CN115494183B - Method for detecting 1,2, 4-triazole in triazole medicines - Google Patents
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Abstract
The application belongs to the technical field of chromatographic analysis, and particularly relates to a method for detecting 1,2, 4-triazole in triazole medicines. The method of the present application comprises: under the condition of a buffer solution, performing derivatization reaction on a triazole drug sample and a fluorenylmethoxycarbonyl chloride acetonitrile solution, and then adding a diluent to obtain a derivatization product solution; detecting the derivatization product solution by adopting a high performance liquid chromatography to obtain a qualitative and quantitative detection result of the 1,2, 4-triazole in the triazole medicines; the conditions are as follows: octadecyl is a chromatographic column of a filling agent; taking a formic acid aqueous solution as a mobile phase A, and taking acetonitrile as a mobile phase B; the method has good specificity, high sensitivity and accurate and reliable result, can be used for controlling the quality of the 1,2, 4-triazole impurity of any step intermediate in the itraconazole process, and simultaneously supports the quality control of the 1,2, 4-triazole impurity in any triazole medicament.
Description
Technical Field
The application belongs to the technical field of chromatographic analysis, and particularly relates to a method for detecting 1,2, 4-triazole in triazole medicines.
Background
Efinaconazole is a novel triazole external drug approved by FDA in 2014, and the action mechanism of efinaconazole is to inhibit fungal 14 a-demethylase, thereby affecting the integrity and function of cell membrane. 1,2, 4-triazole provides an itraconazole triazole ring structure fragment, the polarity of the 1,2, 4-triazole is large, a conventional reverse phase chromatography system is difficult to reserve, the conventional reverse phase chromatography or HILIC mode is mostly used for analyzing and detecting related substances at present, but solvents with similar polarities, such as N, N-dimethylformamide, dimethyl sulfoxide and the like, are required to be considered in the production process when the 1,2, 4-triazole is used for analyzing impurities, the triazole serving as the impurities is difficult to separate from the solvents, the ultraviolet absorption wavelength (195 nm) of the 1,2, 4-triazole in liquid chromatography is low, the baseline interference is large, the absorption is weak, and the sensitivity of the method is insufficient.
Disclosure of Invention
In view of this, the application provides a method for detecting 1,2, 4-triazole in triazole drugs, which adopts high performance liquid chromatography for pre-column derivatization detection of 1,2, 4-triazole in an efinaconazole intermediate, and the derivatized 1,2, 4-triazole can be detected under conventional chromatographic conditions, and has the advantages of good specificity, high sensitivity, accurate and reliable result, and can be used for quality control of 1,2, 4-triazole impurities of the intermediate in any step of the efinaconazole process, and simultaneously supports quality control of 1,2, 4-triazole impurities in any triazole drugs.
The application provides a method for detecting 1,2, 4-triazole in triazole medicines, which comprises the following steps:
under the condition of a buffer solution, performing derivatization reaction on a triazole drug sample and a fluorenylmethoxycarbonyl chloride acetonitrile solution, and then adding a diluent to obtain a derivatization product solution;
detecting the derivatization product solution by adopting a high performance liquid chromatography to obtain a qualitative and quantitative detection result of the 1,2, 4-triazole in the triazole medicines;
the conditions of the high performance liquid chromatography are as follows: octadecyl is used as a chromatographic column of the filling agent; taking a formic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; the elution method comprises the following steps: enabling the mobile phase to pass through a chromatographic column by adopting a gradient elution mode, and then carrying out ultraviolet detection;
in 0-15min, the mass content of the mobile phase A in the mobile phase is reduced from 70-80% to 50-60%, and the mass content of the mobile phase B in the mobile phase is increased from 20-30% to 40-50%;
in 15-20min, the mass content of the mobile phase A in the mobile phase is reduced from 50-60% to 45-55%, and the mass content of the mobile phase B in the mobile phase is increased from 40-50% to 45-55%;
in 20-21min, the mass content of the mobile phase A in the mobile phase is reduced to 20-30% from 45-55%, and the mass content of the mobile phase B in the mobile phase is increased to 70-80% from 45-55%;
and (3) performing isocratic elution on the mobile phase A and the mobile phase B at 21-26min, wherein the mass content of the mobile phase A in the mobile phase is 20-30%, and the mass content of the mobile phase B in the mobile phase is 70-80%.
Specifically, the detection method can detect 1,2, 4-triazole from triazole drugs or triazole drug intermediates, wherein the triazole drugs can be itraconazole, fluconazole, itraconazole, voriconazole or the like; the triazole medicine intermediate is any one step intermediate in the preparation of triazole medicines, such as (2R, 3R) -2- (2, 4-difluorophenyl) -1- (1, 2, 4-triazole-1-yl) -2, 3-dibutanol.
Preferably, the proportion of the mobile phase A is reduced to 50-60% from 70-80% within 0-15min, and the proportion of the mobile phase B is increased to 40-50% from 20-30%; more preferably, the proportion of mobile phase a decreases from 76% to 55% and the proportion of mobile phase B increases from 24% to 45% for gradient elution.
Preferably, the proportion of the mobile phase A is reduced from 50% -60% to 45% -55% in 15-20min, the proportion of the mobile phase B is increased from 40% -50% to 45% -55% for gradient elution, more preferably, the proportion of the mobile phase A is reduced from 55% to 50%, and the proportion of the mobile phase B is increased from 45% to 50% for gradient elution.
Preferably, in 20 to 21min, the proportion of the mobile phase A is reduced from 45-55% to 20-30%, the proportion of the mobile phase B is increased from 45-55% to 70-80% for gradient elution, more preferably, the proportion of the mobile phase A is reduced from 50% to 25%, and the proportion of the mobile phase B is increased from 50% to 75% for gradient elution.
Preferably, the mobile phase A and the mobile phase B are subjected to isocratic elution within 21-26min, the proportion of the mobile phase A is 20% -30%, the proportion of the mobile phase B is 70% -80%, more preferably, the mobile phase A and the mobile phase B are subjected to gradient elution, the proportion of the mobile phase A is 25%, and the proportion of the mobile phase B is 75%.
Preferably, the buffer solution is selected from phosphate buffer salts or/and pyridine. More preferably, pyridine is used as a buffer solution.
Preferably, the buffer solution is borax buffer salt or phosphate buffer salt, and the pH value of the buffer solution is adjusted to 8-12 by adopting an adjusting solution.
Specifically, the adjusting solution may be a common acid solution, such as a solution of hydrochloric acid.
Preferably, the concentration of the fluorenylmethoxycarbonylylchloride in the fluorenylmethoxycarbonylchloroacetonitrile solution is 2 to 8 mg/mL. More preferably, the fluorenylmethoxycarbonylcarbonyl chloride acetonitrile solution has a fluorenylmethoxycarbonylcarbonyl chloride concentration of 5 mg/mL.
Preferably, the mobile phase A is a formic acid aqueous solution with the volume fraction of 0.05% -0.15%. More preferably, the mobile phase a is 0.1% by volume aqueous formic acid.
Preferably, the diluent is an acetonitrile-water solution, and the volume ratio of the acetonitrile to the water is 100:0 to 50:50. more preferably, acetonitrile is used as the diluent.
Preferably, the mass content of the mobile phase A in the mobile phase is reduced from 76% to 55% and the mass content of the mobile phase B in the mobile phase is increased from 24% to 45% in 0-15min;
the mass content of the mobile phase A in the mobile phase is reduced from 55% to 50% and the mass content of the mobile phase B in the mobile phase is increased from 45% to 50% within 15-20min;
at 20-21min, the mass content of the mobile phase A in the mobile phase is reduced from 50% to 25%, and the mass content of the mobile phase B in the mobile phase is increased from 50% to 75%;
and (3) performing isocratic elution on the mobile phase A and the mobile phase B at 21-26min, wherein the mass content of the mobile phase A in the mobile phase is 25%, and the mass content of the mobile phase B in the mobile phase is 75%.
Preferably, the flow rate of the mobile phase in the high performance liquid chromatography detection process is 1.1-1.3mL/min; the temperature of the chromatographic column is 25-35 ℃; the particle size of the chromatographic column is 3.0-5.0 μm.
Preferably, the detection wavelength in the high performance liquid chromatography detection process is 205nm-215nm.
Specifically, the impurity, namely 1,2, 4-triazole detected by the application has a structure shown in a formula 1;
The detection method is different from the conventional method for separating substances through a chromatographic column, and firstly treats a sample in a derivative mode and then separates the sample in a high performance liquid phase mode. The detection method can effectively detect the 1,2, 4-triazole impurities in the triazole medicaments, and the experimental result shows that the method has high detection sensitivity, good specificity and accurate and reliable result, and can be used for quality control of the 1,2, 4-triazole impurities in the triazole medicaments, such as an efinaconazole intermediate. The method refers to and conforms to the specification of high performance liquid chromatography in appendix of the fourth part of China pharmacopoeia 2020, and the detection method has high sensitivity and good specificity.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
Fig. 1 is a high performance liquid chromatogram of a solution after derivatization of an efinaconazole intermediate and a 1,2, 4-triazole mixed solution provided in example 1 of the present application, wherein the peak appearance time of a target peak in fig. 1 is 14.477, 14.980 and 15.467 in sequence from left to right, and the peak of the 1,2, 4-triazole derivative is 14.980 minutes;
fig. 2 is a high performance liquid chromatogram of a solution derived from a mixed solution of an efinaconazole intermediate and 1,2, 4-triazole provided in example 2 of the present application, wherein a target peak leading time in fig. 2 is 12.362;
fig. 3 is a high performance liquid chromatogram of a solution after derivatization of an efinaconazole intermediate and a 1,2, 4-triazole mixed solution provided in example 3 of the present application, wherein the peak appearance time of the target peak in fig. 3 is 10.838 and 11.263 in sequence from left to right, and the peak of the 1,2, 4-triazole derivative is 11.263 minutes;
fig. 4 is a high performance liquid chromatogram of a solution after derivatization of the efinaconazole intermediate provided by the application in comparative example 1 and a 1,2, 4-triazole mixed solution.
Detailed Description
The application provides a method for detecting 1,2, 4-triazole in triazole medicines, which can simultaneously and accurately detect 1,2, 4-triazole impurities in an efinaconazole intermediate and is used for solving the technical defect that the 1,2, 4-triazole in the triazole medicines in the prior art is difficult to accurately detect due to weak ultraviolet absorption of the 1,2, 4-triazole.
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments obtained by a person of ordinary skill in the art based on the embodiments in the present application without making any creative effort belong to the protection scope of the present application.
Wherein, the impurity 1,2, 4-triazole in the following examples is shown as formula 1,
The starting materials or reagents used in the following examples are all commercially available.
The efinaconazole intermediate in the following examples is any intermediate in the efinaconazole process, and the efinaconazole intermediate used in this example is (2r, 3r) -2- (2, 4-difluorophenyl) -1- (1, 2, 4-triazol-1-yl) -2, 3-dibutanol.
Example 1
In the embodiment of the application, the 1,2, 4-triazole control solution in the efinaconazole intermediate is detected and validated by a high performance liquid chromatography pre-column derivatization method, and the method comprises the following specific steps:
(1) Preparation of a solution to be derivatized:
a. test solution: precisely weighing a proper amount of the efinaconazole intermediate, adding a diluent to dilute the mixture to prepare a solution containing 10mg/mL of the efinaconazole intermediate, and shaking up. Wherein the diluent is acetonitrile.
b. Control solution: accurately weighing a proper amount of 1,2, 4-triazole, adding a diluent to dilute and prepare a solution containing 10 micrograms of 1,2, 4-triazole per 1mL, and shaking up.
c. Adding a standard solution into a test sample: precisely taking the efinaconazole intermediate and a proper amount of 1,2, 4-triazole, adding a diluent to dilute the mixture to prepare a solution containing 10mg/mL of the efinaconazole intermediate and 10 mu g of 1,2, 4-triazole, and shaking up.
(2) And (3) a derivation process: precisely measuring 1mL of a solution to be derivatized (a diluent, a reference solution, a test solution or a test and standard solution), placing the solution into an EP tube, precisely adding 0.1mL of a buffer solution, and uniformly mixing by vortex; adding 0.5ml of derivatization agent precisely, and vortexing for at least 15 seconds; add 1.6mL of diluent and vortex mix to give the derivatizing solution. Wherein the diluent is acetonitrile; the derivatization reagent is FOMC-Cl acetonitrile solution with the concentration of 5 mg/ml; the buffer solution is pyridine.
(3) And taking the derivative solution for high performance liquid chromatography test. The sample introduction amount was 10. Mu.L, the flow rate was 1.2mL/min, and the column temperature was 30 ℃. The derivative spectrum of the sample labeling solution is shown in FIG. 1.
Wherein, a high performance liquid chromatograph ultraviolet visible detector is used, the wavelength of the ultraviolet detector is 266nm, and octadecyl bonding silica gel particles with the particle size of 3.5 mu m are selected as a chromatographic column of the filler. Mobile phase a was 0.1% by volume aqueous formic acid and mobile phase B was acetonitrile. In the high performance liquid detection process, a gradient elution mode is adopted to enable the mobile phase to pass through the chromatographic column, and the gradient elution program is set according to the following table 1, namely when the time is 0-15min, the gradient elution is carried out by taking 76% of the mobile phase A and 24% of the mobile phase B as starting points, the proportion of the mobile phase A is reduced to 55%, and the proportion of the mobile phase B is increased to 45%. Mobile phase a (55%) dropped to (50%) and mobile phase B (45%) increased to (50%) 15-20 min; 20-21min mobile phase A (50%) decreased to (25%) and mobile phase B (50%) increased to (75%); the mobile phase A keeps 25% isocratic elution and the mobile phase B keeps 75% isocratic elution for 21-26 min.
Table 1 gradient elution table for example 1
(4) The methodology of the high performance liquid chromatography analysis method of the established 1,2, 4-triazole content analysis method is verified according to the chromatographic parameters. The verification items include system applicability, specificity, quantitation limit, detection limit, linearity, precision (including repeatability and intermediate precision), solution stability and durability.
The methodology was verified by the above method, and the verification results are shown below:
1. the system applicability is that in the detection results of 5 reference substance solutions derived in parallel in the experiment, the RSD is 0.4%; the minimum value of the resolution of the 1,2, 4-triazole derivative peak and the adjacent impurity peak is 1.50.
2. The specificity is as described in table 2.
TABLE 2 specificity
3. The detection limit and the quantification limit, the concentration of 1,2, 4-triazole in the solution of the detection limit is 1.085 mu g/ml, the S/N is 125, and the 1,2, 4-triazole with 100ppm can be detected; the concentration of the quantitative limit solution 1,2, 4-triazole is 2.170 mu g/ml, the minimum S/N is phase 228, the peak area RSD of the 1,2, 4-triazole derivative is 0.4% after 6 times of continuous sample injection, and the 1,2, 4-triazole with 200ppm can be quantitatively detected.
4. Linear, wherein a linear equation Y =34.094X-2.2979 of peak area and concentration of a detection result of a derivative product with the concentration of 1,2, 4-triazole of 2.170-21.7 mu g/ml; the correlation coefficient is 1.0000; the ratio RSD of each concentration to the peak area was 1.5%.
5. Precision, 1,2, 4-triazole is not detected in the test solution; a test solution of 0.1% horizontal concentration 1,2, 4-triazole is added for derivatization experiment, the recovery rate of the detection result is between 98.15% and 101.17%, and the recovery rate RSD is 1.2%. The recovery rate of the intermediate precision experiment is 98.15% -101.17%, and the recovery rate RSD is 1.2%.
6. And (3) accuracy, adding a sample solution of 200ppm to 0.15% horizontal concentration 1,2, 4-triazole for derivatization experiment, wherein the recovery rate of the detection result is 94.37-102.39%, and the recovery rate RSD is 2.7%.
7. And (3) the solution stability is that the content of a sample subjected to a derivatization experiment by adding a test solution of 0.1% horizontal concentration 1,2, 4-triazole is reduced by 5.2% after the sample is placed at 5 ℃ for 54 hours, and the content is less than 10% of an acceptable standard. The samples were considered stable for 54 hours under this condition.
Example 2
In the embodiment of the application, the 1,2, 4-triazole control solution in the efinaconazole intermediate is detected and validated by a high performance liquid chromatography pre-column derivatization method, and the method comprises the following specific steps:
(1) Preparing a sample labeling solution:
precisely taking an appropriate amount of the efinaconazole intermediate and 1,2, 4-triazole, adding a diluent to dilute the mixture to prepare a solution containing 10mg/ml of the efinaconazole intermediate and 10 mu g of 1,2, 4-triazole, and shaking up, wherein the diluent is a solution of acetonitrile-water (1.
(2) And (3) a derivation process: precisely measuring a sample to be tested, adding 0.4mL of standard solution into an EP (EP) tube, precisely adding 0.2mL of buffer solution, and uniformly mixing by vortex; precisely adding 0.2mL of a derivatizing agent, and vortexing for at least 15 seconds; add 0.8mL of diluent and vortex mix to give the derivatizing solution. Wherein the diluent is acetonitrile-water (1), the derivatization agent is 5mg/mL FOMC-Cl acetonitrile solution, and the buffer solution is 100mmol/L disodium hydrogen phosphate aqueous solution.
(3) And taking the derivative solution for high performance liquid chromatography test. The sample introduction amount was 10. Mu.L, the flow rate was 1.0mL/min, and the column temperature was 30 ℃. The derivatization map of the test article with the standard solution is shown in FIG. 2.
(4) An ultraviolet detector of a high performance liquid chromatograph is used, the wavelength of the ultraviolet detector is 210nm, and a chromatographic column with C18 bonded silica gel particles with the particle size of 3.5 mu m as a filling agent is selected. Mobile phase a was 0.1% by volume aqueous formic acid and mobile phase B was acetonitrile. In the high performance liquid detection process, a gradient elution mode is adopted to enable the mobile phase to pass through the chromatographic column, and the gradient elution program is set according to the following table 2, namely when 0-15min is carried out, the gradient elution is carried out by taking 80% of the mobile phase A and 20% of the mobile phase B as starting points, the proportion of the mobile phase A is reduced to 55%, and the proportion of the mobile phase B is increased to 45%. Mobile phase a (55%) dropped to (50%) and mobile phase B (45%) increased to (50%) 15-20 min; 20-21min mobile phase A (50%) dropped to (25%) and mobile phase B (50%) increased to (75%); the mobile phase A keeps 25% isocratic elution and the mobile phase B keeps 75% isocratic elution for 21-26 min.
Table 3 gradient elution table for example 2
Example 3
In the embodiment of the application, the 1,2, 4-triazole control solution in the efinaconazole intermediate is detected and verified by methodology by adopting high performance liquid chromatography pre-column derivatization, and the method comprises the following specific steps:
(1) Preparing a sample labeling solution:
precisely taking the efinaconazole intermediate and a proper amount of 1,2, 4-triazole, adding a diluent to dilute the mixture to prepare a solution containing 10mg/mL of the efinaconazole intermediate and 10 mu g of 1,2, 4-triazole, and shaking up. Wherein the diluent is a solution of acetonitrile-water (1.
(2) And (3) derivation process: precisely measuring a sample to be tested, adding 0.4mL of standard solution, placing the sample into an EP tube, precisely adding 0.2mL of buffer solution, and uniformly mixing by vortex; precisely adding 0.2mL of a derivatizing agent, and vortexing for at least 15 seconds; add 0.8mL of diluent and vortex mix to give the derivatizing solution. Wherein, the diluent is acetonitrile-water (1).
(3) And taking the derivative solution for high performance liquid chromatography test. The sample introduction amount was 10. Mu.L, the flow rate was 1.0mL/min, and the column temperature was 30 ℃. The derivatization pattern of the test article spiking solution is shown in FIG. 3.
(4) An ultraviolet detector of a high performance liquid chromatograph is used, the wavelength of the ultraviolet detector is 210nm, and a chromatographic column with C18 bonded silica gel particles with the particle size of 3.5 mu m as a filling agent is selected. Mobile phase a was 0.1% by volume aqueous formic acid and mobile phase B was acetonitrile. In the high performance liquid detection process, a gradient elution mode is adopted to enable the mobile phase to pass through the chromatographic column, and the gradient elution program is set according to the following table 3, namely when 0-12min is carried out, the gradient elution is carried out by taking 70% of the mobile phase A and 30% of the mobile phase B as starting points, the proportion of the mobile phase A is reduced to 50%, and the proportion of the mobile phase B is increased to 50%.12-17min, wherein the mobile phase A of the mobile phase A keeps 50% isocratic elution, and the mobile phase B keeps 50% isocratic elution; 17-28min mobile phase A (50%) dropped to (25%) and mobile phase B (50%) increased to (75%); the mobile phase A keeps 25% isocratic elution for 18-25min, and the mobile phase B keeps 75% isocratic elution.
Table 4 gradient elution table for example 3
Comparative example 1
The comparative example provides a comparative detection method, and the content of the 1,2, 4-triazole after the derivatization is detected by adopting a high performance liquid chromatography, and the method comprises the following steps:
the method of the comparative example is similar to that of example 2, except that the buffer solution is triethylamine, the rest steps are the same as those of example 2, the derivatization map of the 1,2, 4-triazole solution is shown in fig. 4, as can be seen from fig. 4, no 1,2, 4-triazole peak appears at a specific time, and the triethylamine in fig. 4 as the buffer solution cannot support the derivatization of the 1,2, 4-triazole.
In summary, in the detection method (1) of the present application, the derivatizing agent is FOMC-Cl, the buffer solution is one of pyridine and phosphate buffer solution with pH =8 to pH =12, and the derivatizing is performed before entering the high performance liquid chromatography; (2) Selecting octadecylsilane chemically bonded silica as a chromatographic column of a filling agent; gradient elution is carried out by adopting a mobile phase, wherein the phase A of the mobile phase is a water phase prepared by 0.1 percent formic acid, and the phase B of the mobile phase is acetonitrile; the detector adopted by the high performance liquid chromatography is an ultraviolet detector, the high performance liquid chromatography detection is carried out under proper gradient, flow rate and column temperature, and the 1,2, 4-triazole in the sample solution is analyzed. The method can effectively detect the 1,2, 4-triazole impurities in the efinaconazole, and the detection method has high sensitivity and good specificity.
The foregoing is only a preferred embodiment of the present application and it should be noted that, as will be apparent to those skilled in the art, numerous modifications and adaptations can be made without departing from the principles of the present application and such modifications and adaptations are intended to be considered within the scope of the present application.
Claims (9)
1. A method for detecting 1,2, 4-triazole in triazole medicines is characterized by comprising the following steps:
under the condition of phosphate buffer salt or pyridine or borax buffer salt, performing derivatization reaction on a triazole drug sample and a fluorenylmethoxycarbonyl acyl chloride acetonitrile solution, and then adding a diluent to obtain a derivatization product solution; the triazole drug sample is (2R, 3R) -2- (2, 4-difluorophenyl) -1- (1, 2, 4-triazol-1-yl) -2, 3-dibutanol;
detecting the derivatization product solution by adopting a high performance liquid chromatography to obtain a qualitative and quantitative detection result of the 1,2, 4-triazole in the triazole medicines;
the conditions of the high performance liquid chromatography are as follows: chromatographic column with octadecylsilane chemically bonded silica as filler; taking a formic acid aqueous solution as a mobile phase A, and taking acetonitrile as a mobile phase B; the elution method comprises the following steps: enabling the mobile phase to pass through a chromatographic column by adopting a gradient elution mode, and then carrying out ultraviolet detection;
in 0-15min, the mass content of the mobile phase A in the mobile phase is reduced from 70-80% to 50-60%, and the mass content of the mobile phase B in the mobile phase is increased from 20-30% to 40-50%;
in 15-20min, the mass content of the mobile phase A in the mobile phase is reduced from 50-60% to 45-55%, and the mass content of the mobile phase B in the mobile phase is increased from 40-50% to 45-55%;
in 20-21min, the mass content of the mobile phase A in the mobile phase is reduced from 45-55% to 20-30%, and the mass content of the mobile phase B in the mobile phase is increased from 45-55% to 70-80%;
and (3) performing isocratic elution on the mobile phase A and the mobile phase B at 21-26min, wherein the mass content of the mobile phase A in the mobile phase is 20-30%, and the mass content of the mobile phase B in the mobile phase is 70-80%.
2. The detection method according to claim 1, wherein the pH of the phosphate buffer salt or the borax buffer salt is adjusted to 8 to 12 by using an adjusting solution.
3. The detection method according to claim 1, wherein the concentration of fluorenylmethoxycarbonylylchloride in the fluorenylmethoxycarbonylchloroacetonitrile solution is 2 to 8 mg/mL.
4. The detection method according to claim 1, wherein the concentration of fluorenylmethoxycarbonylylchloride in the fluorenylmethoxycarbonylchloroacetonitrile solution is 5 mg/mL.
5. The detection method according to claim 1, wherein the mobile phase A is a formic acid aqueous solution with a volume fraction of 0.05% -0.15%.
6. The detection method according to claim 1, wherein the diluent is an acetonitrile-water solution, and the volume ratio of the acetonitrile to the water is 100:0 to 50:50.
7. the detection method according to claim 1, wherein the mass content of the mobile phase A in the mobile phase is reduced from 76% to 55% and the mass content of the mobile phase B in the mobile phase is increased from 24% to 45% in 0-15min;
the mass content of the mobile phase A in the mobile phase is reduced from 55% to 50% and the mass content of the mobile phase B in the mobile phase is increased from 45% to 50% within 15-20min;
at 20-21min, the mass content of the mobile phase A in the mobile phase is reduced from 50% to 25%, and the mass content of the mobile phase B in the mobile phase is increased from 50% to 75%;
and (3) carrying out isocratic elution on the mobile phase A and the mobile phase B at 21-26min, wherein the mass content of the mobile phase A in the mobile phase is 25%, and the mass content of the mobile phase B in the mobile phase is 75%.
8. The detection method according to claim 1, wherein the flow rate of the mobile phase in the high performance liquid chromatography detection process is 1.1-1.3mL/min; the temperature of the chromatographic column is 25-35 ℃; the particle size of the chromatographic column is 3.0-5.0 mu m.
9. The detection method according to claim 1, wherein the detection wavelength in the high performance liquid chromatography detection process is 205nm-215nm.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5003076A (en) * | 1988-10-12 | 1991-03-26 | Shionogi & Co., Ltd. | Benzotriazole derivatives and chiral derivatization reagents for carboxylic acids thereof |
| CN106946804A (en) * | 2017-03-30 | 2017-07-14 | 成都绿林科技有限公司 | The preparation technology and its content assaying method of a kind of triazole derivatives preparation |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5003076A (en) * | 1988-10-12 | 1991-03-26 | Shionogi & Co., Ltd. | Benzotriazole derivatives and chiral derivatization reagents for carboxylic acids thereof |
| CN106946804A (en) * | 2017-03-30 | 2017-07-14 | 成都绿林科技有限公司 | The preparation technology and its content assaying method of a kind of triazole derivatives preparation |
Non-Patent Citations (3)
| Title |
|---|
| Determination of Amitrole in Environmental Water Samples with Precolumn Derivatization by High-Performance Liquid Chromatography;YAN SUN等;《J. Agric. Food Chem.》;20090508;第57卷;第4540-4544页 * |
| Ion-pairing high-performance liquid chromatography determination of amitrole in apple after solid-phase extraction and precolumn derivatization;Yan Sun等;《Anal Bioanal Chem》;20090726;第395卷;第465-471页 * |
| 柱前衍生-反相高效液相色谱测定茶叶中19种氨基酸含量;孔谦等;《食品科技》;20181231;第43卷(第10期);第320-325页 * |
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