Analysis method for detecting colistin in bacteria

Abstract

The invention belongs to the technical field of bacteria detection, and discloses an analysis method for detecting colistin in bacteria. The method comprises the following steps: firstly, centrifugally washing bacterial suspension incubated by sub-bacteriostasis colistin, and repeatedly freezing and thawing by adopting liquid nitrogen, so that bacterial cells are cracked; placing the split thalli in an acidic solution, and hydrolyzing at 60-120 DEG C in a protective atmosphere to obtain 2, 4-diaminobutyric acid; adjusting the pH to be neutral by using an alkaline solution to obtain hydrolysate; adding a derivatization buffer solution and a derivatization agent chloroformic acid-9-fluorenyl methyl ester into the hydrolysate, reacting at 20-80 DEG C to obtain a derivatization reaction solution, and treating to obtain filtrate; and separating the filtrate through a C8 reversed-phase chromatographic column, determining by selecting a fluorescence detector, and qualitatively and quantitatively analyzing the colistin in the bacteria by adopting a standard addition method, so that the colistin in the bacteria can be detected. According to the method, the colistin in the bacteria is detected by using a high performance liquid chromatography-fluorescence method, and a technical support is provided for exploring an antibacterial action mechanism of the colistin and a drug resistance mechanism of the bacteria.

Description

Technical Field

[0001] The present invention belongs to the field of bacterial detection, and particularly relates to an analytical method for detecting colistin in bacteria. Background Art

[0002] Colistin, namely polymyxin E, is a lipopeptide cationic antibiotic isolated from Bacillus polymyxa, which has strong antibacterial activity against most Gram-negative bacteria, especially effective in the treatment of multi-drug resistant Gram-negative bacterial infections. In the production of food animals, colistin is mainly used to treat intestinal infections caused by Gram-negative bacteria, such as severe diseases like chicken-derived pathogenic Escherichia coli infection and diarrhea caused by Escherichia coli in suckling pigs. However, the widespread and excessive use of colistin in animal husbandry has led to an increase in its drug resistance. In recent years, the emergence of plasmid-mediated horizontally transferable mcr resistance genes has attracted serious attention from scholars and has become an important issue in the fields of public health and food safety. Constructing a sensitive, accurate and reliable analytical method for the direct detection and quantification of colistin antibiotics in bacterial cells is of great significance for clarifying the antibacterial mechanism and the mechanism of drug resistance generation of colistin.

[0003] At present, there are many reported detection methods for colistin residues in animal-derived foods at home and abroad, such as microbiological method, high performance liquid chromatography, liquid chromatography-tandem mass spectrometry, capillary electrophoresis, etc., but there are few methods for the determination of colistin in bacterial matrices. High-sensitivity and high-selectivity liquid chromatography-tandem mass spectrometry is an ideal method for detecting trace compounds in complex matrices. However, colistin is a compound with surfactant properties with a hydrophilic head and a hydrophobic tail chain, and is easily affected by the environmental medium during the electrospray ionization process, and the ionization efficiency is unstable, making it difficult to obtain accurate and reliable detection results. Summary of the Invention

[0004] The present invention aims to overcome the deficiencies of the existing detection technologies and provides an analytical method for detecting colistin in bacteria. This method is based on the principle of acid hydrolysis combined with a derivatization strategy to establish a high-performance liquid chromatography-fluorescence method for the determination of colistin in bacteria with high sensitivity, good stability and strong operability.

[0005] The above object of the present invention is achieved by the following technical solutions:

[0006] An analytical method for detecting colistin in bacteria, comprising the following steps:

[0007] S1. The bacterial suspension incubated with sub-inhibitory colistin is centrifuged and washed, and repeatedly frozen and thawed with liquid nitrogen to lyse the bacterial cells;

[0008] S2. Place the lysed bacteria in an acidic solution and hydrolyze them at 60 - 120 °C in a protective atmosphere. The colistin in the bacterial cells is hydrolyzed into 2,4 - diaminobutyric acid. Then, adjust the pH to neutral with an alkaline solution and make up the volume to obtain a hydrolyzate.

[0009] S3. Add a derivatization buffer and the derivatizing agent 9 - fluorenylmethyl chloroformate to the hydrolyzate, react at 20 - 80 °C to obtain a derivatization reaction solution, and obtain a filtrate after treatment with a syringe filter membrane.

[0010] S4. Separate the filtrate through a C8 reversed - phase chromatographic column, perform gradient elution using acetonitrile A and acetic acid - ammonium acetate buffer solution B as the mobile phase, select a fluorescence detector for determination, and perform qualitative and quantitative analysis of colistin in bacteria by the standard addition method to achieve the detection of colistin in bacteria.

[0011] Preferably, the bacteria described in step S1 are Gram - negative bacteria.

[0012] More preferably, the Gram - negative bacteria are Escherichia coli, Salmonella, Pseudomonas aeruginosa or Klebsiella pneumoniae.

[0013] Preferably, the concentration of the sub - inhibitory colistin in step S1 is 0.2 - 2 μg / mL; the incubation conditions of the bacterial suspension are shaking in a shaker at 100 - 200 r / m and incubating at a constant temperature of 37 °C for 0 - 10 h; the temperature of the centrifugation is 0 - 20 °C, the speed of the centrifugation is 1000 - 12000 r / m, and the time of the centrifugation is 5 - 30 min; the washing solution is 0.8 - 1 wt% normal saline, and the number of washing times is 1 - 5 times; the time of freeze - thawing is 10 - 60 s, and the number of freeze - thawing times is 2 - 5 times.

[0014] Preferably, the acidic solution in step S2 is hydrochloric acid or sulfuric acid, and the concentration of the acidic solution is 1 - 10 moL / L; the protective atmosphere is nitrogen, helium or argon, and the hydrolysis time is 10 - 30 h.

[0015] Preferably, the alkaline solution in step S2 is sodium hydroxide solution or potassium hydroxide solution, and the concentration of the alkaline solution is 8 - 20 mol / L.

[0016] Preferably, the derivatization buffer in step S3 is 0.01 - 1 mol / L boric acid - borax buffer solution with a pH of 7.5 - 10, the concentration of the derivatizing agent 9 - fluorenylmethyl chloroformate is 1 - 10 mmol / L, and the volume ratio of the derivatization buffer to the derivatizing agent is (2 - 10):(5 - 20); the diameter of the syringe filter membrane is 0.1 - 0.5 μm, and the reaction time is 2 - 20 min.

[0017] Preferably, the gradient elution program in step S4 is 0 - 10 min, 30% A, 70% B; 10 - 30 min, 30 - 50% A, 50 - 70% B; 30 - 37 min, 50% A, 50% B; 37 - 38 min, 30 - 50% A, 50 - 70% B; 38 - 42 min, 30% A, 70% B.

[0018] Preferably, the concentration of ammonium acetate in the acetic acid - ammonium acetate buffer solution in step S4 is 2 - 50 mmol / L, and the pH of the acetic acid - ammonium acetate buffer solution is 3 - 6.5.

[0019] Application of the described analytical method in detecting colistin in bacteria.

[0020] Preferably, the colistin is a trace (1 - 100 ng / mL) veterinary antibiotic.

[0021] Colistin of the present invention is an antibiotic with a hydrophilic head and a hydrophobic tail chain, and is effective in the treatment of multi - drug - resistant Gram - negative bacterial infections. However, due to the chemical structure of colistin itself with surfactant characteristics, the stable, accurate and reliable analysis and detection of it have always been highly concerned. Colistin is hydrolyzed with hydrochloric acid to obtain the characteristic product 2,4 - diaminobutyric acid, which reacts with 9 - fluorenylmethyl chloroformate to form a stable fluorescent derivative. Based on this, on the basis of optimizing the conditions of bacterial lysis and colistin hydrolysis and derivatization reactions, a sensitive, reliable and stable high - performance liquid chromatography - fluorescence method for detecting colistin in bacteria was established. The derivative is separated by a C8 (250 mm × 4.6 mm inner diameter, 5 μm film thickness) chromatographic column and determined by a fluorescence detector.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. In the present invention, bacteria are lysed by liquid nitrogen freezing to completely release colistin. There are 5 2,4 - diaminobutyric acids in the colistin molecule. Strong acids such as hydrochloric acid or sulfuric acid are used to hydrolyze the peptide bonds in the colistin molecule, and colistin is hydrolyzed into the characteristic small molecule 2,4 - diaminobutyric acid in an acidic solution. 2,4 - diaminobutyric acid reacts with the derivatizing agent 9 - fluorenylmethyl chloroformate to form a stable fluorescent derivative 9 - fluorenylmethoxycarbonyl - 2,4 - diaminobutyric acid. The hydrolyzate emits strong fluorescence after derivatization, and the derivatization reaction conditions are mild and have good chromatographic behavior. By monitoring 9 - fluorenylmethoxycarbonyl - 2,4 - diaminobutyric acid with a high - performance liquid chromatography - fluorescence detector, the detection of colistin incubated with bacteria is realized. This method has high sensitivity and strong practical operability, providing new ideas, new means and technical support for the detection research of trace colistin (1 - 100 ng / mL).

[0024] 2. The hydrolysis process of the present invention is simple, the hydrolysis effect is complete, and the hydrolysis product 2,4-diaminobutyric acid is stable.

[0025] 3. The present invention incubates and treats Gram-negative bacteria such as Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Klebsiella pneumoniae with sub-inhibitory concentrations of colistin. On the basis of optimizing the hydrolysis of colistin in bacteria and the derivatization reaction conditions of the characteristic product 2,4-diaminobutyric acid, an analytical method for determining colistin in bacteria by high performance liquid chromatography-fluorescence method based on 9-fluorenylmethoxycarbonyl-2,4-diaminobutyric acid is constructed. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the high performance liquid chromatography diagram of 9-fluorenylmethoxycarbonyl-2,4-diaminobutyric acid;

[0027] Figure 2 is the schematic diagram of acid hydrolysis of colistin;

[0028] Figure 3 is the schematic diagram of the derivatization reaction of 2,4-diaminobutyric acid and 9-fluorenylmethyl chloroformate;

[0029] Figure 4 is the mass spectrometry diagram of the reaction product of 2,4-diaminobutyric acid and 9-fluorenylmethyl chloroformate;

[0030] Figure 5 is the schematic diagram of the mass spectrometry fragmentation pathway of the reaction product of 2,4-diaminobutyric acid and 9-fluorenylmethyl chloroformate. DETAILED DESCRIPTION OF THE INVENTION

[0031] The following further illustrates the content of the present invention with specific examples, but should not be construed as a limitation to the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0032] The instruments used in the examples are as follows: Agilent 1260 series high performance liquid chromatograph, equipped with a fluorescence detector (Agilent Technologies, USA); F-7000 fluorescence spectrophotometer (Hitachi High-Technologies International Trade Co., Ltd.); HHVE-50 autoclave (HIRAYAMA, Japan); SW-CJ double-sided single-person laminar flow hood (Suzhou Sutai Purification Equipment Engineering Co., Ltd.); constant temperature incubator (Thermo Scientific, USA); THZ-C constant temperature shaker (Guangzhou Shenhua Biotechnology Co., Ltd.); Thermo D-37520 high-speed refrigerated centrifuge (Thermo Scientific, USA); MTX-2500 mini mixer (Shanghai Qiqian Electronic Technology Co., Ltd.); SHA-B constant temperature water bath oscillator (Changzhou Aohua Instrument Co., Ltd.); DHG-9055A electric blast drying oven (Shanghai Yiheng Scientific Instrument Co., Ltd.); 208WGHB9D Haier upright freezer (Haier Group, China); Milli-Q Advantage A10 ultrapure water system (Millipore, USA); nylon 66 filter membrane with a pore size of 0.22 μm (Tianjin Jinteng Co., Ltd.).

[0033] The drugs used in the examples are as follows: MacConkey agar powder medium (containing glucose), LB agar powder medium (containing glucose) were purchased from Guangdong Huankai Microbial Science and Technology Co., Ltd.; CAMH broth medium (containing calcium chloride) was purchased from Qingdao Haibo Biotechnology Co., Ltd.; analytical pure sodium chloride, sodium hydroxide, boric acid, sodium tetraborate decahydrate, concentrated hydrochloric acid, hydroxylamine hydrochloride, methanol, n-hexane and ethyl acetate were purchased from Guangzhou Chemical Reagent Factory; chromatographic pure methanol, ammonium acetate and acetic acid were purchased from Shanghai Macklin Biochemical Co., Ltd.; colistin reference substance was purchased from Beijing Puboxin Biotechnology Co., Ltd.; 2,4-diaminobutyric acid dihydrochloride was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; 9-fluorenylmethyl chloroformate was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0034] Gram-negative bacteria such as Escherichia coli, Salmonella, and Pseudomonas aeruginosa were all preserved in our laboratory.

[0035] Selection and optimization of sample pretreatment conditions in Example 1

[0036] 1. Extraction: To explore the antibacterial mechanism of colistin, it is of great value to incubate bacteria with low-level colistin and accurately and reliably determine trace colistin in bacterial cells. The low-temperature freezing method is usually used to break bacterial cells. In this experiment, the effects of two lysis methods, repeated freezing and thawing with liquid nitrogen and freezing at -80 °C, on the detection of the target substance were investigated. The results showed that repeated freezing and thawing with liquid nitrogen could more fully and stably break the cell membrane at different concentrations of 10 ng / mL, 20 ng / mL, and 100 ng / mL, and the content of the 9-fluorenylmethoxycarbonyl-2,4-diaminobutyric acid derivative was high. Therefore, repeated freezing and thawing with liquid nitrogen was used to lyse bacterial cells in this experiment.

[0037] 2. Hydrolysis: Colistin is a lipopeptide compound formed by the condensation of several amino acids such as 2,4-diaminobutyric acid. In this invention, hydrochloric acid solution was used to digest bacteria, and colistin in bacterial cells was hydrolyzed into 2,4-diaminobutyric acid. Then, 9-fluorenylmethyl chloroformate was used to derivatize 2,4-diaminobutyric acid to produce a stable 9-fluorenylmethoxycarbonyl-2,4-diaminobutyric acid derivative with strong fluorescence, and the detection of colistin in bacteria was achieved by high-performance liquid chromatography monitoring. Therefore, to obtain the best hydrolysis effect, the hydrolysis time of colistin in bacterial samples was optimized by referring to the method of hydrolyzing amino acids in biological matrices. Under the hydrolysis conditions of 6 mol / L hydrochloric acid solution and nitrogen filling and sealing at 110 °C, the effects of different hydrolysis times (16 h, 20 h, 24 h, and 32 h) on the hydrolysis effect of colistin were evaluated. The results showed that the hydrolysis time had a great influence on the hydrolysis yield of colistin. As the hydrolysis time increased, the peak area of the derivative of 2,4-diaminobutyric acid gradually increased. When the hydrolysis time was too short (16 h and 20 h), the hydrolysis was insufficient; when hydrolyzed for 24 h, the peak area reached the maximum; when the hydrolysis time was increased (32 h), the peak area of the hydrolysis product no longer increased but decreased instead. In summary, it was finally selected to hydrolyze at 6 mol / L hydrochloric acid, 110 °C, nitrogen filling, and sealed for 24 h.

[0038] 3. Derivatization: The condensation reaction of 2,4-diaminobutyric acid with 9-fluorenylmethyl chloroformate is closely related to the pH of the boric acid-borax buffer solution (concentration 0.01 - 1 mol / L) in the reaction system, the dosage of the derivatizing agent, the derivatization time, and the derivatization temperature. The effects of these four factors on the derivatization reaction were investigated respectively to optimize the derivatization reaction conditions. The results showed that the pH value of the boric acid-borax buffer solution in the reaction system had a great influence on the derivatization reaction. As the pH increased, the derivatization efficiency increased. When the pH of the buffer solution was 8.5, the peak area of the derivatized product 9-fluorenylmethoxycarbonyl-2,4-diaminobutyric acid reached the maximum, and then began to gradually decrease. As the dosage of the derivatizing reagent increased, the peak area of the derivatized product increased almost proportionally until it reached the maximum at 125 μL and remained almost the same at 150 μL. This derivatization reaction was rapid, and the derivatization rate reached the maximum after 10 min of reaction. As the derivatization time increased further, the peak area of the derivative gradually decreased. In the range of 30 - 70 °C, first, as the derivatization temperature increased, the yield of the derivatization reaction increased. When the temperature was 50 °C, the peak area of the derivatized product reached the maximum value. Continuing to increase the reaction temperature, the peak area of the derivatized product decreased slightly, but the derivatization temperature had little effect on this reaction. Considering comprehensively, finally, the pH of the boric acid-borax buffer solution was selected as 8.5, the dosage of the derivatizing agent was 125 μL, the reaction time was 10 min, and the reaction temperature was 50 °C as the derivatization reaction conditions.

[0039] Example 2

[0040] 1. Preparation of standard solutions

[0041] Standard stock solution (1 mg / mL): Weigh an appropriate amount of 2,4-diaminobutyric acid reference substance accurately, dissolve it with a small amount of deionized water, transfer it to a 10 mL brown volumetric flask, and make up the volume with deionized water to prepare a 1 mg / mL standard stock solution. Store it in the dark in a -20 °C refrigerator, and the validity period is 6 months.

[0042] Standard working solution (10 μg / mL): After diluting the above standard stock solution, accurately pipette 1 mL into a 10 mL brown volumetric flask, dilute and make up the volume with chromatographic acetonitrile to prepare a 10 μg / mL standard working solution. Store it in the dark at -20 °C, and the validity period is 1 month. Prepare standard working solutions with appropriate concentrations by successive dilution with chromatographic acetonitrile as needed, and use them immediately after preparation.

[0043] 2. Preparation of solutions

[0044] 9-Fluorenylmethyl chloroformate derivatizing reagent (4 mmol / L): Weigh an appropriate amount of 9-fluorenylmethyl chloroformate fluorescent reagent accurately, dissolve it with a small amount of chromatographic acetonitrile, transfer it to a 10 mL volumetric flask, and make up the volume with acetonitrile to prepare a 4 mmol / L 9-fluorenylmethyl chloroformate derivatizing agent solution. Use it immediately after preparation.

[0045] Hydrochloric acid solution (6 mol / L): Measure 20 mL of concentrated hydrochloric acid, add 20 mL of ultrapure water, mix well, prepare a 6 mol / L hydrochloric acid solution, store it in the dark at -20 °C, and the validity period is 1 month.

[0046] CAMH liquid medium: Weigh 11 g of CAMH solid medium into a conical flask, dissolve it with 500 mL of deionized water, autoclave it at 121 °C, and use it after cooling.

[0047] 3. Sample pretreatment

[0048] Take 500 μL of the bacterial suspension sample (bacteria incubated with 0.5 MIC colistin for 4 h) into a 2 mL centrifuge tube, centrifuge at 5000 rpm for 10 min at 4 °C, and discard the supernatant; gently wash the bacterial pellet 4 times with 0.9% normal saline (pre-cooled at 4 °C), place it in liquid nitrogen and freeze quickly for 30 s, repeat the freeze-thaw cycle 3 times to lyse the bacterial cells; add 500 μL of 6 mol / L hydrochloric acid solution to the lysed bacterial pellet, vortex and mix well, then transfer it to a digestion tube, repeatedly fill it with nitrogen to displace the air, seal it, hydrolyze it at 110 °C for 24 h, take it out and cool it to room temperature, adjust the pH of the hydrolysis solution to neutral with 16 mol / L sodium hydroxide solution, and make up the volume to 1 mL with pure water. Pipette 100 μL of the fixed volume solution into a 2 mL centrifuge tube, add 64 μL of boric acid-borax buffer solution and 136 μL of 9-fluorenylmethyl chloroformate derivatizing agent solution, vortex and mix well, react at 40 °C for 8 min, filter it through a 0.22 μm organic filter membrane, and the filtrate is used for liquid chromatography detection.

[0049] 4. Chromatographic conditions: Use a reversed-phase chromatographic column Agilent Eclipse XDB-C8 (250 mm × 4.6 mm i.d., 5 μm), the excitation wavelength is 271 nm, the emission wavelength is 320 nm, the column temperature is 30 °C, the flow rate is 0.8 mL / min, mobile phase A is 10 mmol / L ammonium acetate solution (adjust the pH to 4.5 with acetic acid), and mobile phase B is acetonitrile. Gradient elution program: 0 - 10 min, 70% A, 30% B; 10 - 30 min, 50 - 70% A, 30 - 50% B; 30 - 37 min, 50% A, 50% B; 37 - 38 min, 50 - 70% A, 30 - 50% B.

[0050] Figure 1 For the high performance liquid chromatography of 9-fluorenylmethoxycarbonyl-2,4-diaminobutyric acid. Among them, a is the reagent blank; b is the reagent blank during the reaction process; c is the derivatizing agent blank; d is the derivatizing agent reaction process blank; e is the 2,4-diaminobutyric acid standard solution; f is the blank bacterial suspension added with 2,4-diaminobutyric acid sample (60 ng / mL). 1, 2 and 3 represent the α-substituted, γ-substituted and α,γ-disubstituted derivative products respectively). From Figure 1As can be seen, 2,4-diaminobutyric acid was derivatized with 9-fluorenylmethyl chloroformate to obtain three derivatives (1, 2, and 3) of 9-fluorenylmethoxycarbonyl-2,4-diaminobutyric acid with α-substitution, γ-substitution, and α,γ-disubstitution. They were completely separated, and the retention times were 7.6 min, 11.1 min, and 35.9 min in sequence. There were no interfering peaks near the three derivatives.

[0051] Example 3 Standard Curve, Detection Limit, and Quantification Limit

[0052] The incubated bacterial cell suspension was prepared into a blank bacterial cell solution according to the steps of bacterial lysis and hydrolysis in the sample pretreatment in Example 2. Several portions of 100 μL were taken and equal volumes of 2,4-diaminobutyric acid standard working solutions with different concentrations were added. The samples were derivatized according to the sample pretreatment in Example 2 to prepare standard working solutions with concentrations of 60, 120, 200, 300, 450, and 600 ng / mL, and then detected by machine. The concentration of the target analyte was set as the abscissa x, and the corresponding chromatographic peak area was set as the ordinate y. The two were subjected to linear regression analysis to obtain the regression equation and the correlation coefficient (r 2 ). The results showed that in the concentration range of 60 - 600 ng / mL, there was a good linear relationship between the concentration of the target analyte and its corresponding peak area. The fitted linear equation was y = 1.38x + 7.85, and the correlation coefficient (r 2 ) was 0.9968.

[0053] A suitable concentration of colistin standard working solution was added to the blank bacterial sludge (0.5 mL bacterial suspension) to prepare a series of low-concentration blank spiked samples. The samples were processed according to the hydrolysis and derivatization steps in the sample pretreatment in Example 2 and then detected by machine. The detection limit and quantification limit of the method were taken as the added concentrations corresponding to the signal-to-noise ratio (S / N) greater than or equal to 3 and 10, respectively. The results showed that the detection limit and quantification limit of the analyte in bacteria were 30 ng / mL and 60 ng / mL, respectively, indicating that the method had high sensitivity and could meet the detection requirements of colistin in bacteria.

[0054] Example 4 Accuracy and Precision

[0055] Add appropriate concentrations of colistin standard working solution to the blank bacterial sludge (0.5 mL bacterial suspension) to prepare spiked samples with three spiking levels of 60 ng / mL, 120 ng / mL, and 600 ng / mL. Treat the samples according to the hydrolysis and derivatization steps in the sample pretreatment of Example 2, and then perform on-machine detection. Using the linear equation y = 1.38x + 7.85, calculate the recovery rate and precision of each concentration spiking level. Set six parallels for each concentration level and repeat three batches. The accuracy of the method is evaluated by calculating the average recovery rate of the samples. The results are shown in Table 1. At the three spiking levels of 60, 120, and 600 ng / mL, the within-batch recovery rate of colistin in the bacterial samples is 83.8 - 112.8%, and the between-batch recovery rate is 84.3 - 103.3%. The relative standard deviations within-batch and between-batch are less than 5.1% and 8.3% respectively, indicating that this method is stable and reliable and can meet the detection requirements for the study of the antibacterial mechanism of colistin.

[0056] Table 1 Recovery rate and precision of colistin in bacterial sludge

[0057]

[0058] Application Example 1

[0059] Twenty-eight strains of Escherichia coli were selected for the application research of this method. Escherichia coli ATCC25922 was used as the quality control bacterium, and the MIC values of the strains were determined by the microbroth dilution method. Among them, the MIC values of 5 drug-resistant strains were 4 - 8 μg / mL, and the MIC values of 23 sensitive strains were 0.5 - 2 μg / mL. Incubate 28 strains of Escherichia coli with the corresponding sub-inhibitory concentration (1 / 2 MIC) of colistin, and analyze the samples according to Step 3 in Example 2.

[0060] According to the measured concentration of the colistin hydrolysis product 2,4-diaminobutyric acid and the proportional relationship between their relative molecular masses, calculate the content of colistin in the bacterial samples. That is:

[0061]

[0062] Where: C represents the mass concentration of colistin, with the unit of ng / mL;

[0063] C Dab represents the mass concentration of the 2,4-diaminobutyric acid derivative measured on the machine, with the unit of ng / mL;

[0064] M CST represents the relative molecular mass of colistin (1155.43), with the unit of g / mol;

[0065] M Dab represents the relative molecular mass of Dab (118.13), with the unit of g / mol;

[0066] K represents the dilution factor.

[0067] Figure 2 It is a schematic diagram of the acid hydrolysis of colistin; take a little colistin sulfate into a digestion tube, add 0.5 mL of 6 mol / L hydrochloric acid solution, vortex and mix well to dissolve, repeatedly fill with nitrogen to displace air, seal, acidify at 110 °C for 24 h, take out and cool to room temperature, adjust the pH to neutral, and make up the volume to 1 mL with deionized water. From Figure 2 It can be seen that there are 5 molecules of 2,4-diaminobutyric acid in the colistin molecule. Therefore, when 1 molecule of colistin is completely hydrolyzed under strong acid conditions, 5 molecules of 2,4-diaminobutyric acid can be obtained.

[0068] Figure 3 It is a schematic diagram of the derivatization reaction of 2,4-diaminobutyric acid with 9-fluorenylmethyl chloroformate; among them, Dab represents 2,4-diaminobutyric acid, FMOC-Cl represents 9-fluorenylmethyl chloroformate, and FMOC-Dab represents the derivative product. 9-fluorenylmethyl chloroformate is a commonly used amino derivatization reagent, which can react with amino groups to form stable amide bonds. Both amino groups at the α and γ positions in the structure of 2,4-diaminobutyric acid can undergo nucleophilic substitution reactions with the carbonyl group of the acyl chloride in 9-fluorenylmethyl chloroformate to generate 1 molecule of 2,4-diaminobutyric acid derivatized with 9-fluorenylmethyl chloroformate and 2 molecules of 2,4-diaminobutyric acid derivatized with 9-fluorenylmethyl chloroformate. Among them, the reaction of the amino group at the α or γ position of 2,4-diaminobutyric acid with 9-fluorenylmethyl chloroformate generates two different monosubstituted derivative products, namely α-substituted and γ-substituted derivative products. The optimized chromatographic conditions of the present invention can completely separate a total of 3 derivative products of mono- and disubstitution. There is a good linear relationship between the peak areas of the 3 peaks and the concentration of the standard solution. Quantitative analysis can be carried out by using the total area of the 3 peaks or taking a certain target peak among them. Quantitative analysis is carried out with the peak of the monosubstituted 9-fluorenylmethyl chloroformate derivative of 2,4-diaminobutyric acid at the γ position.

[0069] Figure 4 It is the mass spectrum of the reaction product of 2,4-diaminobutyric acid with 9-fluorenylmethyl chloroformate. Among them, a represents the mass spectrum of the monosubstituted derivative product, and b represents the mass spectrum of the disubstituted derivative product. The general structural formula of the target derivative generated by the reaction of 2,4-diaminobutyric acid with 9-fluorenylmethyl chloroformate is: the product derivatized with monosubstituted 9-fluorenylmethyl chloroformate is C 19 H 20 N2O4, with a molecular weight of 340.2; the product derivatized with disubstituted 9-fluorenylmethyl chloroformate is C 34 H 30N2O6, with a molecular weight of 562.2. The product obtained from the derivatization reaction of 2,4-diaminobutyric acid and 9-fluorenylmethyl chloroformate was subjected to a primary mass spectrometry scan using a tandem quadrupole mass spectrometer. In the electrospray negative ion mode, the main parent ion obtained for the derivatized product was [M-H] formed by deprotonation of the carboxyl group. - The negative ions, and the mass numbers of the mono-substituted and bis-substituted derivatized products were m / z 339.2 and m / z 561.2, respectively. Further, a full scan of the secondary mass spectrometry was performed on the parent ions m / z 339.2 and m / z 561.2 of the mono-substituted and bis-substituted derivatives. Figure 5 It is a schematic diagram of the mass spectrometry fragmentation pathway of the reaction product of 2,4-diaminobutyric acid and 9-fluorenylmethyl chloroformate. Among them, a and b respectively represent the possible cleavage modes of the α,γ mono-substituted derivatized product; c represents the possible cleavage mode of the bis-substituted derivatized product, and m / z117.0 and m / z 143.0 are the main fragment ions of both the mono-substituted and bis-substituted derivatized products, and the possible pathways and attributions of the corresponding mass spectrometry fragmentation.

[0070] In summary, the present invention is based on the liquid nitrogen freeze-thaw method to lyse bacterial cells, the lysed bacteria are placed in an acidic solution for high-temperature sealed hydrolysis, and derivatized with 9-fluorenylmethyl chloroformate, and a sensitive, stable and reliable liquid chromatography-fluorescence method for detecting trace colistin in bacteria is established. This method is economical and environmentally friendly, can simply and quickly determine the concentration of colistin in bacteria, and helps the research on the antibacterial mechanism of colistin.

[0071] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. An analytical method for detecting colistin in bacteria, characterized in that: The following steps are involved: S1. The bacterial suspension incubated with subinhibitory colistin was centrifuged, washed, and repeatedly frozen and thawed with liquid nitrogen to lyse the bacterial cells; S2. placing the lysed bacterial cells in an acidic solution and hydrolyzing them at 60-120° C. in a protective atmosphere, wherein the colistin in the bacterial cells is hydrolyzed into 2,4-diaminobutyric acid, and then adjusting the pH to neutral with an alkaline solution and fixing the volume to obtain a hydrolyzate; S3. adding a derivatization buffer and a derivatization agent 9-fluorenylmethyl chloroformate to the hydrolyzate, reacting at 20 to 80° C. to obtain a derivatization reaction solution, and treating the solution with a syringe filter to obtain a filtrate; S4. The filtrate is separated by a C8 reverse phase chromatography column, and acetonitrile A and acetic acid-ammonium acetate buffer solution B are used as mobile phases for gradient elution. A fluorescence detector is selected for determination, and the standard addition method is used for qualitative and quantitative analysis of colistin in bacteria, so as to realize the detection of colistin in bacteria.

2. The analytical method for detecting colistin in bacteria according to claim 1, characterized in that: The bacteria described in step S1 are Gram-negative bacteria.

3. The analytical method for detecting colistin in bacteria according to claim 2, characterized in that: The Gram-negative bacteria are Escherichia coli, Salmonella, Pseudomonas aeruginosa or Klebsiella pneumoniae.

4. The analytical method for detecting colistin in bacteria according to claim 1, characterized in that: The concentration of the sub-inhibitory colistin described in step S1 is 0.2-2 μg / mL; the incubation conditions of the bacterial suspension are 100-200 r / m shaker, 37°C constant temperature incubation for 0-10 h; the centrifugation temperature is 0-20°C, the centrifugation rate is 1000-12000 r / m, and the centrifugation time is 5-30 min; the washing solution is 0.8-1wt% physiological saline, and the number of washings is 1-5 times; the freeze-thaw time is 10-60s, and the number of freeze-thaw cycles is 2-5 times.

5. The analytical method for detecting colistin in bacteria according to claim 1, characterized in that: The acidic solution described in step S2 is hydrochloric acid or sulfuric acid, and the concentration of the acidic solution is 1-10 mol / L; the protective atmosphere is nitrogen, helium or argon, and the hydrolysis time is 10-30 hours.

6. The analytical method for detecting colistin in bacteria according to claim 1, characterized in that: The alkaline solution in step S2 is a sodium hydroxide solution or a potassium hydroxide solution, and the concentration of the alkaline solution is 8-20 mol / L.

7. The analytical method for detecting colistin in bacteria according to claim 1, characterized in that: The derivatization buffer in step S3 is a 0.01-1 mol / L boric acid-borax buffer solution with a pH of 7.5-10, the concentration of the derivatization agent 9-fluorenylmethyl chloroformate is 1-10 mmol / L, and the volume ratio of the derivatization buffer to the derivatization agent is (2-10):(5-20); the diameter of the needle filter membrane is 0.1-0.5 μm; and the reaction time is 2-20 min.

8. The analytical method for detecting colistin in bacteria according to claim 1, characterized in that: The gradient elution procedure described in step S4 is 0-10 min, 30% A, 70% B; 10-30 min, 30-50% A, 50-70% B; 30-37 min, 50% A, 50% B; 37-38 min, 30-50% A, 50-70% B; 38-42 min, 30% A, 70% B.

9. The analytical method for detecting colistin in bacteria according to claim 1, characterized in that: The concentration of ammonium acetate in the acetic acid-ammonium acetate buffer solution in step S4 is 2 to 50 mmol / L, and the pH of the acetic acid-ammonium acetate buffer solution is 3 to 6.

5.

10. Use of the analytical method according to any one of claims 1 to 9 in detecting colistin in bacteria.

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