CN111458417A - Method and kit for combined detection of multiple antibiotics in sample to be detected - Google Patents
Method and kit for combined detection of multiple antibiotics in sample to be detected Download PDFInfo
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- CN111458417A CN111458417A CN201910054433.8A CN201910054433A CN111458417A CN 111458417 A CN111458417 A CN 111458417A CN 201910054433 A CN201910054433 A CN 201910054433A CN 111458417 A CN111458417 A CN 111458417A
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Abstract
The invention provides a method for jointly detecting multiple antibiotics in a sample to be detected. According to an embodiment of the invention, the method comprises: 1) extracting the sample to be detected; 2) performing liquid chromatography-mass spectrometry detection on the extracting solution; 3) and determining the content of the multiple antibiotics in the sample to be detected based on the detection result of liquid chromatography-mass spectrometry. The detection method provided by the embodiment of the invention can simultaneously detect the content of multiple antibiotics in a sample to be detected, is used for scientific research or monitoring the level of various antibiotics in a human body, and reasonably avoids the use of the antibiotics, so that the detection cost is reduced, and the efficiency is improved. The method has high sensitivity, strong specificity and high accuracy.
Description
Technical Field
The invention relates to the field of biological detection, in particular to a method and a kit for jointly detecting multiple antibiotics in a sample to be detected.
Background
Antibiotics are substances produced by microorganisms (including bacteria, fungi, actinomycetes), which inhibit or kill other microorganisms. The use of antibiotics has made a great epoch-crossing significance to medicine, and is the most commonly used anti-infective drug in hospitals. However, the phenomenon of abuse of antibiotics is quite common in China at present. The usage rate of the antibiotics of inpatients in China is as high as 80%, wherein the combined usage of more than two antibiotics accounts for 58%, and the usage rate is as high as 30% of the international level. In addition, antibiotics in external environments (food, water body, etc.) are transferred into human body, so that part of antibiotics can be accumulated in human body, and normal physiological functions are affected (see table 2). Even leading to the generation and the variation of drug-resistant bacteria, thereby leading some common antibiotics to lose efficacy and promoting the growth of super bacteria which can not be killed by the antibiotics. In view of this, the monitoring of antibiotics is more and more important in all countries, so that the monitoring of the antibiotic level in human body and the reasonable avoidance of the use of antibiotics have very important significance for infectious disease diagnosis.
Currently, the monitoring of antibiotics mainly aims at the monitoring of antibiotic residues in vitro environments (food, water body environments and the like), and detection technologies comprise an enzyme-linked immunoassay method, a thin-layer chromatography, a radioimmunoassay, a high performance liquid chromatography and the like. The detection of the level of antibiotics in human bodies is rarely reported, and the existing detection of antibiotics has fewer types and higher detection limit.
Therefore, techniques for rapidly monitoring the levels of various antibiotics in humans are of particular importance.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art.
The application provides a liquid chromatography-tandem mass spectrometry (L C-MS/MS) method for simultaneously and accurately and quantitatively measuring the concentration of various antibiotics in urine, has the advantages of high flux and high speed, and has high application value in clinical practice and scientific research practice, and simultaneously provides a kit for detecting the content of the antibiotics in urine by using the liquid chromatography-tandem mass spectrometry (L C-MS/MS), and the kit realizes the rapid and accurate detection of the content of the 40 antibiotics in human urine.
In a first aspect of the invention, a method for the combined detection of multiple antibiotics in a test sample is provided. According to an embodiment of the invention, the method comprises: 1) extracting the sample to be detected; 2) performing liquid chromatography-mass spectrometry detection on the extracting solution; 3) and determining the content of the multiple antibiotics in the sample to be detected based on the detection result of liquid chromatography-mass spectrometry. The detection method provided by the embodiment of the invention can be used for simultaneously detecting the content of multiple antibiotics in a sample to be detected, and can be further used for scientific research based on the detection result of the content of multiple antibiotics in the sample to be detected, if the antibiotics come from an animal to be dosed, the content of the antibiotics in the animal body can be judged to be changed according to the detection result before and after the animal is dosed, so that the medicine with the function of regulating the content of the antibiotics in the animal body can be screened out; the kit can also be further used for monitoring the levels of various antibiotics in a human body, has high detection efficiency and reasonably avoids the use of the antibiotics. According to the detection method provided by the embodiment of the invention, liquid chromatography separation is adopted, so that the interference of isomers can be effectively reduced, and meanwhile, cross reaction can be effectively eliminated by using the resolution of mass spectrum detection, so that the specificity of various antibiotics can be rapidly and quantitatively detected, and the detection method has the advantages of high flux and low cost. The detection method provided by the embodiment of the invention has the advantages of high sensitivity, strong specificity, high stability and high accuracy.
According to an embodiment of the present invention, the method may further have at least one of the following additional technical features:
according to an embodiment of the present invention, the sample to be tested is a urine sample. Generally, antibiotics are gradually eliminated along with metabolism in vivo, but along with the occurrence of conditions such as abuse of the antibiotics and the like, the situation that the antibiotics in vivo remain for a long time occurs, so that the drug resistance is enhanced; the urine is generated by human metabolism, and can reflect the recent residual level of antibiotics in the human body to a certain extent by combining factors such as the administration condition of an examinee and the like. According to the detection method provided by the embodiment of the invention, the detection of the antibiotic content in the urine sample can be realized, and further the noninvasive monitoring of the antibiotic content in a human body can be realized.
According to the embodiment of the invention, the extraction treatment is carried out in the extraction working solution, the extraction working solution comprises the antibiotic isotope internal standard and an organic solvent or an acid reagent, the organic solvent is preferably methanol, the acid reagent is preferably sulfosalicylic acid, the extraction treatment is preferably carried out through an SPE column, the SPE column is Cleanert-PEP-2SPE60mg/3m L.
According to an embodiment of the present invention, the extraction process is performed under the following conditions: and placing the sample to be detected in the extraction working solution, and centrifuging for 20min at 4 ℃ and 4000rpm in a dark condition, wherein the volume ratio of the sample to be detected to the extraction working solution is 1: 3. The inventor finds that the extraction treatment is carried out under the conditions, so that the antibiotics in the urine can be ensured to fully enter the extracting solution, the antibiotics can not be degraded, and the foundation is laid for the liquid chromatography separation-mass spectrometry detection of the subsequent extracting solution.
According to the embodiment of the invention, after the extraction treatment and before the liquid chromatography-mass spectrometry detection, the extracting solution is further subjected to nitrogen blow-drying and redissolution treatment so as to obtain the liquid to be loaded. Further, interferents in the extracting solution can be further removed, and the accuracy of liquid chromatography-mass spectrometry detection is improved.
According to an embodiment of the invention, the redissolution treatment is carried out in a redissolution comprising water and methanol in a volume ratio of 7: 3. And a product obtained after nitrogen blow-drying is redissolved by using a redissolvent comprising water and methanol, so that the introduction of interference ions can be avoided to the maximum extent, and meanwhile, the solubility in a 30% methanol solution is high, so that the accuracy and stability of liquid chromatography separation-mass spectrometry detection can be further improved.
According to an embodiment of the invention, the liquid chromatography separation is carried out under the following conditions:
the sample injection volume was 5u L, the chromatography column was ACE-Excel-1.7-C18-PFP 100 x 2.1mm (1.7 μm) or Waters-ACQ-UP L C-C18-PFP 100 x 2.1mm (1.7 μm) or Waters-ACQ-UP L C-18-BEH 100 x 2.1mm (1.7 μm), the column temperature was 40 ℃, mobile phase A was an aqueous solution containing 0.1% methanol, mobile phase B was a methanol solution containing 0.1% formic acid, the detection time was 6.5min for each sample, and the elution gradient was as shown in Table 1.
Table 1:
| time point min | Flow rate m L/min | ％B | Curve of |
| 0 | 0.4 | 15 | - |
| 0.50 | 0.4 | 15 | 6 |
| 0.55 | 0.4 | 25 | 10 |
| 2.00 | 0.4 | 30 | 10 |
| 2.05 | 0.4 | 45 | 6 |
| 3.05 | 0.4 | 45 | 8 |
| 3.75 | 0.4 | 65 | 6 |
| 3.80 | 0.4 | 100 | 6 |
| 5.00 | 0.4 | 100 | 6 |
| 5.05 | 0.4 | 15 | 6 |
| 6.50 | 0.4 | 15 |
The inventor finds that the separation and detection of the sample solution to be loaded under the liquid chromatography detection conditions have good antibiotic specificity separation and detection effects, the detection signal of each target analysis antibiotic is strong (peak height), and the separation degree and difference between the detection signal of each target analysis antibiotic and the detection signal of other non-target analysis antibiotics are high.
According to an embodiment of the invention, the mass spectrometric detection is performed under the following conditions: the mass spectrometric detection is carried out under the following conditions: electrospray positive ion mode (ESI +) of tandem mass spectrometer, multiple reaction monitoring mode (MRM), ion source temperature 600 ℃, capillary voltage 5500kV, source gas parameters as shown in Table 2, and ion pair information as shown in Table 3.
Table 2: source gas parameters (Source gas parameters)
| Curtain Gas (CUR) Curtain Gas | 35.0 |
| Collision Gas of Collision Gas (CAD) | Medium |
| Ion Spray Voltage of Ion Spray Voltage | 5500.0 |
| Temperature (TEM) temperature | 600.0 |
| Ion Source |
40.0 |
| Ion Source |
40.0 |
Table 3: ion pair information
The inventor finds that the specificity, accuracy and specificity of detection can be further improved by further separating and detecting and confirming the sample solution to be loaded under the mass spectrum detection condition.
According to an embodiment of the present invention, the plurality of antibiotics includes the antibiotics shown in table 4.
Table 4:
the method provided by the embodiment of the invention can realize simultaneous detection of the antibiotics shown in the table 4, and has the advantages of high detection sensitivity, strong specificity and high stability.
According to the embodiment of the invention, the content of the determined antibiotic is determined based on the chromatographic peak area of the determined antibiotic detected by liquid chromatography-mass spectrometry.
According to the embodiment of the invention, the method further comprises the step of simultaneously carrying out antibiotic content detection on the quality control product, wherein the quality control product is a sample containing the antibiotic with known concentration. And further, the quality control can be carried out on the detection result, and the authenticity of the detection result is ensured. Meanwhile, an antibiotic content-peak area standard curve can be drawn according to the liquid chromatography separation-mass spectrometry detection result of the quality control product, and the content of the antibiotic in the sample to be detected can be accurately quantified by using the drawn standard curve.
In a second aspect of the invention, a method for combined detection of multiple antibiotics in a sample to be detected is characterized in that the nucleotide metabolites are derived from a blood sample, and the method comprises 1) placing 100 mu L of the sample to be detected in 300 mu L of extraction working solution, and centrifuging the solution for 20min at 4 ℃ and 4000rpm in the absence of light so as to obtain an extraction solution, wherein the extraction working solution comprises an antibiotic isotope internal standard substance and 100% methanol, the antibiotic isotope internal standard substance comprises antibiotic isotope internal standard substances shown in Table 4, the concentrations of the antibiotic isotope internal standard substances in the extraction working solution are respectively 5ppb, 2) performing nitrogen blow-drying treatment on the extraction solution at 35 ℃, performing re-dissolving treatment on a nitrogen blow-dried treatment product in a re-dissolving agent of 90 mu L, wherein the re-dissolving agent comprises water and methanol, and the volume ratio of the water to the methanol is 7:3 so as to obtain a sample solution to be applied, and 3) performing liquid chromatography-detection on the sample solution to be applied,
wherein the liquid chromatographic separation is carried out under the following conditions:
sample introduction volume was 5u L, chromatography column ACE-Excel-1.7-C18-PFP 100 x 2.1mm (1.7 μm) or Waters-ACQ-UP L C-C18-PFP 100 x 2.1mm (1.7 μm) or Waters-ACQ-UP L C-C18-BEH100 x 2.1mm (1.7 μm), column temperature was 40 ℃, mobile phase a was an aqueous solution containing 0.1% methanol, mobile phase B was a methanol solution containing 0.1% formic acid, detection time was 6.5min per sample, elution gradient as shown in table 1;
the mass spectrometric detection is carried out under the following conditions: electrospray positive ion mode (ESI +) of a tandem mass spectrometer, multiple reaction monitoring mode (MRM), ion source temperature of 600 ℃, capillary voltage of 5500kV, source gas parameters as shown in Table 2, and ion pair information as shown in Table 3;
4) determining the content of the determined antibiotic based on the chromatographic peak area of the determined antibiotic detected by liquid chromatography-mass spectrometry;
the method further comprises the step of simultaneously detecting the content of antibiotics in a quality control product, wherein the quality control product is a sample containing the antibiotics with known concentration;
optionally, the extraction working solution is obtained by:
dissolving an antibiotic isotope internal standard with a concentration of 1ppm at 5 mu L in 100% methanol at 50 mu L to obtain a stock solution of the antibiotic isotope standard;
the isotopic internal standard stock solution was mixed with a methanol precipitant in 100% methanol at a ratio of 1:20 to obtain the extraction working solution.
The detection method provided by the embodiment of the invention can be used for simultaneously detecting the content of 40 antibiotics shown in table 1 in a urine sample, can be further used for scientific research based on the detection result of the content of the 40 antibiotics in the urine, can also be further used for monitoring the level of various antibiotics in a human body, is high in detection efficiency, and reasonably avoids the use of the antibiotics. According to the detection method provided by the embodiment of the invention, liquid chromatography separation is adopted, so that the interference of isomers can be effectively reduced, and meanwhile, cross reaction can be effectively eliminated by using the resolution of mass spectrum detection, so that the specificity of various antibiotics can be rapidly and quantitatively detected, and the detection method has the advantages of high flux and low cost. The detection method provided by the embodiment of the invention has the advantages of high sensitivity, strong specificity, high stability and high accuracy.
In a third aspect of the invention, the invention provides a kit for combined detection of multiple antibiotics in a sample to be detected. According to an embodiment of the invention, the kit comprises: (A) antibiotic isotope internal standard: consists of isotopic internal standards of the antibiotics shown in table 4; (B) quality control product: the quality control product is a sample containing antibiotics with known concentration; and (C) instructions for use, said instructions describing the aforementioned method. The kit provided by the embodiment of the invention is simple and rapid to operate, high in specificity and good in stability, facilitates standardized operation, greatly improves the reliability and stability of a detection result, is also suitable for high-throughput screening, and can reduce the detection cost and improve the efficiency. In addition, the kit adopts a urine sample, is convenient for sampling, and is suitable for monitoring the content of the antibiotics in the human body in a non-invasive and real-time manner.
According to an embodiment of the present invention, the kit may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the kit further comprises: (D) an antibiotic correction product comprising a bile acid standard; (E) void urine, which is urine known to contain no antibiotics; (F) a QA test article comprising a standard of an antibiotic as set forth in Table 4; (G) a mobile phase additive comprising formic acid. The antibiotic correction product is used for carrying out quantitative analysis when drawing a standard, the blank urine is urine which is determined to be free of antibiotic through detection and is used for quantitative analysis and experimental comparison when detecting the antibiotic, the QA test product is used for the adaptability of a detection system, and the mobile phase additive is used for preparing mobile phases A and B in liquid chromatography separation detection. Furthermore, the kit provided by the embodiment of the invention is simpler and quicker to operate, higher in detection specificity, better in stability and more convenient for standardized operation.
Drawings
FIG. 1 shows the results of antibiotic detection in 77 urine samples according to an embodiment of the present invention;
FIG. 2 is a chromatogram of a sample spiked with 40 antibiotics according to example 1 of the present invention;
FIG. 3 is a graph comparing the detected amounts of antibiotics by three types of chromatography columns according to example 2 of the present invention; and
FIG. 4 is a graph comparing the extraction efficiencies of 40 antibiotics according to different extraction modes of example 3 of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In order to achieve the above objects, in one aspect, the present invention provides a kit for rapid quantitative determination of 40 antibiotics in urine (using liquid chromatography-tandem mass spectrometry).
According to the specific embodiment of the invention, the kit comprises 40 isotope mixed internal standard substances (168 persons), a standard curve series (a mixed standard solution of 7 antibiotics with the concentration cal1-cal7 from low to high for drawing a standard curve and carrying out quantitative analysis), quality control substances (a low-concentration quality control substance QC L, a medium-concentration quality control substance QCM and a high-concentration quality control substance QCH), a test substance (QA), a 350 mu L96 well machine plate, a sealing film and an operation instruction.
Kits according to specific embodiments of the present invention are shown in table 5 below.
Table 5:
the kit provided by the invention can be further elaborated, and comprises methanol, a chromatographic column and the like, and the consumable materials can be selected by the technicians in the field, and further description is omitted
In another aspect, the present invention provides a method for the combined detection of 40 antibiotics using the kit of the first aspect.
According to a specific embodiment of the invention, the method comprises: (1) preparing a working solution; (2) extracting a sample; (3) nitrogen blowing and redissolving; (4) and (4) performing mass spectrometry detection.
According to the specific embodiment of the present invention, the method for using the kit for rapidly and quantitatively detecting 40 antibiotics in urine provided by the present invention comprises the following steps:
1. preparation of working fluid
And (3) directly adding 50 mu L methanol into an isotope internal standard product bottle to dissolve the isotope internal standard product, mixing with the precipitator solution at a ratio of 1:20, and performing vortex for 1min to prepare the internal standard working solution.
2. Sample extraction
Adding 100 mu L of each urine sample (or quality control product QC) into a corresponding 96-well upper template, adding 300 mu L internal standard working solution and a closing plate into a sample point, a standard curve point (cal 1-cal7 and 7 mixed antibiotic standard product solvents with different concentrations) and a blank point (a solvent which does not contain an antibiotic standard product but contains the antibiotic internal standard product) except for double blanks (solvent which does not contain the antibiotic internal standard product and antibiotic standard product), carrying out vortex shaking for 30 seconds and 2 times, centrifuging at 4 ℃ for 20min, and taking 360 mu L supernatant at 4000rpm for nitrogen blowing.
3. Nitrogen blowing redissolution
Blowing at 35 deg.C with nitrogen, and dissolving with 90 μ L solvent (V)Water/methanol 70: 30) Redissolving in the upper machine plate, and waiting for the machine.
4. Mass spectrometric detection on computer
Preparing a mobile phase A of 500m L pure water and 0.5m L mobile phase additive;
preparing a mobile phase B of 500m L methanol and 0.5m L mobile phase additive;
0.5m L30% methanol was added to QA bottle and shaken for 1 min.
The suitability of the system is tested using QA.
5. Report of quantitative results: the concentration of each analyte is automatically obtained according to the instrument set program.
According to the present invention, the object of the present invention can be achieved by performing the detection using the kit of the present invention, and the specific operation of the above-mentioned steps can be performed by referring to the conventional operation without any particular requirement.
In a further aspect, the invention provides the use of a kit according to the first aspect for the detection of 40 antibiotics (as shown in table 4). The application of the kit for detecting the liquid chromatography-tandem mass spectrometry in the detection of antibiotics is provided.
The kit has the following beneficial effects: the kit is used for liquid chromatography-tandem mass spectrometry detection, when a morning urine sample is detected, the content of 40 antibiotics can be rapidly, accurately and quantitatively detected at one time only by 100 microliters of urine sample, the antibiotic level of a human body can be effectively diagnosed and detected according to the indexes, prevention is reasonably avoided, and the use of antibiotics is reduced.
The present application will be described in further detail with reference to specific examples. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application.
Example 1
This example is intended to illustrate the liquid chromatography tandem mass spectrometry detection kit of the present invention and its application.
The antibiotic test was carried out on 77 urine samples provided by the company's manufacturing center as follows, and the test conditions are shown in FIG. 1.
Reagent materials: the kit described above;
preparing a specimen: morning urine;
the detection method comprises the following steps:
1. preparation of working fluid
Adding 50 mu L methanol directly into the isotope internal standard bottle to dissolve the isotope internal standard, mixing with precipitator solution (methanol) at a ratio of 1:20, and vortexing for 1min to prepare internal standard working solution.
2. Sample extraction
Adding 100 mu L of each urine sample (or QC) into a corresponding 96-hole upper sample plate, adding 300 mu L internal standard working solution into a sample point, a standard curve point and a blank point except double blanks, and closing a plate, wherein the blank matrix is double distilled water, uniformly swirling for 30 seconds and 2 times, centrifuging at 4 ℃ for 20min, and taking 360 mu L of supernatant at 4000rpm until nitrogen is blown;
3. nitrogen blowing redissolution
Blowing at 35 deg.C with nitrogen, re-dissolving 90 μ L double solvent (V water/methanol 70: 30) in upper plate, and waiting for loading
4. Mass spectrometric detection on computer
Preparing a mobile phase A of 500m L pure water and 0.5m L mobile phase additive;
preparing a mobile phase B of 500m L methanol and 0.5m L mobile phase additive;
the detection chromatographic column adopts: ACE-Excel-1.7-C18-PFP 100 x 2.1mm (1.7 μm);
0.5m L30% methanol is added into the QA bottle, and the mixture is shaken for 1min, and the adaptability of the QA detection system is adopted.
5. Report of quantitative results: the concentration of each analyte is automatically obtained according to the instrument set program.
6. And (3) putting the 96-pore plate into an automatic sample injector, detecting by adopting liquid chromatography-tandem mass spectrometry, and processing data by adopting a set program to obtain the concentrations of the 40 antibiotics.
The quality control product can evaluate whether the method fluctuates or not, wherein CV is less than or equal to 15 percent, which indicates that the method is stable and the collected data is reliable, and FIG. 2 is a chromatogram of 40 antibiotics for sample labeling (morning urine to be detected plus an antibiotic standard sample); table 6 shows the CV of each analyte in the quality control QCM. The CV is less than 20% as can be seen from the data, and the detection requirement is met.
Table 6: stability of antibiotics in quality control Material (sample QCM)
Note: n is the number of repetitions.
Example 2 optimization of separation chromatography columns
The samples (morning urine + mixed standards of various antibiotics) were tested as follows
Reagent materials: the kit described above;
the detection method comprises the following steps: the conditions and operation were the same as in the example 1 method except that the column to be optimized was varied
1. Preparation of working fluid
Adding 50 mu L methanol directly into the isotope internal standard bottle to dissolve the isotope internal standard, mixing with precipitator solution (methanol) at a ratio of 1:20, and vortexing for 1min to prepare internal standard working solution.
2. Sample extraction
Adding 100 mu L of each urine sample (or QC) into a corresponding 96-hole upper sample plate, adding 300 mu L internal standard working solution into a sample point, a standard curve point and a blank point except double blanks, and closing a plate, wherein the blank matrix is double distilled water, uniformly swirling for 30 seconds and 2 times, centrifuging at 4 ℃ for 20min, and taking 360 mu L of supernatant at 4000rpm until nitrogen is blown;
3. nitrogen blowing redissolution
Blowing at 35 deg.C with nitrogen, re-dissolving 90 μ L double solvent (V water/methanol 70: 30) in upper plate, and waiting for loading
4. Mass spectrometric detection on computer
Preparing a mobile phase A of 500m L pure water and 0.5m L mobile phase additive;
preparing a mobile phase B of 500m L methanol and 0.5m L mobile phase additive;
the detection chromatographic columns are respectively as follows:
0.5m L30% methanol is added into the QA bottle, and the mixture is shaken for 1min, and the adaptability of the QA detection system is adopted.
5. Report of quantitative results: the concentration of each analyte is automatically obtained according to the instrument set program.
6. And (3) putting the 96-pore plate into an automatic sample injector, detecting by adopting liquid chromatography-tandem mass spectrometry, and processing data by adopting a set program to obtain the concentrations of the 40 antibiotics.
The results are shown in fig. 3, which shows that the highest amount of antibiotics were detected using column ①, the lowest amount of antibiotics were detected using column ②, and the intermediate amounts of antibiotics were detected using column ③.
Example 3 optimization of extraction of working solution
The samples (morning urine + mixed standards of various antibiotics) were tested as follows
Reagent materials: the kit described above;
the detection method comprises the following steps: the conditions and operation were the same as in the example 1 process, except that the precipitant to be optimized was varied
1. Preparation of working fluid
And (3) directly adding 50 mu L methanol into an isotope internal standard product bottle to dissolve the isotope internal standard product, mixing with the precipitator solution at a ratio of 1:20, and performing vortex for 1min to prepare the internal standard working solution.
The precipitating agents are respectively three:
The extraction method ③ comprises SPE column extraction, Cleanert-PEP-2SPE60mg/3m L
2. Sample extraction
Adding 100 mu L of each urine sample (or QC) into a corresponding 96-hole upper sample plate, adding 300 mu L internal standard working solution into a sample point, a standard curve point and a blank point except double blanks, and closing a plate, wherein the blank matrix is double distilled water, uniformly swirling for 30 seconds and 2 times, centrifuging at 4 ℃ for 20min, and taking 360 mu L of supernatant at 4000rpm until nitrogen is blown;
3. nitrogen blowing redissolution
Blowing at 35 deg.C with nitrogen, re-dissolving 90 μ L double solvent (V water/methanol 70: 30) in upper plate, and waiting for loading
4. Mass spectrometric detection on computer
Preparing a mobile phase A of 500m L pure water and 0.5m L mobile phase additive;
preparing a mobile phase B of 500m L methanol and 0.5m L mobile phase additive;
the detection chromatographic columns are respectively as follows:
0.5m L30% methanol is added into the QA bottle, and the mixture is shaken for 1min, and the adaptability of the QA detection system is adopted.
5. Report of quantitative results: the concentration of each analyte is automatically obtained according to the instrument set program.
6. And (3) putting the 96-pore plate into an automatic sample injector, detecting by adopting liquid chromatography-tandem mass spectrometry, and processing data by adopting a set program to obtain the concentrations of the 40 antibiotics.
The experimental results are shown in fig. 4, the results show that effective extraction and subsequent detection of antibiotics in urine can be realized by adopting the extraction modes of ①, ② and ③, and the 40 antibiotics correspondence table in fig. 4 is shown in table 7.
Table 7:
example 4 limit of quantitation and Limit of detection test
The experimental process comprises the following steps:
adding a mixed standard substance solution of various antibiotics into blank urine for detection; gradually dilute (diluent is blank urine) until no detection can be made.
The following are the quantitation limit and detection limit standards:
and (4) quantitative limit: the assessment method enables the analyte minimum concentration in the sample to be reliably quantified with acceptable accuracy and precision.
Detection limit: the sensitivity and the noise of the evaluation method and the evaluation instrument also indicate the background value of the extracted biological sample.
And (4) qualified standard: the lower limit of the quantification is suitable for the expected concentration and the test purpose, and the S/N is required to be more than or equal to 10; the lower limit of detection should be the lowest concentration detectable in the sample, and S/N is required to be more than or equal to 3.
The quantitative and detection limits of the method for detecting antibiotic content according to the embodiment of the present invention are shown in table 8 below.
Table 8:
the foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.
Furthermore, the terms "first", "second" and "first" 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 at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
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 are not necessarily intended to 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. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (10)
1. A method for detecting multiple antibiotics in a sample, comprising:
1) extracting a sample to be detected;
2) performing liquid chromatography-mass spectrometry detection on the extracting solution;
3) and determining the content of the multiple antibiotics in the sample to be detected based on the detection result of liquid chromatography-mass spectrometry.
2. The method of claim 1, wherein the test sample is a urine sample.
3. The method according to claim 2, characterized in that the extraction treatment is carried out in an extraction working solution comprising an antibiotic isotope internal standard and an organic solvent or acid reagent;
preferably, the organic solvent is methanol;
preferably, the acid agent is sulfosalicylic acid;
preferably, the extraction treatment is performed by SPE column extraction, and the SPE column is Cleanert-PEP-2SPE60mg/3m L.
4. The method according to claim 3, wherein the extraction process is carried out under the following conditions:
and placing the sample to be detected in the extraction working solution, and centrifuging for 20min at 4 ℃ and 4000rpm in a dark condition, wherein the volume ratio of the sample to be detected to the extraction working solution is 1: 3.
5. The method according to claim 1, characterized in that after extraction treatment and before liquid chromatography-mass spectrometry detection, the extract is further subjected to nitrogen blow-drying and redissolution treatment so as to obtain a to-be-loaded solution;
optionally, the redissolution treatment is carried out in a redissolvent comprising water and methanol in a volume ratio of 7: 3.
6. The method according to claim 1, characterized in that the liquid chromatographic separation is carried out under the following conditions:
the sample injection volume was 5u L, the chromatography column was ACE-Excel-1.7-C18-PFP 100 x 2.1mm (1.7 μm) or Waters-ACQ-UP L C-C18-PFP 100 x 2.1mm (1.7 μm) or Waters-ACQ-UP L C-18-BEH 100 x 2.1mm (1.7 μm), the column temperature was 40 ℃, mobile phase A was an aqueous solution containing 0.1% methanol, mobile phase B was a methanol solution containing 0.1% formic acid, the detection time was 6.5min for each sample, and the elution gradient was as shown in Table 1.
7. The method of claim 1, wherein the mass spectrometric detection is performed under the following conditions: electrospray positive ion mode (ESI +) of a tandem mass spectrometer, multiple reaction monitoring mode (MRM), ion source temperature of 600 ℃, capillary voltage of 5500kV, source gas parameters as shown in Table 2, and ion pair information as shown in Table 3;
optionally, the plurality of antibiotics comprises an antibiotic shown in table 4;
optionally, determining the content of the antibiotic based on the chromatographic peak area of the antibiotic detected by liquid chromatography-mass spectrometry;
optionally, the method further comprises the step of simultaneously detecting the content of the antibiotics in a quality control product, wherein the quality control product is a sample containing the antibiotics with known concentration.
8. A method for detecting multiple antibiotics in a sample, comprising:
1) placing a sample to be tested of 100 mu L in the extraction working solution of 300 mu L, and centrifuging for 20min at 4 ℃ and 4000rpm in the dark to obtain an extracting solution, wherein the extraction working solution comprises the antibiotic isotope standard and 100% methanol, the antibiotic isotope standard comprises the antibiotic isotope standard shown in the table 4, and the concentrations of the antibiotic isotope standard in the extraction working solution are respectively 5 ppb;
2) carrying out nitrogen blow-drying treatment on the extracting solution at 35 ℃, and carrying out redissolution treatment on a nitrogen blow-dried treatment product in a redissolution of 90 mu L, wherein the redissolution comprises water and methanol, and the volume ratio of the water to the methanol is 7:3 so as to obtain a to-be-loaded solution;
3) performing liquid chromatography-mass spectrometry detection on the liquid to be loaded,
wherein the liquid chromatographic separation is carried out under the following conditions:
sample introduction volume was 5u L, chromatography column ACE-Excel-1.7-C18-PFP 100 x 2.1mm (1.7 μm) or Waters-ACQ-UP L C-C18-PFP 100 x 2.1mm (1.7 μm) or Waters-ACQ-UP L C-C18-BEH100 x 2.1mm (1.7 μm), column temperature was 40 ℃, mobile phase a was an aqueous solution containing 0.1% methanol, mobile phase B was a methanol solution containing 0.1% formic acid, detection time was 6.5min per sample, elution gradient as shown in table 1;
the mass spectrometric detection is carried out under the following conditions: electrospray positive ion mode (ESI +) of a tandem mass spectrometer, multiple reaction monitoring mode (MRM), ion source temperature of 600 ℃, capillary voltage of 5500kV, source gas parameters as shown in Table 2, and ion pair information as shown in Table 3;
4) determining the content of the antibiotic based on the chromatographic peak area of the antibiotic detected by liquid chromatography-mass spectrometry;
the method further comprises the step of simultaneously detecting the content of antibiotics in a quality control product, wherein the quality control product is a sample containing the antibiotics with known concentration;
optionally, the extraction working solution is obtained by:
dissolving an antibiotic isotope internal standard with a concentration of 1ppm at 5 mu L in 100% methanol at 50 mu L to obtain a stock solution of the antibiotic isotope standard;
the stock solution of the isotope internal standard was mixed with methanol at a ratio of 1:20 using 100% methanol to obtain the extraction working solution.
9. A kit for detecting multiple antibiotics in a sample, comprising:
(A) antibiotic isotope standards: consists of isotope standards of antibiotics shown in table 4;
(B) quality control product: the quality control product is a sample containing antibiotics with known concentration; and
(C) an instruction for use, said instruction describing the method according to any one of claims 1 to 8.
10. The kit of claim 9, further comprising:
(D) an antibiotic correction product comprising a bile acid standard;
(E) void urine, which is urine known to contain no antibiotics;
(F) a QA test article comprising a standard of an antibiotic as set forth in Table 4;
(G) a mobile phase additive comprising formic acid.
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| CN115267016A (en) * | 2022-06-14 | 2022-11-01 | 上海市环境科学研究院 | Method for simultaneously detecting 27 antibiotics in eggs or milk by combining aqueous two-phase extraction with liquid chromatography-mass spectrometry |
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