CN114236022A - Method for detecting kanamycin content in fermentation liquor by pre-column derivatization-HPLC - Google Patents

Abstract

The invention provides a method for detecting kanamycin content in fermentation liquor by pre-column derivatization-HPLC, which comprises the following steps: preparing a kanamycin reference substance solution and a sample solution, preparing a derivative solution, detecting by adopting an HPLC method, recording peak areas, and calculating the content of kanamycin in the sample by adopting an external standard method. The method has the advantages of simple sample pretreatment step, small consumption of the derivatization reagent, mild derivatization condition and simple and controllable operation process; by optimizing chromatographic conditions, the method is more suitable for completely separating the kanamycin from fermentation medium components, and is suitable for simultaneously monitoring the kanamycin content and related components in fermentation liquor, thereby providing guidance for regulating and controlling the fermentation process.

Description

Method for detecting kanamycin content in fermentation liquor by pre-column derivatization-HPLC

Technical Field

The invention belongs to the field of component detection, relates to a method for detecting kanamycin in fermentation liquor, and particularly relates to a method for detecting the kanamycin content in the fermentation liquor by pre-column derivatization-HPLC.

Background

Kanamycin is an aminoglycoside antibiotic and has strong antibacterial action on most gram-negative bacteria, enterobacter, proteus, salmonella, pasteurella multocida, staphylococcus aureus, tubercle bacillus and the like. Kanamycin B has stronger action than kanamycin but has higher neurotoxicity and renal toxicity, so the content of kanamycin B is required to be limited in pharmacopoeias of various countries.

The kanamycin production method is mainly a fermentation method. Because the kanamycin B removal capacity in the fermentation liquor extraction process is limited, the fermentation level is required to be improved as much as possible by the regulation and control of the fermentation process, and meanwhile, the content of the kanamycin B is required to be controlled to ensure that the final finished product is qualified. Therefore, the fermentation process regulation and control needs a detection method capable of simultaneously determining the kanamycin fermentation level and the kanamycin B content.

Since aminoglycosides do not absorb ultraviolet light, separation and detection of kanamycin by HPLC-UV method cannot be carried out directly. Kanamycin and kanamycin B can be detected by using a general HPLC-ELSD method in Chinese pharmacopoeia and Chinese veterinary pharmacopoeia respectively, but the detection object is finished powder and is not suitable for detection of fermentation liquor, kanamycin and saccharides have similarity, and kanamycin is easily interfered by saccharides in a culture medium to influence the detection accuracy when an evaporative light scattering detector is used for detecting fermentation liquor samples. Some enterprises adopt a colorimetric method to detect the intermediate, so that the cost is low, the efficiency is high, but the detection accuracy and stability are poor, and kanamycin B cannot be detected simultaneously. In addition, relevant documents report a pre-column derivatization-high performance liquid chromatography, a post-column derivatization-fluorescence method, an HPLC-pulse amperometric detection method, an enzyme-linked immunosorbent assay, a capillary electrophoresis method, a liquid chromatography-mass spectrometry method and the like, and the pre-column derivatization-HPLC-UV method is a commonly applied intermediate analysis method at present in view of equipment cost and easy operability, and can meet the requirement of quality control in a kanamycin fermentation process.

The pre-column derivatization reagents reported in the literature include 2,4, 6-trinitrobenzenesulfonic acid, ninhydrin-pyridine, o-phthalaldehyde, phenyl isothiocyanate, and the like. The literature, namely HPLC derivatization determination of kanamycin sulfate and injection thereof, uses phenyl isothiocyanate as a pre-column derivatization reagent, can simultaneously determine kanamycin and kanamycin B in a sample, but research objects of the kanamycin sulfate and the injection are finished product powder and injection, the operation process comprises a water bath heating step, the reaction conditions are complex, and a normalization method is adopted for determining the content of kanamycin B, so that the guiding significance is not great.

Disclosure of Invention

In order to solve the problems, the invention aims to develop and improve a fermentation broth sample treatment method and a derivative process respectively on the basis of documents so as to provide a method suitable for simultaneously detecting kanamycin content and related components in fermentation broth, thereby providing guidance for regulating and controlling the fermentation process.

The technical scheme adopted by the invention for realizing the purpose is that the method for detecting the kanamycin content in the fermentation liquor by the pre-column derivatization-HPLC comprises the following steps:

a. preparing a kanamycin reference substance solution and a sample solution;

b. preparation of the derivatization solution: mixing the kanamycin reference substance solution and the sample solution with the derivative reagent solution respectively, shaking uniformly, and standing at room temperature for reaction for 0.5-1 h; then adding water into the reaction solution for dilution by 5 times, filtering by using a microporous filter membrane to respectively obtain a kanamycin reference substance derivative solution and a sample derivative solution to be detected;

c. detection by HPLC: and respectively injecting the kanamycin reference substance derivative solution and the sample derivative solution into HPLC (high performance liquid chromatography), recording peak areas, and calculating the kanamycin content in the sample by adopting an external standard method. Meanwhile, the correction factor of kanamycin is used for approximately calculating the content of kanamycin B.

Further, the sample is kanamycin fermentation broth.

Further, the concentration of the kanamycin reference solution is 0.4-2.0 mg/mL; the preparation method of the kanamycin sample solution comprises the following steps: taking a uniform kanamycin fermentation liquid sample, centrifuging for 10-40min at 3000-.

Further, the preparation process of the derivative solution comprises the steps of taking 1mL of each of the kanamycin reference solution and the sample solution, adding the kanamycin reference solution and the sample solution into 10mL of colorimetric tubes with plugs, adding the derivative reagent solution in an equal amount, shaking uniformly, and standing at room temperature for reaction for 0.5-1 h. And (3) adding water into the reaction solution for dilution by 5 times, and filtering by using a microporous filter membrane to obtain a kanamycin reference substance derivative solution and a sample derivative solution.

Further, the derivatization reagent solution is a mixed solution of phenyl isothiocyanate, triethylamine and acetonitrile, wherein the concentration of the phenyl isothiocyanate is 0.5%, and the concentration of the triethylamine is 2% -8%.

Further, the chromatographic conditions were as follows:

mobile phase A: 80% acetonitrile;

mobile phase B: 0.1mol/L sodium acetate solution (pH adjusted to 6.5 with glacial acetic acid) -acetonitrile (93: 7);

a chromatographic column: 4.6 × 250mm C₁₈；

Sample introduction amount: 10-50 μ L;

column temperature: 30-40 ℃;

flow rate: 0.9-1.1 mL/min;

wavelength: 254 nm;

the gradient elution procedure was:

time (minutes)	Mobile phase A%	Mobile phase B%
			0	0	100
14	20	80
			24	27	73
24.05	100	0
			31.3	100	0
31.35	0	100
			35	0	100

The invention has the beneficial effects that: the method has the advantages of simple sample pretreatment step, small consumption of the derivatization reagent, mild derivatization condition and simple and controllable operation process; by optimizing chromatographic conditions, the method is more suitable for completely separating the kanamycin from fermentation medium components, and is suitable for simultaneously monitoring the kanamycin content and related components in fermentation liquor, thereby providing guidance for regulating and controlling the fermentation process.

Drawings

FIG. 1 is a chromatogram of a control-derived solution of example 1 (kanamycin tR: 15.9 min; kanamycin B: 23.5 min);

FIG. 2 is a sample derived solution chromatogram of example 1.

Detailed Description

The technical solution of the present invention is illustrated below by specific examples, but the scope of the present invention is not limited thereto:

the first embodiment is as follows:

chromatographic conditions are as follows:

sample introduction amount: 10 mu L of the solution;

column temperature: 40 ℃;

flow rate: 1.0 mL/min;

wavelength: 254nm

a. Preparation of kanamycin control solution: 20mg of kanamycin control was weighed out precisely, dissolved in 50mL of water and shaken up.

Preparation of kanamycin sample solution: taking a uniform kanamycin fermentation broth sample, centrifuging for 40min at 3000r/min, taking supernate, and diluting by 10 times with purified water to obtain a kanamycin sample solution.

b. Preparing a derivatization reagent solution (preparing before use): mixing phenyl isothiocyanate, triethylamine and acetonitrile in proportion; wherein the concentration of phenyl isothiocyanate is 0.5 percent and the concentration of triethylamine is 2 percent.

c. Preparation of the derivatization solution: accurately transferring 1.0mL of each of the reference substance solution and the sample solution, respectively adding the reference substance solution and the sample solution into a 10mL colorimetric tube with a plug, respectively adding 1.0mL of the derivative reagent solution, shaking up, standing at room temperature for reaction for 1h, respectively adding 8mL of purified water, shaking up, respectively filtering by using a microporous filter membrane to obtain the kanamycin reference substance derivative solution and the kanamycin sample derivative solution, and respectively filling the kanamycin reference substance derivative solution and the kanamycin sample derivative solution into small bottles to be detected.

d. And (3) determination: respectively injecting the reference substance derivative solution and the sample derivative solution into a liquid chromatograph for separation and determination; and parallelly injecting the reference substance derivative solution for 3 times, injecting the sample derivative solution for 1 time, and recording the peak area.

e. And (4) calculating a result: concentration (weighed mass W x purity P%/volume V) and peak area A of the solution derived from kanamycin control_{To pair}Calculating a correction factor F, and then respectively calculating the contents of kanamycin and kanamycin B in the fermentation liquid sample by using the correction factor, the target peak area A and the sample dilution multiple n:

kanamycin content in the sample is F multiplied by A_{Sample (A)}×n＝8.35mg/mL。

Kanamycin B content ═ FxA_B×n＝0.33mg/mL。

The second embodiment is as follows:

chromatographic conditions are as follows:

sample introduction amount: 50 mu L of the solution;

column temperature: 35 ℃;

flow rate: 0.9 mL/min;

wavelength: 254nm

a. Preparation of kanamycin control solution: kanamycin control 50mg is precisely weighed, dissolved in 25mL water and shaken up.

Preparation of kanamycin sample solution: taking a uniform kanamycin fermentation broth sample, centrifuging for 10min at 8000r/min, taking supernate, and diluting by 5 times with purified water to obtain a kanamycin sample solution.

b. Preparing a derivatization reagent solution (preparing before use): mixing phenyl isothiocyanate, triethylamine and acetonitrile in proportion; wherein the concentration of phenyl isothiocyanate is 0.5 percent and the concentration of triethylamine is 8 percent.

c. Preparation of the derivatization solution: accurately transferring 1.0mL of each of the reference substance solution and the sample solution, respectively adding the reference substance solution and the sample solution into a 10mL colorimetric tube with a plug, respectively adding 1.0mL of the derivative reagent solution, shaking uniformly, standing at room temperature for reaction for 0.5h, respectively adding 8mL of purified water, shaking uniformly, respectively filtering by using a microporous filter membrane to obtain the kanamycin reference substance derivative solution and the kanamycin sample derivative solution, and respectively filling the kanamycin reference substance derivative solution and the kanamycin sample derivative solution into small bottles to be detected.

d. And (3) determination: respectively injecting the reference substance derivative solution and the sample derivative solution into a liquid chromatograph for separation and determination; and parallelly injecting the reference substance derivative solution for 3 times, injecting the sample derivative solution for 1 time, and recording the peak area.

e. And (4) calculating a result: concentration (weighed mass W x purity P%/volume V) and peak area A of the solution derived from kanamycin control_{To pair}Calculating a correction factor F, and then respectively calculating the contents of kanamycin and kanamycin B in the fermentation liquid sample by using the correction factor, the target peak area A and the sample dilution multiple n:

kanamycin content in the sample is F multiplied by A_{Sample (A)}×n＝2.16mg/mL。

Kanamycin B content ═ FxA_B×n＝0.12mg/mL。

The third concrete embodiment:

chromatographic conditions are as follows:

sample introduction amount: 20 mu L of the solution;

column temperature: 30 ℃;

flow rate: 1.1 mL/min;

wavelength: 254nm

a. Preparation of kanamycin control solution: kanamycin control 25mg is weighed precisely, dissolved in 25mL water and shaken up.

Preparation of kanamycin sample solution: taking a uniform kanamycin fermentation broth sample, centrifuging for 20min at 5000r/min, taking supernate, and diluting the supernate by 25 times with purified water to obtain a kanamycin sample solution.

b. Preparing a derivatization reagent solution (preparing before use): mixing phenyl isothiocyanate, triethylamine and acetonitrile in proportion; wherein the concentration of phenyl isothiocyanate is 0.5 percent and the concentration of triethylamine is 7 percent.

c. Preparation of the derivatization solution: accurately transferring 1.0mL of each of the reference substance solution and the sample solution, respectively adding the reference substance solution and the sample solution into a 10mL colorimetric tube with a plug, respectively adding 1.0mL of the derivative reagent solution, shaking up, standing at room temperature for reaction for 50min, respectively adding 8mL of purified water, shaking up, respectively filtering by using a microporous filter membrane to obtain a kanamycin reference substance derivative solution and a kanamycin sample derivative solution, and respectively filling the kanamycin reference substance derivative solution and the kanamycin sample derivative solution into small bottles to be detected.

d. And (3) determination: respectively injecting the reference substance derivative solution and the sample derivative solution into a liquid chromatograph for separation and determination; and parallelly injecting the reference substance derivative solution for 3 times, injecting the sample derivative solution for 1 time, and recording the peak area.

e. And (4) calculating a result: concentration (weighed mass W x purity P%/volume V) and peak area A of the solution derived from kanamycin control_{To pair}Calculating a correction factor F, and then respectively calculating the contents of kanamycin and kanamycin B in the fermentation liquid sample by using the correction factor, the target peak area A and the sample dilution multiple n:

kanamycin content in the sample is F multiplied by A_{Sample (A)}×n＝10.87mg/mL。

Kanamycin B content ═ FxA_B×n＝0.45mg/mL。

Claims (6)

1. A method for detecting the kanamycin content in fermentation liquor by pre-column derivatization-HPLC is characterized in that: the method comprises the following steps:

a. preparing a kanamycin reference substance solution and a kanamycin sample solution;

b. preparation of the derivatization solution: mixing the kanamycin reference substance solution and the kanamycin sample solution with the derivative reagent solution respectively, shaking up, and standing at room temperature for reaction for 0.5-1 h; then adding water into the reaction solution for dilution by 5 times, filtering by using a microporous filter membrane to respectively obtain a kanamycin reference substance derivative solution and a kanamycin sample derivative solution to be detected;

c. detection by HPLC: and respectively injecting the kanamycin reference substance derivative solution and the kanamycin sample derivative solution into HPLC (high performance liquid chromatography), recording peak areas, and calculating the kanamycin content in the sample by adopting an external standard method. Meanwhile, the correction factor of kanamycin is used for approximately calculating the content of kanamycin B.

2. The method for detecting kanamycin content in fermentation broth by pre-column derivatization-HPLC according to claim 1, wherein the method comprises the following steps: the kanamycin sample solution is kanamycin fermentation liquor.

3. The method for detecting kanamycin content in fermentation broth by pre-column derivatization-HPLC according to claim 1, wherein the method comprises the following steps: the concentration of the kanamycin reference substance solution is 0.4-2.0 mg/mL; the preparation method of the kanamycin sample solution comprises the following steps: taking a uniform kanamycin fermentation liquid sample, centrifuging for 10-40min at 3000-.

4. The method for detecting kanamycin content in fermentation broth by pre-column derivatization-HPLC according to claim 1, wherein the method comprises the following steps: the preparation process of the derivative solution comprises the steps of taking 1mL of each kanamycin reference solution and 1mL of each kanamycin sample solution, adding the kanamycin reference solution and the kanamycin sample solution into a 10mL colorimetric tube with a plug respectively, adding the derivative reagent solution in an equal amount, shaking uniformly, and standing at room temperature for reaction for 0.5-1 h. And (3) adding water into the reaction solution for dilution by 5 times, and filtering by using a microporous filter membrane to obtain a kanamycin reference substance derivative solution and a kanamycin sample derivative solution.

5. The method for detecting kanamycin content in fermentation broth by pre-column derivatization-HPLC according to claim 4, wherein the method comprises the following steps: the derivatization reagent solution is a mixed solution of phenyl isothiocyanate, triethylamine and acetonitrile, wherein the concentration of the phenyl isothiocyanate is 0.5%, and the concentration of the triethylamine is 2% -8%.

6. The method for detecting kanamycin content in fermentation broth by pre-column derivatization-HPLC according to claim 1, wherein the method comprises the following steps: the chromatographic conditions were as follows:

mobile phase A: 80% acetonitrile;

mobile phase B: 0.1mol/L sodium acetate solution (pH adjusted to 6.5 with glacial acetic acid) -acetonitrile (93: 7);

a chromatographic column: 4.6 × 250mm C₁₈；

Sample introduction amount: 10-50 μ L;

column temperature: 30-40 ℃;

flow rate: 0.9-1.1 mL/min;

wavelength: 254 nm;

the gradient elution procedure was:

time (minutes) Mobile phase A% Mobile phase B% 0 0 100 14 20 80 24 27 73 24.05 100 0 31.3 100 0 31.35 0 100 35 0 100

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