CN111458433A - Method for detecting kasugamycin by high performance liquid chromatography - Google Patents
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- PVTHJAPFENJVNC-MHRBZPPQSA-N kasugamycin Chemical compound N[C@H]1C[C@H](NC(=N)C(O)=O)[C@@H](C)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)[C@@H]1O PVTHJAPFENJVNC-MHRBZPPQSA-N 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 20
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 9
- 239000012071 phase Substances 0.000 claims abstract description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 239000012074 organic phase Substances 0.000 claims abstract description 8
- 238000010828 elution Methods 0.000 claims abstract description 7
- 241001251200 Agelas Species 0.000 claims abstract description 5
- 238000010829 isocratic elution Methods 0.000 claims abstract description 5
- 238000002347 injection Methods 0.000 claims abstract description 4
- 239000007924 injection Substances 0.000 claims abstract description 4
- 239000000523 sample Substances 0.000 claims description 32
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- 239000012488 sample solution Substances 0.000 claims description 16
- 239000007853 buffer solution Substances 0.000 claims description 9
- ADCGAPKUMAQOLJ-UHFFFAOYSA-N azane;formic acid Chemical compound N.OC=O.OC=O ADCGAPKUMAQOLJ-UHFFFAOYSA-N 0.000 claims description 7
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
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- 239000012086 standard solution Substances 0.000 claims description 6
- 238000005303 weighing Methods 0.000 claims description 6
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- 239000000203 mixture Substances 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 3
- 230000004044 response Effects 0.000 claims description 3
- UNXNGGMLCSMSLH-UHFFFAOYSA-N dihydrogen phosphate;triethylazanium Chemical compound OP(O)(O)=O.CCN(CC)CC UNXNGGMLCSMSLH-UHFFFAOYSA-N 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 7
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- 239000000126 substance Substances 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- ZDRBJJNXJOSCLR-YZKQBBCCSA-N 2-amino-2-[(2r,3s,5s,6r)-5-amino-2-methyl-6-[(2r,3s,5s,6s)-2,3,4,5,6-pentahydroxycyclohexyl]oxyoxan-3-yl]iminoacetic acid;hydron;chloride Chemical compound Cl.N[C@H]1C[C@H](N=C(N)C(O)=O)[C@@H](C)O[C@@H]1OC1[C@H](O)[C@@H](O)C(O)[C@H](O)[C@@H]1O ZDRBJJNXJOSCLR-YZKQBBCCSA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
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- 244000144730 Amygdalus persica Species 0.000 description 1
- 244000241235 Citrullus lanatus Species 0.000 description 1
- 235000012828 Citrullus lanatus var citroides Nutrition 0.000 description 1
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- 238000003908 quality control method Methods 0.000 description 1
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- 230000000717 retained effect Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000012421 spiking Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
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- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
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- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
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- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
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Abstract
The invention relates to a method for detecting kasugamycin by a high performance liquid chromatography, which belongs to the technical field of drug analysis and comprises the following steps of using a chromatographic column, namely Ejel Venusil HI L IC (Agela, 4.6mm, × 150mm and 5 mu m), using a mobile phase, namely an organic phase and a water phase, using an elution mode, namely isocratic elution, using a flow rate of 1.0m L/min, using a detection wavelength of 220 and 245nm, using a column temperature of 40 ℃, using a sample injection amount of 10 mu L, using a diode array detector for isocratic elution.
Description
Technical Field
The invention belongs to the technical field of drug analysis, and particularly relates to a method for detecting kasugamycin by high performance liquid chromatography.
Background
Kasugamycin is a low-toxicity and systemic bactericide. Has excellent control effect and treatment effect on rice blast on rice. Has special effects of preventing and treating diseases such as bacterial angular leaf spot of watermelon, gummosis of peach trees, scab and perforation.
The kasugamycin hydrochloride is characterized by not remaining in a liquid chromatographic column due to large polarity and uncharacteristic ultraviolet absorption, and often produces peaks simultaneously with a peak inversion or a solvent peak and an impurity peak, thereby bringing difficulty to analysis and detection.
The structural formula of kasugamycin hydrochloride is as follows:
the existing detection methods mostly need a large amount of pretreatment, and a hydrophobic chromophore is modified on molecules, so that the chromatographic behavior of kasugamycin is changed, the retention time is prolonged, and the ultraviolet absorption is characterized. However, the method generates HF gas in the derivatization reaction, has high toxicity, needs special refrigeration equipment in order to prevent the derivative from being decomposed, and has complex operation, more required steps, time consumption and labor waste; in some methods, kasugamycin is not retained on a chromatographic column, an ion pair is required to be used as a mobile phase, the chromatographic column is damaged, the operation is complicated, and the inconvenience is brought to analysis work. Therefore, a method for measuring kasugamycin with good separation degree and high column stability is urgently needed.
Disclosure of Invention
Aiming at the problem that ion pairs are required to be used as a mobile phase in the kasugamycin detection process, the invention provides a method for detecting kasugamycin by using a high performance liquid chromatography, so as to solve the problem.
The technical scheme of the invention is as follows:
a method for detecting kasugamycin by high performance liquid chromatography comprises the following steps:
the chromatographic column is Aijel Venusil HI L IC (Agela, 4.6mm, × 150mm, 5 mu m), the mobile phase is organic phase-aqueous phase, the elution mode is isocratic elution, the flow rate is 1.0m, L/min, the detection wavelength is 220 and 245nm, the column temperature is 40 ℃, the sample injection amount is 10 mu, L, the detector adopts a diode array detector, and the elution time is 5-15 min.
Preferably, the volume ratio of the organic phase to the aqueous phase in the mobile phase is 70-98: 30-2.
Preferably, the organic phase in the mobile phase is a mixed solution of one of methanol and acetonitrile; the water phase is one of water, phosphoric acid-triethylamine buffer solution and ammonium formate-formic acid buffer solution.
Preferably, the ratio of acetonitrile in the mobile phase: the volume ratio of the ammonium formate-formic acid buffer solution is 70: 30.
preferably, the detection wavelength is 220 nm.
Preferably, the organic phase and the aqueous phase are separately injected by a double pump.
A method for detecting kasugamycin by high performance liquid chromatography comprises the following specific operation steps:
(1) weighing 0.05-0.10 g of kasugamycin standard, placing the weighed standard in a 100ml volumetric flask, adding 90ml of water phase, dissolving the standard by ultrasonic oscillation, cooling to water temperature, diluting to a scale by using the water phase, and shaking up to obtain a kasugamycin standard solution for later use;
(2) weighing 0.05-0.10 g of kasugamycin sample to be detected, placing the sample in a volumetric flask with the volume of 100ml, adding 90ml of water phase, dissolving the sample to be detected by ultrasonic oscillation, cooling to the water temperature, diluting the sample to the scale by the water phase, and shaking up to obtain a kasugamycin sample solution for later use;
(3) setting chromatographic conditions, continuously injecting a plurality of needles of standard sample solution after the baseline of the instrument is stable, injecting 10 mu L sample each time until the relative change of the kasugamycin response values of two adjacent needles is less than 0.5%, and then measuring according to the sequence of the standard solution, the sample solution and the standard solution.
The invention has the beneficial effects that:
compared with the prior art, the method for detecting kasugamycin by the high performance liquid chromatography provided by the invention has the obvious advantages that: the Aijier Hilic chromatographic column is creatively used, so that ion pairs are prevented from being used as a mobile phase, and the chromatographic column is prevented from being damaged; the kasugamycin can be effectively reserved, so that the interference of solvent peaks, peak inversion and impurity peaks is avoided, and the rapid and accurate detection can be realized; the pure water is selected as the mobile phase, so that the damage to the chromatographic column can be effectively avoided, the interference of the absorption of the mobile phase or solvent under short wave on the detection of the sample can be effectively avoided, the quality control of kasugamycin can be better realized, and the accuracy of analysis is improved; the tests of linearity and range, recovery rate, precision and the like of detection are good; the stability of the selected chromatographic column is good; and the flowing phase is simple to prepare and the detection cost is low.
The Venusil HI L IC chromatographic column has greater advantages than a reversed-phase C18 chromatographic column in the aspect of processing strong polar compounds, can retain or separate strong polar, water-soluble and alkaline organic compounds by utilizing a hydrophilic action chromatographic principle, has the characteristic of complementation with reversed-phase chromatography, can use a volatile mobile phase, increases L C/MS analytical sensitivity of the chromatogram, reduces the separation pressure, abandons an ion pair reagent in HI L IC chromatogram, greatly facilitates the development of a preparation chromatographic method, has better reproducibility and service life compared with the traditional amino column and silica gel column, and is an excellent choice for replacing the amino column and the silica gel column.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 example 1 white sample chromatogram 1;
FIG. 2 partial chromatogram of an empty white sample of example 1;
FIG. 3 first detection chromatogram of a standard sample of example 1;
FIG. 4 second detection chromatogram of a standard sample of example 1;
FIG. 5 first detection chromatogram of a test sample of example 1;
FIG. 6 second detection chromatogram of a test sample of example 1;
FIG. 7 is a linear relationship between the concentration (mg/100m L) and the peak area according to the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Chromatographic conditions are as follows:
the chromatographic column is Aijel Venusil HI L IC (Agela, 4.6mm × 150mm, 5 mu m), the mobile phase is acetonitrile and ammonium formate-formic acid buffer solution, the volume ratio is 70: 30, the elution mode is isocratic elution, the flow rate is 1.0m L/min, the detection wavelength is 220 and 245nm, the column temperature is 40 ℃, the sample feeding amount is 10 mu L, the detector adopts a diode array detector, and the elution time is 8 min.
The specific operation steps are as follows:
(1) weighing 0.0629g of kasugamycin standard substance, placing in a 100ml volumetric flask, adding 90ml of water phase, dissolving the standard substance by ultrasonic oscillation, cooling to water temperature, diluting with the water phase to scale, and shaking up to obtain a kasugamycin standard substance solution for later use;
(2) weighing 0.0649g of kasugamycin sample to be detected, placing the sample in a volumetric flask with 100ml, adding 90ml of water phase, dissolving the sample to be detected by ultrasonic oscillation, cooling to water temperature, diluting the sample to scale by using the water phase, and shaking up to obtain a kasugamycin sample solution for later use;
(3) setting chromatographic conditions, continuously injecting 6-needle standard sample solution after the baseline of the instrument is stable, injecting 10 mu L sample each time, and measuring according to the sequence of the standard sample solution, the sample solution and the standard sample solution, wherein the relative change of the response values of the fifth needle and the sixth needle kasugamycin standard sample solution is less than 0.5%.
The areas of kasugamycin peaks of the two-pin sample solutions measured in example 1 and the two-pin standard sample solutions before and after the sample were respectively averaged. Mass fraction X of kasugamycin1(%), calculated according to the following formula:
in the above formula:
A1the average value of the kasugamycin peak area in the standard sample solution is shown;
A2the average value of the kasugamycin peak area in the sample solution is shown;
m1the mass of the kasugamycin standard sample, g;
m2is the mass of the sample, g;
p is the mass fraction of kasugamycin.
The test data are shown in the following table 1:
TABLE 1 test data
And substituting the detection data into a formula, and calculating to obtain the content of the sample of 94.79%.
Example 2
The composition of the mobile phase was investigated according to the test method of example 1, and the specific mobile phase composition and the peak time relationship are shown in table 2 below:
TABLE 2 relationship of mobile phase composition to time to peak
According to detection maps, the detection wavelength range is 220-245nm, and the detection method has the characteristics that the kasugamycin peak area is called to be in a descending trend along with the wavelength red shift, and the base line is deteriorated along with the wavelength blue shift.
Example 3
In order to verify the feasibility and accuracy of the detection method of the present invention, the following verification tests were performed as an example in example 1:
(1) accuracy and precision of sample
Chromatographic conditions are as follows:
a chromatographic column, namely Ejel Venusil HI L IC (Agela, 4.6mm × 150mm, 5 mu m), a mobile phase, namely acetonitrile, ammonium formate-formic acid buffer solution, wherein the volume ratio of acetonitrile to ammonium formate-formic acid buffer solution is 70: 30, the flow rate is 1.0m L/min, the detection wavelength is 220 and 245nm, the column temperature is 40 ℃, the sample injection amount is 10 mu L, and a diode array detector is adopted;
under the stable chromatographic condition, 6 repeated tests are carried out on the kasugamycin sample to be tested with the same content in the example 1, and the precision of the method is tested. The results of the precision test of the present invention are shown in the following table 3:
TABLE 3 precision test results
From the experimental results in table 3, it is understood that the analytical method of the present invention has a standard deviation of 0.12% and a relative standard deviation of 0.13%, indicating that the analytical method of the present invention has high precision.
(2) Test for recovery with addition of standard
The results of the spiking recovery experiments of the present invention are shown in Table 4 below
TABLE 4 recovery test results
From the experimental results of table 4, the average recovery rate of kasugamycin was 99.61%, indicating that the accuracy of the present invention is good.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (7)
1. A method for detecting kasugamycin by high performance liquid chromatography is characterized in that the chromatographic conditions are as follows:
the chromatographic column comprises Aijel Venusil HI L IC (Agela, 4.6mm, × 150mm, 5 mu m), a mobile phase, an organic phase and a water phase, an elution mode comprises isocratic elution, the flow rate is 1.0m, L/min, the detection wavelength is 220 and 245nm, the column temperature is 40 ℃, the sample injection amount is 10 mu, L, and a diode array detector is adopted as the detector, and the elution time is 5-15 min.
2. The method for detecting kasugamycin according to claim 1, wherein the volume ratio of the organic phase to the aqueous phase in the mobile phase is 70-98: 30-2.
3. The method for detecting kasugamycin according to claim 1, wherein the organic phase in the mobile phase is a mixture of one of methanol and acetonitrile; the water phase is one of water, phosphoric acid-triethylamine buffer solution and ammonium formate-formic acid buffer solution.
4. The method for detecting kasugamycin according to claim 1, wherein the ratio of acetonitrile in the mobile phase: the volume ratio of the ammonium formate-formic acid buffer solution is 70: 30.
5. the method for detecting kasugamycin according to claim 1, wherein said detection wavelength is 220 nm.
6. The method for detecting kasugamycin according to claim 1, wherein the organic phase and the aqueous phase are separately injected by using a double pump.
7. The method for detecting kasugamycin according to claim 1, which comprises the following specific steps:
(1) weighing 0.05-0.10 g of kasugamycin standard, placing the weighed standard in a 100ml volumetric flask, adding 90ml of water phase, dissolving the standard by ultrasonic oscillation, cooling to water temperature, diluting to a scale by using the water phase, and shaking up to obtain a kasugamycin standard solution for later use;
(2) weighing 0.05-0.10 g of kasugamycin sample to be detected, placing the sample in a volumetric flask with the volume of 100ml, adding 90ml of water phase, dissolving the sample to be detected by ultrasonic oscillation, cooling to the water temperature, diluting the sample to the scale by the water phase, and shaking up to obtain a kasugamycin sample solution for later use;
(3) setting chromatographic conditions, continuously injecting a plurality of needles of standard sample solution after the baseline of the instrument is stable, injecting 10 mu L sample each time until the relative change of the kasugamycin response values of two adjacent needles is less than 0.5%, and then measuring according to the sequence of the standard solution, the sample solution and the standard solution.
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| CN107764915A (en) * | 2017-08-22 | 2018-03-06 | 浙江省农业科学院 | Detection method of the kasugarnycin in citrus |
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| CN107764915A (en) * | 2017-08-22 | 2018-03-06 | 浙江省农业科学院 | Detection method of the kasugarnycin in citrus |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113588840A (en) * | 2021-08-17 | 2021-11-02 | 丽珠集团福州福兴医药有限公司 | Method for detecting content of milbemycins |
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