CN111474278B - Method and kit for detecting metabolites of macrolide compounds - Google Patents

Abstract

The invention discloses a method and a kit for detecting a metabolite of a macrolide compound, wherein the method for detecting the metabolite of the macrolide compound comprises the following steps: extracting and purifying a sample to be detected so as to obtain a liquid to be detected; detecting by using an ultra-high performance liquid chromatography and quadrupole electrostatic field orbit trap Fourier transform combined mass spectrometry system (UHPLC-Q-active-Orbitrap mass spectrometry combined system) so as to perform qualitative analysis and/or quantitative analysis on the macrolide metabolites. The method adopts a combination system of ultra-high performance liquid chromatography and quadrupole electrostatic field orbit trap Fourier transform for detection, has high sensitivity, recovery rate and reproducibility, and can be used for accurately measuring the residue content of macrolide metabolites in substances such as food and the like.

Description

Method and kit for detecting metabolites of macrolide compounds

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 for 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 consumers. 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 metabolites is as high as 20.1 mug/L, and the metabolites can indirectly pollute crops and food. Includes N-demethyl rokitamycin, N-demethyl clarithromycin, erythromycin A oxime. These 3 metabolites also have some toxic and ecotoxicological effects due to their structural similarity to the parent drug. To date, no residual assay method has been developed to study these 3 metabolites in food products.

Although the drug concentration of these 3 emerging metabolites in agricultural and food products 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, there is provided a method of detecting a metabolite of a macrolide compound. According to an embodiment of the invention, the method comprises: extracting and purifying a sample to be detected so as to obtain a liquid to be detected; detecting by using an ultra-high performance liquid chromatography and quadrupole electrostatic field orbit trap Fourier transform combined mass spectrometry system (UHPLC-Q-active-Orbitrap mass spectrometry combined system) so as to perform qualitative analysis and/or quantitative analysis on the macrolide metabolites.

The term "metabolite of macrolide compound" as used herein refers to a macrolide antibiotic compound which undergoes a chemical structural change under the action of various drug-metabolizing enzymes (especially liver enzymes) in vivo and is then biotransformed into pharmacologically active metabolites or toxic metabolites in vivo, for example, erythromycin A oxime, N-desmethylclarithromycin and N-desmethylroxithromycin are the metabolites of three macrolide compounds, erythromycin, clarithromycin and roxithromycin.

According to the method for detecting the metabolites of the macrolide compounds, disclosed by the embodiment of the invention, an ultra-high performance liquid chromatography and quadrupole electrostatic field orbitrap Fourier transform combined mass spectrometry system is adopted for detection, 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 a 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 food and other substances.

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 so as to obtain the liquid to be detected. Therefore, the sensitivity and the recovery rate of detection are improved.

According to an embodiment of the invention, the extractant is a mixture of 0.05% to 0.1% formic acid-acetonitrile, anhydrous magnesium sulfate and sodium chloride. Wherein, 0.05% -0.1% formic acid-acetonitrile can quickly precipitate protein in milk sample, magnesium sulfate can remove most water in milk, and sodium chloride can separate residual water in milk from acetonitrile solution. When the three are used as an extracting agent together, the target in the sample can be quickly extracted, and the extraction time is shortened. Meanwhile, the price of the reagent is low. According to an example of the present invention, formic acid-acetonitrile was contained in an amount of 300 to 500mg of anhydrous magnesium sulfate and 1 to 3g of sodium chloride based on 10mL of 0.05% to 0.1% (v/v). Therefore, the extraction is carried out by using the extractant with the proportion, the extraction effect is good, and the speed is high.

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 purificant and discovers that the purification effect and the recovery rate of sodium acetate, N-propyl ethylenediamine and C18 are better for macrolide metabolites, and the sensitivity, the accuracy and the recovery rate of 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 an embodiment of the present invention, the chromatographic conditions of the ultra high performance liquid chromatography comprise: a chromatographic column: c18 chromatographic column with specification of 2.1 × 100mm and 3.5 μm; sample introduction temperature: 30 ℃; temperature of the sample: 10 ℃; sample introduction amount: 5.0 mu L; flow rate: 0.2mL/min. Therefore, 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: 3.6KV; sheath gas: 40psi; auxiliary gas: 10arb; temperature of the heater: 350 ℃; a mass spectrum detection mode: full-ms and dd-ms2. 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 an embodiment of the invention, the eluent of the chromatography is a:0.05% -0.15% formic acid-water (10 mmol/L ammonium acetate), B: and (3) acetonitrile. Therefore, the analysis time is short, the chromatographic peak shape is good, and the resolution is high. The ionization efficiency of the object to be detected obviously improves the detection sensitivity. Therefore, the metabolites of macrolide antibiotics can be separated within 10-15min, the peak shape is relatively good, and qualitative and quantitative analysis of target substances is facilitated. The results are shown in FIG. 2.

According to an embodiment of the invention, the elution conditions of the chromatography are: 10% B,0-1 min; 10% -98% b,1-15 minutes; 98% by weight B,15-20 minutes; 98% -10% of B,20-21 minutes; 10% by weight B,21-25 minutes. 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 a further aspect of the invention, there is provided a kit for use in the aforementioned method of detecting a metabolite of a macrolide compound. According to an embodiment of the invention, the kit 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; a second purifying agent: n-propylethylenediamine; the third purifying agent: c18; and (3) chromatographic column: c18 chromatographic column with specification of 2.1X 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 detection is carried out by combining the ultra-high performance liquid chromatography and the quadrupole electrostatic field orbital trap Fourier transform coupling mass spectrometry system, 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. According to some embodiments of the invention, the kit provided by the embodiments of the invention is used for detecting 3 macrolide antibiotic metabolites, the linearity is good in the range of 1-200 [ mu ] g/L, the correlation coefficient is greater than 0.99, the recovery rate of the target can reach 83.4% -117.7%, the detection limit and the quantification limit are 0.10-0.5 [ mu ] g/kg and 0.5-2.0 [ mu ] g/kg respectively, and the method precision is 2.6% -14.6%. The results are shown in Table 2.

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 macrolide metabolites (100. Mu.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 a 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 ℃,10000 r/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) 0.1,0.2,0.5,1,2,5, 10,20,50,100, 200. Mu.L of 10. Mu.g/mL mixing standard A to 10mL volumetric flasks, respectively, were precision pipetted.

(b) Diluting with methanol, diluting to 10mL, and mixing to obtain 0.1,0.2,0.5,1,2,5, 10,20,50,100,200ng/mL standard curve solution.

(3) The standard solution and the purified sample were mixed and subjected to UHPLC-Q-Orbitrap mass spectrometry.

(a) The chromatographic conditions are as follows: a chromatographic column: xbridge-C18 (2.1X 100mm,3.5 μm, waters); the sample introduction temperature is 30 ℃; the sample temperature was maintained at 10 ℃. Sample introduction amount: 5.0 mu L; flow rate: 0.2mL/min. The liquid phase gradient elution procedure was: 10% of B (0-1 min), 10% -98% of B (1-15 min), 98% of B (15-20 min), 98% -10% of B (20-21 min)), 10% 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 spray voltage was set to 3.6KV; sheath gas 40psi; the auxiliary gas is 10arb; the heater temperature was 350 ℃. And (3) a mass spectrum detection mode: full-ms and dd-ms2.

(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 50mL centrifuge tube, adding an extraction reagent (0.1% formic acid-acetonitrile, 10mL, anhydrous magnesium sulfate, 400mg, sodium chloride, 2.0 g), vortexing for 1min, mixing uniformly, performing ultrasonic treatment for one minute, and centrifuging (4 ℃,10000 r/min) for 10min.

(b) 10mL of the supernatant was extracted into a second 50mL centrifuge tube, then sodium acetate (1.0 g), N-propylethylenediamine (10 mg) and C18 (120 mg) were added to the second tube, vortexed for 1min and mixed well.

(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

Chromatographic conditions are as follows: and (3) chromatographic column: and (3) chromatographic column: XBridge-C18 (2.1X 100mm,3.5 μm, waters); the sample introduction temperature is 30 ℃; the sample temperature was maintained at 10 ℃. Sample injection amount: 5.0 mu L; flow rate: 0.2mL/min. Eluent for liquid phase gradient is a:0.1% formic acid, B: acetonitrile, elution procedure was: 10% by weight B (0-1 min), 10% -98% by weight B (1-15 min), 98% by weight B (15-20 min), 98% -10% by weight B (20-21 min), 10% by weight 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 3.6KV; sheath gas 40psi; the auxiliary gas is 10arb; the temperature of the heater is 350 ℃; and (3) a mass spectrum detection mode: full-ms and dd-ms2.

(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 a 10mL volumetric flask, adding a little methanol, uniformly mixing, adding methanol until the standard substance is dissolved, and fixing the volume to 10mL to obtain 1mg/mL single macrolide metabolite standard substance stock solution. Precisely transferring the single macrolide metabolite standard stock solution into 10 mu L to 10mL volumetric flasks respectively, diluting with methanol and diluting to 10mL to obtain a macrolide metabolite mixed standard solution A with the concentration of 1 mu g/mL, and storing in a refrigerator at 4 ℃ for later use.

Precisely transferring 0.1,0.2,0.5,1,2,5, 10,20,50,100,200 μ L of 10 μ g/mL mixed standard A to 10mL volumetric flasks, diluting with methanol, diluting to a constant volume of 10mL, and mixing to obtain 0.1,0.2,0.5,1,2,5, 10,20,50,100,200ng/mL standard curve solutions. Injecting into UHPLC-Q-active-Orbitrap mass spectrum analysis respectively to obtain a standard curve. The LOD was determined by injecting the lowest concentration (in spiked blank milk) and yielded a signal-to-noise ratio (S/N) equal to 3, while the LOQ was calculated with S/N equal to 10.

Adding standard working solution with mixed mass concentration into blank milk, wherein the addition amount is the addition level of LOQ,2LOQ and 4LOQ. Each addition concentration was processed and analyzed as described above for 6 parallel samples, and the recovery rate and day-to-day precision were calculated.

2. Results and analysis:

(1) Selection of chromatographic conditions

In the process of establishing a quantitative method, a mobile phase is one of the most important factors which cannot be ignored. Suitable mobile phases not only shorten the analysis time and improve the chromatographic peak shape, but also achieve good resolution. At the same time, the mobile phase is ionized with the analyte under electrospray ionization, and therefore, the choice of the mobile phase affects the ionization efficiency of the analyte, which affects the sensitivity of mass spectrometry. To improve the detection sensitivity of the analyte, methanol-water solution and acetonitrile-water solution, methanol-0.1% (v/v) formic acid-water solution and acetonitrile-0.1% (v/v) formic acid-water solution, acetonitrile-0.1% (v/v) formic acid-water solution (2, 5, 10,20 mmol/L ammonium acetate) were used for the experiment.

When methanol-water and acetonitrile-water are used as mobile phases, the retention time of the target can be significantly advanced due to the strong elution ability of acetonitrile-water, and the peak width is reduced. However, the response of the 3 metabolites is not very high. Therefore, if formic acid is further added 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 the 0.1% (v/v) formic acid-acetonitrile solution was higher than that of the 0.1% (v/v) formic acid-methanol solution, the 0.1% (v/v) formic acid-acetonitrile solution was selected as the mobile phase. Under these conditions, N-desmethylclarithromycin has a slight peakeleading phenomenon, and therefore, in order to improve the peak shape, ammonium acetate (2, 5, 10,20 mmol/L) was added to the mobile phase at a range of concentrations. The peak front phenomenon is improved significantly and the peak shape is better with increasing ammonium acetate concentration. The chromatographic peak of each target was relatively good at an ammonium acetate concentration of 10mmol/L, so 0.1% (v/v) formic acid-acetonitrile (containing 10mmol/L ammonium acetate) was chosen as the preferred experimental condition in this experiment, and the optimized chromatogram of the extracted ion streams of 3 compounds in a milk sample is shown in FIG. 1.

TABLE 1 Mass Spectrometry parameters for macrolide metabolite Compounds

(3) Optimization of sample extraction solvent

In the development of analytical methods, the selection of a suitable extraction solvent is very important to improve the recovery. In the analytical procedure of this example, acetonitrile (ACN) containing 0.1% formic acid, methanol, acetonitrile containing 10mM ammonium acetate were tested. The recovery results of the extraction are shown in FIG. 2. It can be observed that the recovery rate of 3 macrolide metabolites in methanol is lower than 50%, and after 0.1% formic acid or 10mmol/L ammonium acetate is added, the recovery rates of the three compounds are improved, and the recovery rates of the three compounds are the highest and are all more than 85.3% when 0.05% -0.15% formic acid-acetonitrile is used as an extraction solvent. Therefore, the preferred extraction solvent is selected to contain 0.1% formic acid-acetonitrile.

(4) Optimization of sample extraction solvent volume

The volume of the extraction solvent is an important factor affecting the efficiency of compound extraction. The extraction efficiencies of 5, 10, 15 and 20mL of 0.1% formic acid and acetonitrile solutions were studied, and as a result, as shown in FIG. 3, the extraction efficiency of 10-15mL of acetonitrile was reasonable, and 10mL 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. To reduce the interference of matrix effects on recovery, various purification materials were selected based on macrolide structure, 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 (2 FI), quadratic and cubic polynomials of response, the coefficient constants X1, X2, X3X1²，X2²，X3²Is significant at the 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+9.20724 x1+0.029725 x2+5.33897e-003 x3-1.9955 x1 x2+3.4159 x1 x3+1.8300 x2 x3-18.122 x12-1.9776 x22-3.0315 x32. Based on the regression coefficients and the 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

In the detection of complex matrix samples by liquid chromatography-mass spectrometry, a matrix co-extract has a matrix-enhancing or matrix-inhibiting effect on the ionization of a target substance, thereby affecting the quantitative determination of the target substance. According to the formula: ME (%) = (1-Ss/Sm). Times.100 calculate the Matric Effect (ME). When ME = -20%, weak matrix effect is achieved; medium stroma effect is obtained when ME is = -50% -20% or 20% -50%; a strong matrix effect when ME = < -50% or > 50%. Wherein the response value of the Ss and Sm in pure solvent is the response value of the target substance with the same content added in the sample matrix. In the results, 3 macrolide metabolites had relatively significant matrix effects. Therefore, the matrix matching method is used for the quantification of macrolide metabolites, as shown in Table 2.

(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, 200ng/ml)). The linear regression coefficients (r) of all matrix-matched calibration curves were in each case greater than 0.9995, see table 2.

(8) Precision and recovery rate of detection method

Recovery and accuracy were assessed by treating three levels (20,50,100. Mu.g/kg) of independently 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

At the end 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 shown in Table 3. Traces of macrolides (< LOQ) were observed in 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 a UHPLC-Q-active-Orbitrap mass spectrometry combined technology 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 QuEChERS for detecting macrolide medicines in a milk sample. Optimized sample processing conditions were established by RSM and used to optimize QuEChERS extraction and removal of 3 macrolide metabolites from milk. For these macrolide metabolites, the modified QuEChERS method gave acceptable results with observed recoveries > 85.3%. The method has the advantages of low detection cost, short period, good stability and high accuracy, and is suitable for rapid batch screening of foods in the market, such as milk and the like.

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 (6)

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;

detecting by using an ultra-high performance liquid chromatography and quadrupole electrostatic field orbit trap Fourier transform coupled mass spectrometry system so as to carry out qualitative analysis and/or quantitative analysis on the metabolites of the macrolide compounds, wherein a chromatographic column of the ultra-high performance liquid chromatography is a C18 chromatographic column with the specification of 2.1 multiplied by 100mm and 3.5 mu m,

wherein the extraction process comprises:

mixing the sample to be detected with an extracting agent to obtain a first mixture, wherein the extracting agent is a mixture of 0.05-0.1% formic acid-acetonitrile, anhydrous magnesium sulfate and sodium chloride;

subjecting the first mixture to ultrasonication so as to obtain a second mixture;

subjecting the second mixture to centrifugation to obtain a first supernatant,

wherein the purification treatment is carried out by using a purifying agent which is a mixture of sodium acetate, N-propyl ethylenediamine and C18,

wherein the eluent for the chromatography is A:0.1% formic acid, B: the reaction mixture of acetonitrile and water is mixed,

the elution conditions of the chromatogram were: 10% by weight B,0-1 minute; 10% -98% by weight B,1-15 minutes; 98% by weight B,15-20 minutes; 98% -10% by weight B,20-21 minutes; 10% by weight B,21-25 minutes,

the metabolites include: erythromycin A oxime, N-desmethylclarithromycin, and N-desmethylroxithromycin.

2. The method of claim 1, 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.

3. The method of claim 1, wherein the aqueous solution contains 300 to 500mg of anhydrous magnesium sulfate and 1 to 3g of sodium chloride based on 10ml of 0.05% to 0.15% formic acid-acetonitrile.

4. The method as claimed in claim 1, wherein the mass ratio of sodium acetate, N-propyl ethylenediamine and C18 in the scavenger is (10-20): 1: (1-4).

5. The method of claim 1, wherein the chromatographic conditions of ultra-high performance liquid chromatography comprise:

sample introduction temperature: 30 ℃;

sample temperature: 10 ℃;

sample introduction amount: 5.0 Mu L;

flow rate: 0.2mL/min.

6. 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 ℃;

spray voltage: 3.6KV;

sheath gas: 40psi;

auxiliary gas: 10arb;

temperature of the heater: 350 ℃;

and (3) a mass spectrum detection mode: full-ms and dd-ms2.

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