until the diluent is dissolved, metering the volume by using the diluent, and uniformly mixing. Named I-ID.
Accurately weighing 10mg of the intermediate II reference substance into a 200mL volumetric flask, adding 150mL of diluent, carrying out ultrasonic treatment (if necessary) until the diluent is dissolved, metering the volume by using the diluent, and uniformly mixing. Named Pheno II-ID.
Accurately weighing 10mg of phenol reference substance into a 200mL volumetric flask, adding 150mL of diluent, performing ultrasonic treatment (if necessary) until the phenol reference substance is dissolved, metering the volume by using the diluent, and uniformly mixing. Named Phenol-ID.
Acceptance criteria
The blank solution chromatogram has no obvious interference at the target peak intermediate I, the intermediate II and the phenol; if interference exists, the peak area of the interference peak is not larger than the peak area of the LOQ.
The intermediate I, the intermediate II and phenol in the chromatogram of the reference solution and the 0.1 percent horizontal standard addition recovery rate solution can be completely separated, and the separation degree is more than or equal to 1.5.
As a result: and the blank solution chromatogram has no obvious interference at the target peak intermediate I, the intermediate II and the phenol. The resolution between the intermediate I, the intermediate II and phenol in the chromatogram of the control solution is detailed in table 28, and the resolution between the intermediate I, the intermediate II and phenol in the chromatogram of the 0.1% level standard recovery solution is detailed in table 29. The results are shown in FIG. 26, and the results of the intermediate I localization solution, the intermediate II localization solution and the phenol localization solution are shown in FIGS. 27 to 29. The 0.1% level normalized recovery solution is detailed in FIG. 30. The results met the acceptance criteria.
Watch 28
Name (R)
Degree of separation
Phenol-ID (Phenol)
28.3
I-ID (intermediate I)
28.3,15.7
II-ID (intermediate II)
15.7
Watch 29
Name (R)
Degree of separation
Phenol-ID (Phenol)
27.9
I-ID (intermediate I)
27.9,15.6
II-ID (intermediate II)
15.6
Test example 2 limit of quantitation (LOQ), Limit of detection (LOD)
(1) Limit of quantitation solution (LOQ): preparing a 0.002% quantitative limiting solution (LOQ), injecting 6 needles of the quantitative limiting solution (LOQ), and respectively calculating the signal-to-noise ratio, the Relative Standard Deviation (RSD) of peak areas and the Relative Standard Deviation (RSD) of retention time of the intermediate I, the intermediate II and phenol in the quantitative limiting solution.
Limit of detection solution (LOD): a0.001% detection limit solution (LOD) was prepared, and the detection limit solution (LOD) was obtained by two-step dilution from a 0.002% quantitative limit solution (LOQ). The specific process is as follows: the quantitative limiting solution (LOQ) was diluted twice (5mL → 10mL) to obtain the detection limiting solution (LOD). And (3) injecting a sample into a detection limit solution (LOD), and respectively calculating the average value of the respective signal-to-noise ratios of the intermediate I, the intermediate II and the phenol in the quantitative limit solution.
(2) Acceptance criteria
The detection limit is that the average value of the respective signal-to-noise ratios of the intermediate I, the intermediate II and phenol in the LOD LOD solution is not less than 3.
The quantitative limit is that the average value of the respective signal-to-noise ratios of the intermediate I, the intermediate II and the phenol in the LOQ LOQ solution is not less than 10. RSD of respective peak areas of an intermediate I, an intermediate II and phenol in 6 continuous needles of LOQ is less than or equal to 15 percent, and RSD of retention time is less than or equal to 2.0 percent.
(3) As a result: the results of limit of detection (LOD) and limit of quantitation (LOQ) are shown in tables 30-31. The results all meet the acceptance criteria.
Watch 30 detection Limit (LOD)
Name (R)
Intermediate I
Intermediate II
Phenol and its preparation
First pin signal-to-noise ratio
5
4
1
Second needle signal to noise ratio
13
10
6
Third needle signal-to-noise ratio
3
2
1
Average signal-to-noise ratio (n ═ 3)
7
5
3
TABLE 31 quantitative limit solution (LOQ)
Name (R)
Intermediate I
Intermediate II
Phenol and its preparation
First pin signal-to-noise ratio
37
27
15
Second needle signal to noise ratio
51
40
21
Third needle signal-to-noise ratio
13
10
5
Fourth pin signal to noise ratio
5
4
2
Signal to noise ratio of the fifth needle
11
7
4
Sixth needle signal to noise ratio
28
21
11
Average signal-to-noise ratio (n ═ 6)
24
18
10
Peak area RSD (n ═ 6)
2％
9％
8％
Retention time RSD (n ═ 6)
0.1％
0.1％
0.0％
Test example 3 linearity
Respectively preparing a series of linear solutions, wherein the concentration levels of the intermediate I, the intermediate II and the phenol are respectively 0.002%, 0.01%, 0.05%, 0.1% and 0.2% of the target concentration, injecting 1 needle into each solution, and calculating the respective linear correlation coefficient (r), slope, intercept and residual square sum of the intermediate I, the intermediate II and the phenol.
A portion of 0.05mg/mL of the mixed control solution was prepared as follows: for example: accurately weighing 10mg of intermediate I, intermediate II and phenol reference substance into the same 200mL volumetric flask, adding about 150mL of diluent, performing ultrasonic treatment (if necessary) until the diluent is dissolved, metering the volume by using the diluent, and uniformly mixing, wherein the names are as follows: stock # 1.
Preparation of a linear solution: each of 0.002%, 0.01%, 0.05%, 0.1% and 0.2% horizontal linear solutions was prepared in one portion. The preparation process is as follows:
watch 32
Name of Linear solution
Name of stock solution
Two-step dilution
Theoretical concentration (mg/mL)
0.002％
0.1% horizontal Linear solution
2mL→100mL
0.0002
0.01％
Stock solution #1
2mL→100mL
0.001
0.05％
Stock solution #1
5mL→50mL
0.005
0.1％
Stock solution #1
5mL→25mL
0.01
0.2％
Stock solution #1
10mL→25mL
0.02
Acceptance criteria:
the correlation coefficient (r) of each of intermediate I, intermediate II and phenol is not less than 0.995.
The slope, intercept and residual sum of squares are reported.
Results the results are detailed in table 33. The results met the acceptance criteria.
Watch 33
Name (R)
Intermediate I
Intermediate II
Phenol and its preparation
Linear coefficient (r)
1.000
1.000
1.000
Slope of
17596727.097
13872713.910
8798348.199
Intercept of a beam
67.311
220.604
-142.773
Sum of squares of residuals
195637.496
255476.251
24577.779
Test example 4 accuracy
High piperazine bulk drug sample control solution: in parallel, 3 sample control solutions were prepared.
Preparing a mixed reference substance solution: a0.05 mg/mL portion of the mixed control solution (stock #2) was prepared as follows:
for example: accurately weighing 10mg of intermediate I, intermediate II and phenol reference substance into the same 200mL volumetric flask, adding about 150mL of diluent, performing ultrasonic treatment (if necessary) until the diluent is dissolved, metering the volume by using the diluent, and uniformly mixing, wherein the names are as follows: stock # 2.
A0.005 mg/mL portion of the mixed control solution (stock #3) was prepared as follows: taking 10mL of stock solution #2, adding the stock solution #2 into a 100mL volumetric flask, adding a diluent, fixing the volume, uniformly mixing, and naming as: stock # 3.
Preparing a standard recovery rate solution: respectively preparing a series of solutions with standard addition recovery rates, wherein the standard addition concentrations are 0.002%, 0.05%, 0.1% and 0.2% of the target concentration, 3 parts of the solutions with standard addition recovery rates are respectively prepared at 0.002%, 0.05% and 0.2%, and 6 parts of the solutions with standard addition recovery rates are prepared at 0.1%, and the specific preparation process is as follows:
watch 34
For each solution, 1 needle was injected and the individual recovery results and average values for each level of intermediate I, intermediate II and phenol were calculated.
Acceptance criteria
For the 0.002% and 0.05% level normalized recoveries, the average recovery of each of intermediate I, intermediate II, and phenol should be between 60% and 140%. For the 0.1%, 0.2% level normalized recoveries, the average of the respective recoveries of intermediate I, intermediate II and phenol should be between 80% and 120%.
As a result, the actual configured concentration of phenol spiked solution:
watch 35
Actual preparation concentration of intermediate I spiked solution:
watch 36
Actual preparation concentration of intermediate II spiked solution:
watch 37
The accuracy results are shown in table 38. The results met the acceptance criteria. Note that: the titer of the intermediate I, the intermediate II and the phenol of the reference substance is not mentioned in the actually measured concentration formula in the verification scheme, and the titer of each of the intermediates participates in the calculation of the accuracy in the actual calculation process, so that the requirement of the calculation result of the accuracy is met, and the verification result is not influenced.
Table 38: accuracy results
Name (R)
Intermediate I
Intermediate II
Phenol and its preparation
Average value of 0.002% horizontal standard recovery
100％
102％
110％
0.05% horizontal normalized recovery mean
101％
99％
102％
0.1% horizontal normalized recovery mean
101％
99％
102％
0.2% horizontal normalized recovery mean
101％
99％
101％
Precision of test example 5
Precision of the instrument
A control solution (STD #1) was prepared, 6 samples of the control solution (STD #1) were taken, and the Relative Standard Deviations (RSD) of the peak areas of the intermediate I, the intermediate II and the phenol were calculated, respectively.
Precision of the method
6 parts of 0.1% horizontal spiked recovery solution were prepared in parallel according to the validation protocol, and 1 pin was run for each solution and the Relative Standard Deviation (RSD) of the results for intermediate I, intermediate II and phenol recovery was calculated.
Acceptance criteria: RSD of the peak areas of the intermediate I, the intermediate II and the phenol in the control solution is 5.0 percent.
RSD 15% of results for recovery of intermediate I, intermediate II and phenol in 0.1% level spiked recovery solution.
As a result: instrument precision and method precision results are shown in tables 39, 40. The results meet the acceptance criteria.
Watch 39
Name (R)
Intermediate I
Intermediate II
Phenol and its preparation
01
175819.596
138874.837
88990.123
02
175625.691
139229.992
89170.853
03
175173.269
139281.870
88672.074
04
175612.341
139330.697
88984.977
05
175704.569
139404.501
89018.064
06
175671.088
141203.432
88871.857
Crest plane
0.1％
0.6％
0.2％
Watch 40
Name (R)
Recovery of intermediate I (%)
Recovery of intermediate II (%)
Phenol recovery (%)
01
101
99
101
02
102
100
102
03
101
99
101
04
102
100
102
05
101
100
102
06
101
99
101
RSD at 0.1% horizontal spiked recovery
0％
0％
0％
Test example 6 working Range
The working range will be established according to an appropriate level of accuracy, precision and linearity.
Acceptance criteria report work scope
Results the working range results are shown in table 41. The results meet the acceptance criteria.
Table 41
Test example 7 resistance
Solution preparation: the following representative solutions were formulated for tolerability evaluation: blank solution (diluent), 0.002% quantitation limit solution (LOQ), control solution (STD #1), 0.1% level spiked recovery solution.
The tolerability results were evaluated by varying the following parameters of the liquid phase, only one parameter per test.
1. Initial mobile phase ratio, B%: 7% (5% + 2%), mobile phase gradient program as shown in Table 42, other liquid phase parameters as referenced in Table 19:
watch 42
2. Initial mobile phase ratio, B%: 7% (5% to 2%), mobile phase gradient program as shown in Table 43, other liquid phase parameters refer to Table 19:
watch 43
3. The pH of the mobile phase is 5.2(5.0+0.2), the pH value of acetic acid in liquid phase parameters is adjusted to 5.2 +/-0.05, and other liquid phase parameters refer to a table 19;
4. the pH of the mobile phase is 4.8(5.0-0.2), the pH value of acetic acid in liquid phase parameters is adjusted to 4.8 +/-0.05, and other liquid phase parameters refer to a table 19;
5. column temperature 33 deg.C (30 deg.C +3 deg.C), column temperature of 33 deg.C in liquid phase parameters, and other liquid phase parameters referring to Table 19;
6. the column temperature is 27 ℃ (30 ℃ -3 ℃), the column temperature in the liquid phase parameters is 27 ℃, and other liquid phase parameters refer to table 19;
7. the flow rate was 1.2mL/min (1.0mL/min + 20%), the flow rate was 1.2mL/min among the liquid phase parameters, and the other liquid phase parameters are referred to Table 19;
8. the flow rate was 0.8mL/min (1.0 mL/min-20%), the flow rate was 0.8mL/min among the liquid phase parameters, and the other liquid phase parameters were as shown in Table 19.
Acceptance criteria
No obvious interference exists at a target peak in a blank solution chromatogram; if interference exists, the peak area of the interference peak is not larger than the peak area of the LOQ.
The intermediate I, the intermediate II and phenol in the chromatogram of the first needle reference solution and the 0.1% level standard addition recovery rate solution can be completely separated, and the separation degree is more than or equal to 1.5.
Remarking: if the above two requirements cannot be met under a certain condition of liquid phase parameters, the variable is controlled in actual test and strictly kept consistent with the method.
Results
1. Mobile phase pH4.8 (5.0-0.2) test results:
the blank solution chromatogram has no obvious interference at the target peak intermediate I, intermediate II and phenol (the chromatogram is not provided and is described). The intermediate I, the intermediate II and phenol in the chromatogram of the first needle control solution and the 0.1% level standard addition recovery solution can be completely separated, and the separation degree is more than 1.5. A blank solution, a 0.002% quantitation limit solution (LOQ), a control solution, and a 0.1% level spiked recovery solution were used for the tolerance test, and the test results at ph4.8 mobile phase all met the acceptance criteria.
2. Mobile phase pH 5.2(5.0+0.2) test results:
and the blank solution chromatogram has no obvious interference at the target peak intermediate I, the intermediate II and the phenol. The intermediate I, the intermediate II and phenol in the chromatogram of the first needle reference solution and the 0.1% level standard addition recovery rate solution can be completely separated, and the separation degree is more than or equal to 1.5. The blank solution, 0.002% quantitative limit solution (LOQ), control solution and 0.1% level spiked recovery solution were used for tolerance testing, and the results of the remaining parameters met the acceptance criteria at pH 5.2 mobile phase.
3. Initial mobile phase ratio, B%: 7% (5% + 2%), the results of the test are as follows:
and the blank solution chromatogram has no obvious interference at the target peak intermediate I, the intermediate II and the phenol. The intermediate I, the intermediate II and phenol in the chromatogram of the first needle control solution and the 0.1% level standard addition recovery solution can be completely separated, and the separation degree is more than 1.5. The blank solution, 0.002% limiting solution (LOQ), the control solution and the 0.1% level spiked recovery solution were used for the tolerance test, and the test results met the acceptance criteria under varying mobile phase B (B%: 7% (5% + 2%) parameters.
4. Initial mobile phase ratio, B%: 3% (5% -2%), the test results are as follows:
and the blank solution chromatogram has no obvious interference at the target peak intermediate I, the intermediate II and the phenol. The intermediate I, the intermediate II and phenol in the chromatogram of the first needle reference solution and the 0.1% level standard addition recovery rate solution can be completely separated, and the separation degree is more than or equal to 1.5. The blank solution, 0.002% limiting solution (LOQ), the control solution and the 0.1% level spiked recovery solution were used for the tolerance test, and the test results met the acceptance criteria under varying mobile phase B (B%: 3% (5% -2%) parameters.
5. Column temperature 33 deg.C (30 deg.C +3 deg.C)
And the blank solution chromatogram has no obvious interference at the target peak intermediate I, the intermediate II and the phenol. The intermediate I, the intermediate II and phenol in the chromatogram of the first needle reference solution and the 0.1% level standard addition recovery rate solution can be completely separated, and the separation degree is more than or equal to 1.5. The blank solution, 0.002% quantitative limiting solution (LOQ), the control solution and the 0.1% level spiked recovery solution were used for the tolerance test, and the test results at 33 deg.C (30 deg.C +3 deg.C) column temperature met the acceptance criteria.
6. Column temperature 27 deg.C (30 deg.C-3 deg.C)
And the blank solution chromatogram has no obvious interference at the target peak intermediate I, the intermediate II and the phenol. The intermediate I, the intermediate II and phenol in the chromatogram of the first needle control solution and the 0.1% level standard addition recovery solution can be completely separated, and the separation degree is more than 1.5. The blank solution, 0.002% quantitative limiting solution (LOQ), the control solution and the 0.1% level spiking recovery solution were used for the tolerance test, and the test results at 27 deg.C (30 deg.C-3 deg.C) column temperature all met the acceptance criteria.
7. Flow rate 1.2mL/min (1.0mL/min + 20%)
And the blank solution chromatogram has no obvious interference at the target peak intermediate I, the intermediate II and the phenol. The intermediate I, the intermediate II and phenol in the chromatogram of the first needle control solution and the 0.1% level standard addition recovery solution can be completely separated, and the separation degree is more than 1.5. The blank solution, the 0.002% limiting solution (LOQ), the control solution and the 0.1% level spiked recovery solution were used for the tolerance test, and the test results at a flow rate of 1.2mL/min (1.0mL/min + 20%) all met the acceptance criteria.
8. Flow rate 0.8mL/min (1.0 mL/min-20%)
And the blank solution chromatogram has no obvious interference at the target peak intermediate I, the intermediate II and the phenol. The intermediate I, the intermediate II and phenol in the chromatogram of the first needle control solution and the 0.1% level standard addition recovery solution can be completely separated, and the separation degree is more than 1.5. The blank solution, the 0.002% limiting solution (LOQ), the control solution and the 0.1% level spiked recovery solution were used for the tolerance test, and the test results at a flow rate of 0.8mL/min (1.0 mL/min-20%) all met the acceptance criteria.
Test example 8 solution stability
Stability of control solutions
A control solution was prepared. Storing under appropriate conditions (such as normal temperature condition, 2-8 deg.C condition). Control solutions are prepared fresh and analyzed after standing for specified periods of time (e.g., 24 hours, 48 hours, and 72 hours). The recovery of the control solution after the specified time of standing relative to the freshly prepared control solution was calculated and compared to the recovery at the initial time.
Stability of spiked recovery solution
1 part of 0.1% standard addition level recovery solution is prepared according to the verification scheme, and the 0.1% level recovery solution is stored under appropriate conditions (such as normal temperature conditions and 2-8 ℃).
Control solutions were freshly prepared and analyzed for recovery of 0.1% level recovery solutions after standing for specified periods (e.g., 24 hours, 48 hours, and 72 hours). The recovery results for the 0.1% level recovery solution after the specified time of standing were compared to the ratio of the recovery results for the initial time.
Acceptance criteria
The results of the recovery of the peak areas of the intermediate I, the intermediate II and the phenol in the control solution after the solution is placed for the specified time are between 95.0 and 105.0 percent compared with the initial value.
The recovery of intermediate I, intermediate II and phenol in the solution after the specified time of standing resulted between 95.0-105.0% compared to the initial values.
Results
The stability results for the control solutions are detailed in Table 44, and the stability results for the solutions at recovery levels with added standards are detailed in Table 45. The results all meet the acceptance criteria.
Watch 44
Name (R)
Intermediate I
Intermediate II
Phenol and its preparation
Stability of control solution at room temperature for 1 day (31h)
100％
101％
99％
Control solution stability at room temperature for 5 days (129.5h)
99％
100％
99％
TABLE 45
Name (R)
Intermediate I
Intermediate II
Phenol and its preparation
0.1% horizontal spiked recovery solution stability at room temperature for 1 day (20.5h)
100％
101％
99％
0.1% horizontal spiked recovery solution stability at room temperature for 5 days (119h)
99％
100％
99％
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.