CN113804792A - Method for detecting 5-p-nitrophenyl furfural related substances - Google Patents
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Abstract
The invention provides a method for detecting 5-p-nitrophenyl furfural related substances, which comprises the following steps: (a) dissolving a test sample and a reference sample in a diluent to obtain a test sample solution and a reference sample solution; (b) respectively carrying out high performance liquid chromatography detection on the test solution and the reference solution; wherein, the conditions of the high performance liquid chromatography detection comprise: under the detection wavelengths of 365nm and 275nm, phenyl silane bonded silica gel is used as a filler of a chromatographic column, ammonium acetate aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and elution is carried out by adopting a gradient elution mode. The method provided by the invention has the advantages of high detection sensitivity, good repeatability, high accuracy, good separation degree of related substances and main components, and less used organic phases, thereby effectively saving the detection cost and remarkably reducing the detection waste liquid.
Description
Technical Field
The invention belongs to the field of analytical chemistry. Specifically, the invention relates to a method for detecting 5-p-nitrophenyl furfural related substances.
Background
5-p-nitrophenyl furfural with molecular weight of 217.17 and molecular formula of C11H7NO4The structural formula is as follows:
5-p-nitrophenylfurfural, as a starting material for the synthesis of dantrolene sodium, may produce process impurities during the production process, such as p-nitrophenol, p-nitroaniline, p-nitrochlorobenzene, and furfural. In the prior art, the purity of 5-p-nitrophenyl furfural is mostly detected by liquid chromatography. However, the existing method often has the problem that the difference between the peak retention time of impurities such as p-nitrophenol, p-nitroaniline, p-nitrochlorobenzene, furfural and the like and the peak retention time of 5-p-nitrophenyl furfural serving as a main component is not large, namely the separation degree is not good, so that the content of impurities such as p-nitrophenol, p-nitroaniline, p-nitrochlorobenzene, furfural and the like in 5-p-nitrophenyl furfural cannot be accurately measured, and further the subsequent drug synthesis is adversely affected.
In view of the above, it is necessary to develop a method for detecting related substances in 5-p-nitrophenyl furfural more reliably and accurately.
Disclosure of Invention
The invention aims to provide a method for detecting 5-p-nitrophenylfurfural-related substances, which has the advantages of high detection sensitivity, good repeatability, high accuracy, good separation degree of the related substances from main components, and less used organic phases, thereby effectively saving the detection cost and remarkably reducing the detection waste liquid.
The above purpose of the invention is realized by the following technical scheme:
a method for detecting 5-p-nitrophenylfurfural-related substances comprises the following steps:
(a) dissolving a test sample and a reference sample in a diluent to obtain a test sample solution and a reference sample solution;
(b) respectively carrying out high performance liquid chromatography detection on the test solution and the reference solution;
wherein, the conditions of the high performance liquid chromatography detection comprise:
under the detection wavelengths of 365nm and 275nm, phenyl silane bonded silica gel is used as a filler of a chromatographic column, ammonium acetate aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and elution is carried out by adopting a gradient elution mode.
Preferably, in step (a), the diluent is a mixture of DMF, acetic acid, acetone, and 60 wt.% aqueous acetonitrile.
Preferably, the concentration of the ammonium acetate aqueous solution is 3.5g/L to 4.0g/L, more preferably 3.85 g/L.
In the present invention, the concentration of the aqueous ammonium acetate solution may be selected from any value within a range of 3.5g/L, 3.6g/L, 3.7g/L, 3.8g/L, 3.85g/L, 3.9g/L, 3.95g/L, 4.0g/L, and any two of the above.
Preferably, the pH of the ammonium acetate aqueous solution is 8.4-8.6, and more preferably 8.5.
Preferably, the column temperature of the high performance liquid chromatography column is 20 ℃ to 30 ℃, more preferably 25 ℃.
In the present invention, the column temperature of the high performance liquid chromatography column may be selected from, for example, 20 ℃, 21 ℃, 22 ℃, 23 ℃, 24 ℃, 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, or 30 ℃.
Preferably, the flow rate of the mobile phase is between 0.9mL/min and 1.1mL/min, preferably 1.0 mL/min.
Preferably, the procedure of gradient elution comprises, in volume fraction of the mobile phase a and the mobile phase B:
eluting with 50-60% of mobile phase A-50-40% of mobile phase B for 0-6 min;
eluting with 25-35% of mobile phase A-75-65% of mobile phase B for 6-7 min;
eluting with 1-3% of mobile phase A-99-97% of mobile phase B for 7-13 min;
eluting with 50-60% of mobile phase A-50-40% of mobile phase B for 13-15 min.
More preferably, the procedure of gradient elution comprises, in volume fraction of the mobile phase a and the mobile phase B:
eluting with 55% mobile phase A-45% mobile phase B for 0-6 min;
eluting with 30% mobile phase A-70% mobile phase B for 6-7 min;
eluting with 2% mobile phase A-98% mobile phase B for 7-13 min;
eluting with 55% mobile phase A-45% mobile phase B for 13-15 min.
Preferably, the related substances are selected from one or more of p-nitrophenol, p-nitroaniline, p-nitrochlorobenzene and furfural.
Preferably, in step (a), the reference solution is a mixed reference solution of a 5-p-nitrophenylfurfural reference and a reference of the related substance.
Preferably, 5-p-nitrophenyl furfural, p-nitrophenol, and p-nitroaniline were detected at a detection wavelength of 365nm, and p-nitrochlorobenzene and furfural were detected at a detection wavelength of 275 nm.
Preferably, the concentration of the test solution is 0.2mg/mL to 0.6mg/mL, preferably 0.25 mg/mL.
Preferably, the concentration of each substance in the control solution is 40ng/mL to 120ng/mL, preferably 50 ng/mL.
Preferably, the high performance liquid chromatography column is an ACE phenyl 4.6mm × 250mm × 5.0 μm column.
Preferably, the content of the related substances is calculated by an external standard method.
As a preferred embodiment, the present invention provides a method for detecting a 5-p-nitrophenylfurfural-related substance, which comprises:
(a) dissolving a test sample and a reference sample in a diluent to obtain a test sample solution and a reference sample solution; (b) respectively carrying out high performance liquid chromatography detection on the test solution and the reference solution;
wherein, the conditions of the high performance liquid chromatography detection comprise:
under the detection wavelength of 365nm and 275nm, phenyl silane bonded silica gel is used as a filler of a chromatographic column, ammonium acetate aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B at the column temperature of 20-30 ℃, and the following gradient elution mode is adopted for elution:
eluting with 50-60% of mobile phase A-50-40% of mobile phase B for 0-6 min;
eluting with 25-35% of mobile phase A-75-65% of mobile phase B for 6-7 min;
eluting with 1-3% of mobile phase A-99-97% of mobile phase B for 7-13 min;
eluting with 50-60% of mobile phase A-50-40% of mobile phase B for 13-15 min;
wherein, 5-p-nitrophenyl furfural, p-nitrophenol and p-nitroaniline are detected at a detection wavelength of 365nm, and p-nitrochlorobenzene and furfural are detected at a detection wavelength of 275 nm; and calculating the content of the related substances by adopting an external standard method.
As a preferred embodiment, the present invention provides a method for detecting a 5-p-nitrophenylfurfural-related substance, which comprises:
(a) dissolving a test sample and a reference sample in a diluent to obtain a test sample solution and a reference sample solution; (b) respectively carrying out high performance liquid chromatography detection on the test solution and the reference solution;
wherein, the conditions of the high performance liquid chromatography detection comprise: under the detection wavelength of 365nm and 275nm, phenyl silane bonded silica gel is used as a filler of a chromatographic column, ammonium acetate aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B at the column temperature of 20-30 ℃, and the following gradient elution mode is adopted for elution:
eluting with 55% mobile phase A-45% mobile phase B for 0-6 min;
eluting with 30% mobile phase A-70% mobile phase B for 6-7 min;
eluting with 2% mobile phase A-98% mobile phase B for 7-13 min;
eluting with 55% mobile phase A-45% mobile phase B for 13-15 min;
wherein, 5-p-nitrophenyl furfural, p-nitrophenol and p-nitroaniline are detected at a detection wavelength of 365nm, and p-nitrochlorobenzene and furfural are detected at a detection wavelength of 275 nm; and calculating the content of the related substances by adopting an impurity external standard method.
The invention has at least the following beneficial effects:
the invention provides a method for detecting 5-p-nitrophenylfurfural-related substances, which has the advantages of high detection sensitivity, good repeatability, high accuracy, good separation degree of the related substances from main components, effective separation between different impurities in the related substances, reduction of used organic phases, effective saving of detection cost and remarkable reduction of detection waste liquid.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is an HPLC chromatogram obtained at pH 6.8 of mobile phase A in example 2;
FIG. 2 is an HPLC chromatogram obtained at pH 5.5 of mobile phase A in example 2;
FIG. 3 is an HPLC chromatogram obtained at pH 8.5 of mobile phase A in example 2;
FIG. 4 is an HPLC chromatogram obtained at pH 9.0 of mobile phase A in example 2;
FIG. 5 is a HPLC spectrum obtained at different detection wavelengths in example 2;
fig. 6 is an HPLC spectrum obtained in comparative example 1.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.
EXAMPLE 1 preparation of the solution
Preparing a test solution:
precisely weighing 50mg of a sample to be tested, placing the sample in a 20mL measuring flask, adding 1mL of DMF and 1mL of acetic acid, then adding acetone to a position 1cm below the scale, carrying out ultrasonic treatment to fully dissolve the acetone, diluting the acetone to the scale, and shaking up to be used as a stock solution; precisely measuring 2mL of the stock solution, placing the stock solution into a 20mL measuring flask, diluting the stock solution to a scale with 60 wt.% of acetonitrile aqueous solution, and shaking up to obtain a sample solution.
Preparing a reference substance solution:
precisely weighing 10mg of each of a 5-p-nitrophenyl furfural reference substance, a p-nitrophenol reference substance, a p-nitroaniline reference substance, a p-nitrochlorobenzene reference substance and a furfural reference substance, putting the 5-p-nitrophenyl furfural reference substance, the p-nitrophenol reference substance, the p-nitroaniline reference substance, the p-nitrochlorobenzene reference substance and the furfural reference substance into a 20mL measuring flask, adding 1mL of DMF (dimethyl formamide) and 1mL of acetic acid, adding acetone to a position 1cm below a scale, fully dissolving the acetone by ultrasonic, diluting the acetone to the scale by using acetone, shaking up, and taking the solution as a reference substance stock solution; precisely measuring 1mL of the stock solution, placing the stock solution into a 100mL measuring flask, diluting the stock solution to a scale with 60 wt.% of acetonitrile aqueous solution, and shaking up to be used as a reference intermediate stock solution; precisely measuring 1mL of the control intermediate stock solution, placing the control intermediate stock solution into a 100mL measuring flask, diluting the control intermediate stock solution to a scale with 60 wt.% of acetonitrile aqueous solution, and shaking the test intermediate stock solution uniformly to serve as a control solution.
Example 2 high performance liquid chromatography sample injection
Detection conditions of high performance liquid chromatography: gradient elution was performed according to the elution procedure in Table 1 using phenylsilane bonded silica as the packing material for ACE phenyl 250X 4.6mm X5 μm chromatography column at 365nm and 275nm double detection wavelength, ammonium acetate solution (3.85 g ammonium acetate dissolved in 1L water, pH adjusted with ammonia) as mobile phase A, acetonitrile as mobile phase B, and other injection conditions and variables summarized in the following tables. Precisely measuring 20 μ L of the sample solution, injecting into a liquid chromatograph, and recording chromatogram. And calculating the impurity content by an impurity external standard method.
TABLE 1 gradient elution procedure
| t(min) | Mobile phase A (%, v/v) | Mobile phase B (%, v/v) |
| 0～6 | 55 | 45 |
| 6～7 | 30 | 70 |
| 7～13 | 2 | 98 |
| 13～15 | 55 | 45 |
pH selection of Mobile phase A
The effect of pH change of selected mobile phase a in table 2 and table 3 on the assay results was examined.
TABLE 2 HPLC INJECTION CONDITIONS, PH FINE-REGULATION OF MOBILE PHASE A
TABLE 3 HPLC INJECTION CONDITIONS, pH VARIATION OF MOBILE PHASE A
From the results in fig. 1 to fig. 4 and tables 2 and 3, it can be seen that when the pH of mobile phase a is 8.5, the separation degree of furfural from p-nitrophenol is 2.95, the separation degree of p-nitrophenol from p-nitroaniline is 2.40, and the separation degree of 5-p-nitrophenylfurfural from p-nitrochlorobenzene is 1.65, which is greater than the minimum separation degree requirement 1.5. It can be seen that each impurity peak can be effectively separated from the main component peak with good results, as shown in fig. 3.
When the pH of the mobile phase A is finely adjusted to be 8.5 +/-0.1, the content of furfural, p-nitrophenol, p-nitroaniline and p-nitrochlorobenzene in the test solution and the RSD value of the test solution at the pH of 8.5 are respectively 0.65%, 9.98%, 4.15% and 2.04%, which shows that under the finely adjusted pH range, the separation and detection effects of related substances are feasible and stable.
However, when the pH of mobile phase a deviates significantly from the range of 8.5 ± 0.1, the following deterioration results occur:
when the pH of the mobile phase A is 6.8, the separation degree of p-nitrophenol and p-nitroaniline is 0.71, the separation degree of 5-p-nitrophenyl furfural and p-nitrochlorobenzene is 1.30, which are both less than the minimum separation degree requirement of 1.5, and the separation effect is poor, as shown in figure 1;
when the pH of the mobile phase A is 5.5, the peak of p-nitrophenol and the peak of p-nitroaniline are almost overlapped, the separation degree of 5-p-nitrophenyl furfural and p-nitrochlorobenzene is 1.27, no obvious difference is generated from the pH of the mobile phase A at 6.8, and the separation effect is not ideal, as shown in FIG. 2;
when the pH value of the mobile phase A is 9.0, the separation degree of p-nitrophenol and p-nitroaniline is improved to 4.95, the separation degree of 5-p-nitrophenyl furfural and p-nitrochlorobenzene is improved to 2.59 which is more than the minimum separation degree requirement of 1.5, and the requirement is met; however, furfural and p-nitrophenol almost overlap, and complete and effective separation of the relevant substances cannot be realized, as shown in fig. 4.
Investigation of column temperature
The pH of the mobile phase A is selected to be 8.5, the flow rate of the mobile phase is selected to be 1.0mL/min, other conditions are unchanged, the change condition of the column temperature is listed in the table 4, and the influence of the change of the column temperature on the detection result is examined.
TABLE 4 column temperature Change and results
From the results in table 4, it can be seen that when the column temperature is varied within a range of 25 ℃ ± 5 ℃, the contents of furfural, p-nitrophenol, p-nitroaniline, and p-nitrochlorobenzene in the sample solution and the RSD values at 25 ℃ of the column temperature are 0.52%, 3.61%, 4.89%, and 1.00%, respectively, and the RSD values are within a range of 10.0%, indicating that the separation detection effect of the relevant substances is feasible and stable within the column temperature range.
Investigation of flow Rate
The pH of the mobile phase A was selected to be 8.5, the column temperature was selected to be 25 ℃, other conditions were unchanged, and the change in the flow rate of the mobile phase was as listed in Table 5, and the influence of the change in the flow rate of the mobile phase on the detection results was examined.
TABLE 5 flow rate changes of mobile phase and results
From the results in table 5, when the flow rate of the mobile phase is varied within a range of 1.0mL/min ± 0.1mL/min, the content of furfural, p-nitrophenol, p-nitroaniline, and p-nitrochlorobenzene in the sample solution and the RSD value of the mobile phase at 1.0mL/min are respectively 2.61%, 3.91%, 2.55%, and 2.63%, and the RSD value is within a range of 10.0%, indicating that the separation and detection effect of the relevant substances is feasible and stable in this flow rate range.
Screening of chromatography columns
The pH of the mobile phase A is selected to be 8.5, the column temperature is selected to be 25 ℃, the flow rate of the mobile phase is selected to be 1.0mL/min, other conditions are not changed, the change condition of the chromatographic column is listed in the table 6, and the influence of the change of the chromatographic column on the detection result is examined.
TABLE 6 column changes and results
Selection of detection wavelength
The pH of the mobile phase A was selected to be 8.5, the column temperature was selected to be 25 ℃, the flow rate of the mobile phase was selected to be 1.0mL/min, the ACE phenyl 4.6mm × 250mm × 5.0 μm column was selected as the column, the conditions were otherwise unchanged, the change in the detection wavelength was as listed in Table 7, and the influence of the change in the detection wavelength on the detection results was examined.
TABLE 7 detection of wavelength changes and results
The HPLC spectra obtained at different detection wavelengths are shown in FIG. 5. From the results in fig. 5 and table 7, it can be seen that:
under the detection wavelength of 275nm, the areas of the furfural and p-nitrochlorobenzene peaks are large; under the detection wavelengths of 318nm and 365nm, the areas of the furfural and p-nitrochlorobenzene peaks are very small.
Under the detection wavelength of 318nm, the peak area of p-nitrophenol is large, and the difference is not large under the other three detection wavelengths.
Under the detection wavelength of 365nm, the area of the p-nitroaniline peak is large, and the difference of the other three detection wavelengths is small and small.
Under the detection wavelength of 365nm, the peak area of 5-p-nitrophenyl furfural is larger, and under the other three detection wavelengths, the peak area is smaller when the wavelength is smaller.
By combining the above conditions, the detection of p-nitrophenol, p-nitroaniline and 5-p-nitrophenyl furfural is finally determined under the detection wavelength of 365nm, furfural and p-nitrochlorobenzene are detected under the detection wavelength of 275nm, and under the dual-wavelength detection, each target substance has a peak area as large as possible, so that the detection sensitivity is improved.
Comparative example 1
Preparation of the solution:
control solution: accurately weighing 30mg of the reference substance, placing the reference substance in a 100mL measuring flask, dissolving the reference substance with methanol, diluting the reference substance to a scale, and shaking up to obtain the product.
Test solution: accurately weighing 30mg of the product, placing the product in a 100mL measuring flask, dissolving the product with methanol, diluting the product to a scale, and shaking the product uniformly to obtain the product.
Detection conditions of high performance liquid chromatography: octadecyl bonded silica gel is used as filler (C18, 250mm multiplied by 3.9 μm, or chromatographic column with equivalent efficiency), methanol is used as mobile phase, and isocratic elution is carried out; the flow rate was 1.0mL/min and the detection wavelength was 254 nm. Precisely measuring 10 μ L of the test solution, injecting into a liquid chromatograph, and recording chromatogram. And calculating the impurity content by using an area normalization method.
As shown in fig. 6, it was found that the peak retention times of p-nitrophenol, p-nitroaniline, p-nitrochlorobenzene, and furfural were not much different from the peak retention time of 5-p-nitrophenyl furfural, which was the main component, and thus, the separation could not be performed efficiently. The method of the comparative example can not accurately separate and detect related substances such as p-nitrophenol, p-nitroaniline, p-nitrochlorobenzene, furfural and the like in the 5-p-nitrophenyl furfural.
The foregoing is merely exemplary of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to preferred embodiments, it is not intended to be limited thereto. Those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the scope of the invention as defined by the appended claims.
Claims (10)
1. A method for detecting 5-p-nitrophenylfurfural-related substances comprises the following steps:
(a) dissolving a test sample and a reference sample in a diluent to obtain a test sample solution and a reference sample solution;
(b) respectively carrying out high performance liquid chromatography detection on the test solution and the reference solution;
wherein, the conditions of the high performance liquid chromatography detection comprise:
under the detection wavelengths of 365nm and 275nm, phenyl silane bonded silica gel is used as a filler of a chromatographic column, ammonium acetate aqueous solution is used as a mobile phase A, acetonitrile is used as a mobile phase B, and elution is carried out by adopting a gradient elution mode.
2. The process of claim 1, wherein, in step (a), the diluent is a mixture of DMF, acetic acid, acetone, and 60 wt.% aqueous acetonitrile.
3. The process according to claim 1 or 2, wherein the concentration of the aqueous ammonium acetate solution is between 3.5 and 4.0g/L, preferably 3.85 g/L;
preferably, the pH of the ammonium acetate aqueous solution is 8.4-8.6, and more preferably 8.5.
4. The method according to any one of claims 1 to 3, wherein the high performance liquid chromatography column has a column temperature of 20 ℃ to 30 ℃, preferably 25 ℃.
5. The method according to any one of claims 1 to 4, wherein the flow rate of the mobile phase is between 0.9 and 1.1mL/min, preferably 1.0 mL/min.
6. The method of any one of claims 1 to 5, wherein the procedure of gradient elution comprises, in volume fractions of the mobile phase A and the mobile phase B:
eluting with 50-60% of mobile phase A-50-40% of mobile phase B for 0-6 min;
eluting with 25-35% of mobile phase A-75-65% of mobile phase B for 6-7 min;
eluting with 1-3% of mobile phase A-99-97% of mobile phase B for 7-13 min;
eluting with 50-60% of mobile phase A-50-40% of mobile phase B for 13-15 min.
7. The method of any one of claims 1 to 6, wherein the procedure of gradient elution comprises, in volume fractions of the mobile phase A and the mobile phase B:
eluting with 55% mobile phase A-45% mobile phase B for 0-6 min;
eluting with 30% mobile phase A-70% mobile phase B for 6-7 min;
eluting with 2% mobile phase A-98% mobile phase B for 7-13 min;
eluting with 55% mobile phase A-45% mobile phase B for 13-15 min.
8. The method of any one of claims 1 to 7, wherein the substance of interest is selected from one or more of p-nitrophenol, p-nitroaniline, p-nitrochlorobenzene, and furfural;
preferably, in step (a), the reference solution is a mixed reference solution of a 5-p-nitrophenylfurfural reference and a reference of the related substance;
preferably, 5-p-nitrophenyl furfural, p-nitrophenol, and p-nitroaniline were detected at a detection wavelength of 365nm, and p-nitrochlorobenzene and furfural were detected at a detection wavelength of 275 nm.
9. The method according to any one of claims 1 to 8, wherein the concentration of the test solution is 0.2mg/mL to 0.6mg/mL, preferably 0.25 mg/mL;
preferably, the concentration of each substance in the control solution is 40ng/mL to 120ng/mL, preferably 50 ng/mL.
10. The method of any one of claims 1 to 9, wherein the high performance liquid chromatography column is an ACE phenyl 4.6mm x 250mm x 5.0 μ ι η chromatography column;
preferably, the content of the related substances is calculated by an external standard method.
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Cited By (2)
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| CN115166095A (en) * | 2022-07-20 | 2022-10-11 | 江苏省食品药品监督检验研究院 | Method for detecting related substances of egg yolk lecithin |
| CN115326969A (en) * | 2022-08-17 | 2022-11-11 | 广东众和高新科技股份公司 | Method for measuring contents of p-nitrophenol and dimethylformamide in p-nitrophenyl-beta-hydroxyethyl sulfide |
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| US20100210577A1 (en) * | 2006-06-08 | 2010-08-19 | Cornell Research Foundation, Inc. | Cyclic carboxylic acid rhodanine derivatives for the treatment and prevention of tuberculosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115166095A (en) * | 2022-07-20 | 2022-10-11 | 江苏省食品药品监督检验研究院 | Method for detecting related substances of egg yolk lecithin |
| CN115326969A (en) * | 2022-08-17 | 2022-11-11 | 广东众和高新科技股份公司 | Method for measuring contents of p-nitrophenol and dimethylformamide in p-nitrophenyl-beta-hydroxyethyl sulfide |
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