CN111474278A - Method and kit for detecting metabolites of macrolide compounds - Google Patents
Method and kit for detecting metabolites of macrolide compounds Download PDFInfo
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- G—PHYSICS
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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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The invention discloses a method and a kit for detecting metabolites of macrolide compounds, wherein the method for detecting the metabolites of the macrolide compounds comprises the steps of extracting and purifying a sample to be detected so as to obtain a liquid to be detected, and detecting by using an ultra-high performance liquid chromatography and quadrupole electrostatic field orbital trap Fourier transform mass spectrometry (UHP L C-Q-active-Orbitrap mass spectrometry) system so as to qualitatively and/or quantitatively analyze the macrolide metabolites.
Description
Technical Field
The present invention relates to the field of analytical chemistry, in particular to methods and kits for detecting metabolites of macrolide compounds.
Background
Macrolides are broad spectrum antibiotics produced by streptomyces, primarily used in clinical and animal bacterial infections. However, abuse or improper addition can result in a variety of toxic side effects, such as: digestive tract symptoms, hepatotoxicity, allergies, cardiotoxicity, arrhythmia and condyloma acuminata. These compounds readily accumulate in animals. Moreover, it is easily transmitted into the human body through the food chain, and poses a threat to the consumer. Macrolide antibiotics in food have become a major concern for food safety research today.
At present, 3 metabolites are reported to be found in domestic and industrial wastewater for the first time, the concentration of the 3 metabolites is as high as 20.1 mu g/L, and the 3 metabolites may indirectly pollute crops and food, wherein the 3 metabolites comprise N-demethyl romycin, N-demethyl clarithromycin and erythromycin A oxime, and have toxicity and ecological toxicological effects due to the similarity of the structure of the parent drug.
Although the drug concentration of the 3 emerging metabolites in agricultural products and food is low, the toxicity is significant. At the same time, coupled with the complexity of the matrix, sample purification methods and high sensitivity instrumental analysis methods are extremely challenging. Therefore, there is a need to develop a method to study the possible migration residues of these 3 compounds to effectively control the migration of antibiotics in food products.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. To this end, it is an object of the present invention to propose a method for detecting metabolites of macrolide compounds, which is simple to operate, has high sensitivity and recovery and reproducibility, and is particularly suitable for the metabolites of 3 emerging macrolide compounds (erythromycin, clarithromycin and roxithromycin).
According to one aspect of the invention, the method comprises the steps of carrying out extraction treatment and purification treatment on a sample to be detected so as to obtain a liquid to be detected, and carrying out detection by using an ultra-high performance liquid chromatography and quadrupole electrostatic field orbital trap Fourier transform mass spectrometry (UHP L C-Q-active-Orbitrap mass spectrometry) system so as to carry out qualitative analysis and/or quantitative analysis on the macrolide metabolites.
The term "metabolites of macrolide compounds" as used herein means that the chemical structure of macrolide antibiotic compounds is changed by various drug-metabolizing enzymes (especially liver enzymes) in vivo, and then the compounds are biologically converted into pharmacologically active metabolites or toxic metabolites in vivo, for example, three metabolites of erythromycin A oxime, N-desmethylclarithromycin and N-desmethylroxithromycin are the metabolites of three macrolide compounds of erythromycin, clarithromycin and roxithromycin.
According to the method for detecting the metabolites of the macrolide compounds, which is disclosed by the embodiment of the invention, an ultra-high performance liquid chromatography and quadrupole electrostatic field orbit trap Fourier transform combined mass spectrometry system are adopted for detection, so that the metabolites of the macrolide compounds are well detected, the sensitivity, the recovery rate and the reproducibility are high, the analysis speed is high, the using amount of a solvent is small, the cost is low, the operation is simple and convenient, a device used in the sample preparation process is simple, and the method is particularly suitable for accurately determining the residual content of the metabolites of the macrolide compounds in substances such as food and the like.
According to an embodiment of the present invention, the sample to be tested is a complex substrate sample of food, such as milk. For a sample with a complex matrix, the inventor finds that impurities are adsorbed by extracting treatment and purification treatment and utilizing the interaction between an adsorbent and the impurities in the matrix, so that impurity removal and purification are achieved, the interference of the complex matrix on the detection of a compound to be detected can be quickly and simply removed, the sample treatment procedure is simplified, and the sensitivity and the accuracy of the detection are improved.
According to an embodiment of the present invention, the extraction process includes: mixing the sample to be tested with an extracting agent so as to obtain a first mixture; subjecting the first mixture to ultrasonication so as to obtain a second mixture; the second mixture is centrifuged to obtain a first supernatant. Therefore, the sensitivity and the recovery rate of detection are improved.
According to an embodiment of the invention, the purification process comprises: mixing the first supernatant with a scavenger so as to obtain a third mixture; subjecting the third mixture to ultrasonication so as to obtain a fourth mixture; subjecting the fourth mixture to standing separation to obtain a second supernatant; and drying and filtering the second supernatant to obtain the liquid to be detected. Therefore, the sensitivity and the recovery rate of detection are improved.
According to the embodiment of the invention, the extracting agent is a mixture of 0.05-0.1% formic acid-acetonitrile, anhydrous magnesium sulfate and sodium chloride, wherein the 0.05-0.1% formic acid-acetonitrile can quickly precipitate proteins in a milk sample, the magnesium sulfate can remove most of water in the milk, and the sodium chloride can separate the water remained in the milk from an acetonitrile solution.
According to an embodiment of the invention, the scavenger is a mixture of sodium acetate, N-propylethylenediamine and C18. The inventor screens a plurality of purifiers, and researches show that the purifying effect and the recovery rate of the sodium acetate, the N-propyl ethylenediamine and the C18 are better for macrolide metabolites, and the sensitivity, the accuracy and the recovery rate of the detection are favorably improved.
According to the embodiment of the invention, the mass ratio of the sodium acetate, the N-propyl ethylene diamine and the C18 is 10-20: 1-4. When the three adsorbent materials are used in the amount, the sample matrix effect can be obviously reduced, impurities can be effectively removed, the target object is not adsorbed, and meanwhile, the three adsorbents are relatively minimum in amount, and the recovery rate of the target object is high.
According to the embodiment of the invention, the chromatographic conditions of the ultra-high performance liquid chromatography comprise a chromatographic column C18 chromatographic column with the specification of 2.1 × 100mm and 3.5 microns, the sample injection temperature of 30 ℃, the sample temperature of 10 ℃, the sample injection amount of 5.0 microns L and the flow rate of 0.2m L/min, so that the chromatographic separation effect is good, and the detection accuracy and sensitivity are high.
According to an embodiment of the invention, the mass spectrometric conditions of the mass spectrum comprise: an ion source: a source of HESI; capillary temperature: 350 ℃; mist voltage: 3.6 KV; sheath gas: 40 psi; auxiliary gas: 10 arb; temperature of the heater: 350 ℃; and (3) a mass spectrum detection mode: full-ms and dd-ms 2. Wherein, the resolution of the primary mass spectrum is 70000, and the scanning resolution of the secondary mass spectrum is 35000. Meanwhile, Full scanning can be carried out on the target in the sample by Full scanning in Full time in Full-time, the ionization mode of the target under the ionization condition of the HESI source is found out, the scanning mass number of the parent ion is determined, and dd-ms2Further ionizing the mass-to-charge ratio of the target object, selecting 5 daughter ions with the highest mass-to-charge response, and simultaneously reducing mutual interference of ions with similar mass numbers due to higher mass-to-charge resolution, wherein the detection results of some embodiments of the invention can be shown in Table 1
According to the embodiment of the invention, the eluent of the chromatogram is A: 0.05-0.15% formic acid-water (10 mmol/L ammonium acetate), B: acetonitrile, so that the analysis time is short, the chromatographic peak shape is good, the resolution is high, the ionization efficiency of the object to be detected is improved, the detection sensitivity is improved obviously, therefore, the metabolite of the macrolide antibiotic can be separated within 10-15min, the peak shape is relatively good, and the qualitative and quantitative analysis of the target object is facilitated, and the result is shown in figure 2.
According to an embodiment of the invention, the elution conditions of the chromatography are: 10% of B, 0-1 min; 10% -98% of B, 1-15 minutes; 98% B, 15-20 minutes; 98-10% of B, 20-21 minutes; 10% B, 21-25 min. Therefore, the macrolide metabolites have good separation effect and appropriate detection time.
According to an embodiment of the invention, the metabolites comprise: erythromycin A oxime, N-demethylclarithromycin and N-demethylroxithromycin, and the three metabolites are metabolites of three macrolides, namely erythromycin, clarithromycin and roxithromycin.
According to another aspect of the invention, the invention provides a kit for the aforementioned method for detecting metabolites of macrolide compounds, according to an embodiment of the invention, the kit comprises a first extractant of 0.05-0.1% formic acid-acetonitrile, a second extractant of anhydrous magnesium sulfate, a third extractant of sodium chloride, a first purifying agent of sodium acetate, a second purifying agent of N-propyl ethylenediamine, a third purifying agent of C18, a chromatographic column of C18, the specification of which is 2.1 × 100mm and 3.5 μm.
According to the kit provided by the embodiment of the invention, the extracting agent and the purifying agent are adopted to carry out extraction and purification treatment on a sample, the treatment is simple, the speed is high, the interference of a complex matrix on detection can be effectively removed, the ultra-high performance liquid chromatography and a quadrupole electrostatic field orbit trap Fourier transform coupling mass spectrometry system are combined for detection, the detection effect on macrolide metabolites is good, the sensitivity, the recovery rate and the reproducibility are high, and the kit can be used for accurately determining the residual content of the macrolide metabolites in substances such as food and the like.
According to an embodiment of the invention, the kit further comprises: redissolving a reagent: methanol; standard solution: the standard solution is selected from at least one of erythromycin A oxime, N-demethyl roxithromycin and N-demethyl clarithromycin.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows a single channel chromatographic schematic of a macrolide metabolite (100 μ g/L) according to one embodiment of the present invention;
FIG. 2 is a graph showing the effect of different extraction solvents on the extraction of macrolide metabolite components according to an embodiment of the present invention;
FIG. 3 shows a comparative schematic of target recovery for extraction solvent at different volumes, according to one embodiment of the present invention;
fig. 4 is a graph showing comparison of the adsorption effect of 3 adsorbents according to one embodiment of the present invention on a target.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
It should be noted that the terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Further, in the description of the present invention, "a plurality" means two or more unless otherwise specified.
To facilitate an understanding of the foregoing method for detecting macrolide metabolites, the general steps of the method are provided herein, specifically as follows:
(1) sample extraction and purification:
(a) the extraction method comprises the following steps: accurately weighing 5.00 +/-0.05 g of milk sample, placing the milk sample in a centrifuge tube, adding an extraction reagent A, an extraction reagent B and an extraction reagent C, carrying out vortex, ultrasonic treatment and centrifugation (4 ℃, 10000r/min) to obtain supernatant.
(b) The purification method comprises the following steps: transferring the supernatant to a second centrifuge tube, adding a purifying reagent A, a purifying reagent B and a purifying reagent C, performing vortex for 1min, performing ultrasonic treatment for 1min, blowing the obtained supernatant with nitrogen at 40 ℃, and filtering with a filter membrane.
(2) Preparing a macrolide metabolite mixed standard solution with a concentration gradient:
(a) precision pipetting 0.1, 0.2, 0.5, 1, 2, 5, 10,20,50,100,200 μ L into 10 μ g/m L mix standard a to 10m L volumetric flasks, respectively.
(b) Diluting with methanol, diluting to 10m L, and mixing to obtain 0.1, 0.2, 0.5, 1, 2, 5, 10,20,50,100,200ng/m L standard curve solution.
(3) The standard solution and the purified sample were combined for UHP L C-Q-Orbitrap mass spectrometry.
(a) The chromatographic conditions are that the chromatographic column is XBridge-C18(2.1 × 100mm, 3.5 mu m, Waters), the sample injection temperature is 30 ℃, the sample temperature is kept at 10 ℃, the sample injection amount is 5.0 mu L, the flow rate is 0.2m L/min, and the liquid phase gradient elution procedure is that 10 percent of B (0-1min), 10 percent of B (1-15min), 98 percent of B (15-20min), 98 percent of B (20-21min) and 10 percent of B (21-25 min).
(b) The mass spectrum conditions are as follows: the ion source is a HESI source; the capillary temperature is 350 ℃; the spraying voltage is set to be 3.6 KV; sheath gas 40 psi; the auxiliary gas is 10 arb; the heater temperature was 350 ℃. And (3) a mass spectrum detection mode: full-ms and dd-ms 2.
(4) The macrolide metabolite compounds in the samples were quantified using a standard curve.
(a) And taking the peak area (Y) of the extracted ion chromatographic peak of the standard substance mixed solution as a vertical coordinate, and taking the mass concentration (X) as a horizontal coordinate to establish a linear equation.
(b) And (3) carrying out quantitative determination on the actual sample by adopting an external standard method, namely carrying out quantitative analysis on the macrolide metabolite compounds in the sample by using a prepared standard curve.
The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or apparatus used are conventional products which are commercially available, e.g. from Sigma, without reference to the manufacturer.
Example 1
In this embodiment, the milk sample is detected by detecting 3 macrolide metabolites according to the embodiment of the present invention, and the 3 macrolide compounds are erythromycin a oxime, N-desmethylclarithromycin, and N-desmethylroxithromycin, respectively, according to the following specific methods:
1. experimental methods
(1) Extraction and purification of samples:
(a) accurately weighing 5g of milk sample, placing in a 50m L centrifuge tube, adding extraction reagent (0.1% formic acid-acetonitrile, 10m L, anhydrous magnesium sulfate, 400mg, sodium chloride, 2.0g), vortexing for 1min, mixing, performing ultrasonic treatment for one minute, and centrifuging (4 ℃, 10000r/min) for 10 min.
(b) The supernatant was extracted 10m L into a second 50m L centrifuge tube, then sodium acetate (1.0g), N-propylethylenediamine (10mg) and C18(120mg) were added to the second centrifuge tube, vortexed for 1min and mixed.
(c) At 40 ℃ nitrogen was blown and redissolved in 1ml methanol.
(2) Testing on a machine: filtering with 0.22 μm nylon filter membrane, and detecting.
(3) Conditions of the apparatus
The chromatographic conditions comprise that the chromatographic column comprises XBridge-C18(2.1 × 100mm, 3.5 mu m and Waters), the sample injection temperature is 30 ℃, the sample temperature is kept at 10 ℃, the sample injection amount is 5.0 mu L, the flow rate is 0.2m L/min, the eluent of liquid phase gradient comprises A, 0.1 percent of formic acid and B, acetonitrile, and the elution procedures comprise 10 percent of B (0-1min), 10 percent of B (1-15min), 98 percent of B (15-20min), 98 percent of B (20-21min) and 10 percent of B (21-25 min).
Mass spectrum conditions: mass spectrum conditions: the ion source is a HESI source; the capillary temperature is 350 ℃; the spraying voltage is set to be 3.6 KV; sheath gas 40 psi; the auxiliary gas is 10 arb; the temperature of the heater is 350 ℃; and (3) a mass spectrum detection mode: full-ms and dd-ms 2.
(4) Preparing a standard solution, and measuring a standard curve, a detection limit and a quantification limit.
Accurately weighing 10.0 +/-0.1 mg of single macrolide metabolite standard substance, respectively placing the single macrolide metabolite standard substance into 10m L volumetric flasks, adding a little methanol, uniformly mixing, dissolving the standard substance, adding methanol to a constant volume of 10m L to obtain a single macrolide metabolite standard substance stock solution of 1mg/m L, respectively precisely transferring the single macrolide metabolite standard substance stock solution into 10 mu L-10 m L volumetric flasks, diluting with methanol and fixing the volume to 10m L to obtain a macrolide metabolite mixed standard solution A of 1 mu g/m L, and placing the macrolide metabolite mixed standard solution A in a refrigerator at 4 ℃ for storage for later use.
A mixed mass concentration standard working solution is added into blank milk, so that the addition levels of L OQ, 2L OQ and 4L OQ are the addition levels of three mass concentrations, each addition concentration is processed and analyzed by the method, and the recovery rate and the day-to-day precision are calculated.
2. Results and analysis:
(1) selection of chromatographic conditions
In order to increase the detection sensitivity of analytes, methanol-water and acetonitrile-water solutions, methanol-0.1% (v/v) formic acid-water and acetonitrile-0.1% (v/v) formic acid-water, acetonitrile-0.1% (v/v) formic acid-water (2, 5, 10,20 mmol/L ammonium acetate) are used for the experiments.
Since the strong elution power of acetonitrile-water can be significantly advanced by the retention time of the target and the peak width is reduced when methanol-water and acetonitrile-water are used as mobile phases, however, the response of 3 metabolites is not very high, therefore, this example further adds formic acid to enhance the mass spectral response, 0.1% (v/v) formic acid-methanol solution and 0.1% (v/v) formic acid-acetonitrile solution are used to optimize the liquid phase conditions, and the results show that the mass spectral response is increased by one order of magnitude, since the mass spectral response of 0.1% (v/v) formic acid-acetonitrile solution is higher than that of 0.1% (v/v) formic acid-methanol solution, 0.1% (v/v) formic acid-acetonitrile solution is selected as mobile phase under which N-desmethylclarithromycin has a slight micro-peaked phenomenon, and therefore in order to improve the peak shape, a series of concentrations of ammonium acetate (2, 5, 10,20 mmol/L) are added to the mobile phase as the concentration of ammonium acetate increases, the peak is significantly improved, and the peak shape is better extracted as the ion flow chromatogram of the target ammonium acetate (0.10 mmol) in the milk sample, and the ion flow chromatogram is preferably selected as shown in fig. 3-acetonitrile-3 sample.
TABLE 13 Mass Spectrometry parameters for macrolide metabolite Compounds
(3) Optimization of sample extraction solvent
In the analytical procedure of this example, Acetonitrile (ACN) containing 0.1% formic acid, methanol, acetonitrile containing 10mM ammonium acetate, and the results of recovery from extraction are shown in FIG. 2. it can be observed that the recovery of 3 macrolide metabolites in methanol is less than 50%, and that the recovery of these three compounds is improved after addition of 0.1% formic acid or 10 mmol/L ammonium acetate, since 0.05% -0.15% formic acid-acetonitrile is the extraction solvent, the recovery of the three compounds is the highest, all above 85.3%, and therefore, 0.1% formic acid-acetonitrile is selected as the preferred extraction solvent.
(4) Optimization of sample extraction solvent volume
The extraction efficiency of 5, 10, 15 and 20m L0.1% formic acid and acetonitrile solution was investigated, and as a result, as shown in fig. 3, the extraction efficiency of 10-15m L acetonitrile was reasonable, and 10m L was selected for solvent saving.
(5) Optimization of sample purification conditions
RSM has been used to assess the effect of various factors and levels on response variables. The CCD of RSM is used to design and optimize some extraction and purification methods for macrolides. In order to reduce the interference of matrix effects on recovery, various purification materials were selected according to the structure of macrolides, such as C18, PSA and NaAC. In order to determine the optimal conditions for the purification of macrolide samples and the relationship between recovery and these two variables, analysis of variance was performed by a combination test of two parameters. Erythromycin a oxime was chosen as an example to calculate the regression coefficient values. In linear, two-factor interaction (2FI), quadratic and cubic polynomials of response, the coefficient constants X1, X2, X3X12，X22，X32Is significant at a level of p < 0.05. Thus, after determining the recovery of response under 13 sets of conditions, the regression coefficients for recovery were calculated by RSREG analysis and a polynomial regression model equation was fitted as follows: y ═ 92.311+ 9.20724X 1+ 0.029725X 2+ 5.33897E-003X 3-1.9955X 1X 2+ 3.4159X 1X 3+ 1.8300X 2X 3-18.122X 12-1.9776X 22-3.0315X 32. Based on the regression coefficients and p-values, we concluded that the linear and quadratic terms of the amounts of C18(X1), PSA (X2), NaAC (X3), the maximum recovery corresponds to C18, PSA and NaAC with different variables, the results are shown in fig. 4.
(6) Matrix effect
Matrix co-extracts have matrix enhancing or matrix inhibiting effect on the ionization of target substances in complex matrix samples detected by liquid chromatography-mass spectrometry, thus affecting the quantitative determination of target substances according to the formula ME (%) (1-Ss/Sm) × 100 Matrix Effect (ME) is calculated as weak matrix effect when ME-20% to 20%, medium matrix effect when ME-50% to-20% or 20% to 50%, strong matrix effect when ME ═ 50% or > 50%, wherein Ss and Sm have response values for target in pure solvent and for the same content of target added to the sample matrix.
(7) Linear range, detection limit and quantitation limit of detection methods
The method uses a matrix-matched calibration standard linearly, over 11 concentration levels, over a range of 1-200ng/ml curves (0.1, 0.2, 0.5, 1, 2, 5, 10,20,50,100,200 ng/ml)). The linear regression coefficients (r) of all matrix-matched calibration curves were in each case greater than 0.9995, see table 2.
(8) Detection method precision and recovery rate
Recovery and accuracy were assessed by treating three levels (20,50, 100. mu.g/kg) of independent spiked samples on three different days. As shown in table 2, the recovery of all analytes was 85.3% -117.7% with a precision of less than 4.5%, indicating good reproducibility and reproducibility. See table 2 for details.
TABLE 2 verification parameters of the milk sample method developed
(9) Determination of actual samples
In the final phase of the study, the developed method was applied to the examination of 20 milks obtained from the retail market in China the results obtained are listed in Table 3. traces of macrolides (< L OQ) were observed in the three samples.
TABLE 3 residual concentrations of macrolide antibiotics and metabolites found in the actual samples (. mu.g/kg)
TABLE 3 concentration of macrolide metabolites found in the samples (. mu.g/kg)
The embodiment of the invention adopts UHP L C-Q-active-Orbitrap mass spectrometry to develop an analysis method, which is used for simultaneously extracting 3 macrolide metabolites in milk in a positive ion mode and a negative ion mode and expanding the application range of detecting the macrolide drugs in the milk sample by the QuEChERS, establishes optimized sample processing conditions by RSM and is used for optimizing the extraction and removal of the 3 macrolide metabolites in the milk.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (10)
1. A method of detecting a metabolite of a macrolide compound, comprising:
extracting and purifying a sample to be detected so as to obtain a liquid to be detected;
and detecting by using an ultra-high performance liquid chromatography and quadrupole electrostatic field orbit trap Fourier transform coupled mass spectrometry system so as to perform qualitative analysis and/or quantitative analysis on the macrolide metabolites.
2. The method of claim 1, wherein the extraction process comprises:
mixing the sample to be tested with an extracting agent so as to obtain a first mixture;
subjecting the first mixture to ultrasonication so as to obtain a second mixture;
the second mixture is centrifuged to obtain a first supernatant.
3. The method of claim 2, wherein the decontamination process comprises:
mixing the first supernatant with a scavenger so as to obtain a third mixture;
subjecting the third mixture to ultrasonication so as to obtain a fourth mixture;
subjecting the fourth mixture to standing separation to obtain a second supernatant;
and drying and filtering the second supernatant to obtain the liquid to be detected.
4. The method of claim 2, wherein the extractant is a mixture of 0.05% to 0.1% formic acid-acetonitrile, anhydrous magnesium sulfate, and sodium chloride,
optionally, 300-500mg anhydrous magnesium sulfate and 1-3g sodium chloride based on 10m L0.05-0.05% -0.15% formic acid-acetonitrile.
5. The method of claim 2, wherein the scavenger is a mixture of sodium acetate, N-propylethylenediamine and C18,
optionally, the mass ratio of the sodium acetate to the N-propyl ethylene diamine to the C18 is (10-20) to 1 to (1-4).
6. The method of claim 1, wherein the chromatographic conditions of ultra-high performance liquid chromatography comprise:
the chromatographic column is a C18 chromatographic column with the specification of 2.1 × 100mm and the diameter of 3.5 mu m;
sample introduction temperature: 30 ℃;
sample temperature: 10 ℃;
the sample injection amount is 5.0 mu L;
the flow rate is 0.2m L/min.
7. The method of claim 1, wherein the mass spectrometry conditions of the mass spectrometer comprise:
an ion source: a source of HESI;
capillary temperature: 350 ℃;
spraying voltage: 3.6 KV;
sheath gas: 40 psi;
auxiliary gas: 10 arb;
temperature of the heater: 350 ℃;
and (3) a mass spectrum detection mode: full-ms and dd-ms 2.
8. The method of claim 4, wherein the eluent for chromatography is A: 0.1% formic acid, B: the reaction mixture of acetonitrile and water is mixed,
optionally, the elution conditions of the chromatography are: 10% of B, 0-1 min; 10% -98% of B, 1-15 minutes; 98% B, 15-20 minutes; 98-10% of B, 20-21 minutes; 10% B, 21-25 min.
9. The method of claim 1, wherein the metabolites comprise: erythromycin A oxime, N-desmethylclarithromycin, and N-desmethylroxithromycin.
10. A kit for use in a method for detecting a metabolite of a macrolide compound according to any one of claims 1 to 9, characterized in that it comprises:
a first extractant: 0.05% -0.1% formic acid-acetonitrile;
a second extractant: anhydrous magnesium sulfate;
a third extractant: sodium chloride;
the first purifying agent: sodium acetate;
the second purifying agent: n-propyl ethylenediamine;
the third purifying agent: c18;
chromatographic column C18 chromatographic column with specification of 2.1 × 100mm and 3.5 μm,
optionally, further comprising:
redissolving a reagent: methanol;
standard solution: the standard solution is selected from at least one of erythromycin A oxime, N-demethyl roxithromycin, N-demethyl clarithromycin.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114858953A (en) * | 2022-05-20 | 2022-08-05 | 大连工业大学 | Detection kit and detection method for erythromycin and degradation products in animal-derived food |
| CN114858953B (en) * | 2022-05-20 | 2023-11-21 | 大连工业大学 | Detection kit and detection method for erythromycin and degradation products in animal-derived food |
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| CN111474278B (en) | 2022-11-01 |
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