Method for detecting 17 perfluorinated compounds and 25 antibiotics in marine biological samples

Abstract

The present application relates to a kind of methods for detecting 17 kinds of perfluorinated compounds and 25 kinds of antibiotics in marine biological samples, as follows: marine biological sample powder is weighed, each sample is added with mixed internal standard, and the sample is added with mixed external standard, after adding ultrapure water, acetonitrile and NaCl, vortex ultrasonic centrifugation is carried out, the supernatant is taken in a new centrifuge tube, the remaining part is added with acetonitrile, vortexed, ultrasonic, centrifuged, and the supernatant is taken, the supernatant of two times is combined, the supernatant is frozen, and the supernatant is taken again, the supernatant is blown to near dry under nitrogen flow, and is redissolved in methanol; centrifugation is carried out, and the supernatant is taken into sample vial for testing; high performance liquid chromatography mass spectrometry is used for detection.The method of the present application is simple to operate, reduces the use of extraction reagent, reduces raw material consumption, saves experimental links, reduces experimental cost, simultaneously adopts internal standard correction, external standard quantification to jointly realize the quality control of experimental data, and ensures the precision and accuracy of experimental results.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for detecting 17 kinds of perfluorinated compounds and 25 kinds of antibiotics in marine biological samples, belonging to the technical field of detection. BACKGROUND

[0002] The traditional pretreatment method for detecting trace organic pollutants in complex biological matrix usually needs to use solid phase extraction column or dispersive solid phase extraction agent to extract and enrich the detected substances in the sample and purify the system to reduce matrix interference. When detecting perfluorinated compounds in fish, Gao et al. used acetonitrile to complete two-step extraction, and then used WAX solid phase extraction column to purify the extract (Gao, Li, et al., 2018). In another experiment, QuEChERS method was used for extraction. In addition to adding magnesium sulfate and sodium chloride during extraction, the supernatant was transferred into a new centrifuge tube containing dispersive solid phase extraction agent after being combined and shaken and centrifuged to remove impurities (Gao, Zhang, et al., 2018). Michela Mazzoni et al. added magnesium sulfate and sodium chloride during the extraction of perfluorinated compounds in fish, and the extract was passed through a phospholipid solid phase extraction column again to remove impurities (Mazzoni et al., 2019). Effrosyni Zafeiraki et al. used methanol as an extractant when detecting perfluorinated compounds in shark and skate tissue samples, and also used ASE Dionex 300 equipment (accelerated solvent extraction) to extract and remove impurities (Zafeiraki et al., 2019). Li et al. detected sulfonamide antibiotics in aquatic products using an online SPE system for extraction and purification, and two columns for purification and concentration (Li et al., 2020). Hua et al. detected antibiotic levels in fish using a modified QuEChERS method, and added two salts and a dispersive extraction agent to the extraction system to purify the supernatant (Hua et al., 2022). The above methods are limited to existing research, and the same treatment method is used for different matrix samples, ignoring the characteristics of the sample itself. Although SPE solid phase extraction column and dispersive solid phase extraction agent can remove various impurities, their operation is complex and requires the addition of more substances to the test system.

[0003] The prior art often causes loss of the analyte and signal fluctuation when processing and analyzing biological samples, which may occur in the processes of sample transfer, adsorption, evaporation and injection, and especially in the process of mass spectrometric detection, the detection signal of the analyte may change due to the matrix effect, by adding an equal amount of an internal standard having similar physical and chemical properties as the analyte into all samples in the same analysis batch, and using the response value ratio of the analyte and the internal standard for quantitative calculation, the signal fluctuation and loss of most of the analyte can be corrected, and the accuracy and precision of the analysis results and the reliability of the method can be greatly improved by using a suitable internal standard, but for detection of multiple perfluorinated compounds and multiple antibiotics, the internal standard is not easy to select and determine, and is easily affected by the interference of the sample itself, resulting in greatly reduced accuracy and reliability, and high detection cost. SUMMARY

[0004] (One) technical problems to be solved

[0005] In order to solve the above problems of the prior art, the present application provides a method for detecting 17 kinds of perfluorinated compounds and 25 kinds of antibiotics in marine biological samples.

[0006] (Two) technical solutions

[0007] In order to achieve the above purpose, the main technical solutions adopted by the present application include:

[0008] The method for detecting 17 kinds of perfluorinated compounds and 25 kinds of antibiotics in marine biological samples comprises the following steps:

[0009] S1, weigh marine biological sample powder, and add an internal standard to each sample;

[0010] S2, weigh another sample from any two marine biological samples in S1, and add an internal standard according to step S1, and then add a mixed external standard as a spiked sample;

[0011] S3, vortex mix the above samples after adding ultrapure water, and stand at room temperature for a period of time to balance the system, then vortex mix after adding acetonitrile, ultrasonic, vortex mix after adding NaCl, and ultrasonic;

[0012] S4, then centrifuge, and take the supernatant in a new centrifuge tube;

[0013] Add acetonitrile in the remaining part, vortex, ultrasonic, and centrifuge, take the supernatant, combine the supernatants of the two times, stand the supernatant in a-20℃ refrigerator, take the supernatant again, blow the supernatant to near dryness, and re-dissolve in methanol;

[0014] S5. After vortexing the reconstituted system until homogeneous, centrifuge, collect the supernatant and let it stand at -20℃, then centrifuge again and collect the supernatant into a sample vial for testing.

[0015] S6. Detection was performed using high performance liquid chromatography-mass spectrometry;

[0016] S7. Prepare external standard standards of different concentrations for plotting standard curves. An internal standard is added to each standard, and the results are detected by high performance liquid chromatography-mass spectrometry.

[0017] S8. Based on the chromatogram of the obtained standard, determine the corresponding chromatogram of the sample group to be tested, thereby obtaining the qualitative and quantitative results of perfluorinated compounds and antibiotics in the sample to be tested.

[0018] In a preferred embodiment, the internal standard is sodium perfluoro-1-hexane[18O2]sulfonate, sodium perfluorooctanesulfonate-13C8, perfluorooctanoic acid-13C8, perfluorononanoic acid-13C9, roxithromycin-D7, azithromycin-D3, enrofloxacin-D5, sulfamethoxazole-D4, and sulfadiazine-D4.

[0019] In a preferred embodiment, in step S1, the amount of sample weighed is 0.1 to 1 g, the concentration of each internal standard is 50 to 100 ng / ml, and the total amount of internal standard added is 50 to 100 μl.

[0020] In a preferred embodiment, in step S2, the external standard includes perfluorinated compounds: perfluorobutyric acid, perfluorovaleric acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluorooctadecanic acid, potassium perfluorobutylsulfonate, sodium perfluorohexylsulfonate, sodium perfluorooctylsulfonate, sodium perfluorononylsulfonate, sodium perfluorodecylsulfonate; macrolide antibiotics: azithromycin, clarithromycin. Medications include: erythromycin, roxithromycin; quinolone antibiotics: enrofloxacin, flumethin, nalidixic acid, sinofloxacin, difluorofloxacin hydrochloride; sulfonamide antibiotics: sulfathiazole, sulfapyridine, sulfadiazine, sulfadimethoxazole, sulfotriazine, sulfadimethoxazole, sulfamethazine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfadiazine, sulfamethoxypyrimidine, sulfadiazine, sulfamethoxypyrimidine, sulfadiazine, sulfamethoxypyrimidine, sulfadiazine, sulfamethoxypyrimidine, sulfadiazine, sulfamethoxypyrimidine, sulfadiazine, sulfamethoxypyrimidine, sulfadiazine.

[0021] In a preferred embodiment, in step S2, the concentration of each external standard added to the spiked sample is 50-100 ng / ml, and the total amount added is 50-100 μl.

[0022] In a preferred embodiment, in step S3, the amount of ultrapure water and acetonitrile added is equal, and the amount of NaCl added is 10% mass volume percentage by g / ml in the mixed solution; the ultrasonic time is 5-15 min.

[0023] In a preferred embodiment, in step S4, the centrifugation conditions are 4°C, 9000-12000 rpm for 5-10 min. In a preferred embodiment, in step S4, the amount of methanol used is 0.5 ml.

[0024] In a preferred embodiment, in step S5, the centrifugation conditions are 4°C, 12000-15000 rpm for 10-20 min.

[0025] In a preferred embodiment, in step S6, when the high performance liquid chromatography mass spectrometry instrument is used for detection, the conditions for qualitative and quantitative detection of perfluorinated compounds are as follows: dynamic multiple reaction monitoring in AJS-ESI negative ion mode, mobile phase A and B are ultrapure water and methanol respectively, injection amount is 5 μl, flow rate is 0.2 ml / min, column temperature is 40°C constant temperature;

[0026] The elution gradient of the mobile phase is as follows: 0 min, 30% B; 2 min, 40% B; 5 min, 80% B; 7 min, 95% B; 9 min, 100% B; 10 min, 100% B; 11 min, 30% B; re-equilibrate for 2 min;

[0027] The gas and gas shift temperatures are 325°C and 350°C respectively, the flow rates are 10 and 11 ml / min respectively, and the nebulizer pressure is 20 psi.

[0028] In a preferred embodiment, in step S6, when detection is performed, the conditions for qualitative and quantitative detection of antibiotics are as follows: dynamic multiple reaction monitoring in AJS-ESI positive ion mode, mobile phase A and B are ultrapure water containing 0.1% formic acid and methanol containing 0.1% formic acid respectively, injection amount is 2 μl, flow rate is 0.2 ml / min, column temperature is room temperature;

[0029] The elution gradient of the mobile phase is as follows: 0 min, 10% B; 3.5 min, 40% B; 4 min, 48% B; 4.5 min, 30% B; 6 min, 100% B; 10 min, 100% B; 11 min, 10% B; re-equilibrate for 2 min;

[0030] The gas and gas shift temperatures are 325°C and 350°C respectively, the flow rates are 10 and 11 ml / min respectively, and the nebulizer pressure is 20 psi.

[0031] Further, in step S7, the standard samples of different concentrations of external standards are sequentially decreased in the following concentrations: 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, 2 ng / ml, 1 ng / ml, 0.5 ng / ml, 0.2 ng / ml, and 0.1 ng / ml, and the internal standard is added in the same concentration and volume as the internal standard in S1.

[0032] Further, in step S8, the measured concentration of the spiked sample is subtracted from the measured concentration of the corresponding sample without the external standard, and the ratio of the theoretical value of the standard substance added in the spiked sample is the recovery rate of the spike, and the recovery rate of the spike is in the interval of 60-130%, indicating that the detection result is accurate and reliable; if it is not in the interval, it indicates that an error has occurred in the experiment, and the result obtained is unreliable and needs to be retested.

[0033] The average of the measured response values of all samples in the standard curve sample is taken as the standard, and the measured response value of the internal standard in each sample is divided by the average value, and the result is the internal standard recovery rate of each sample, which is used as a quality control index as the external standard recovery rate, and needs to be in the interval of 60-130%, indicating that the detection result is accurate and reliable; if it is not in the interval, it needs to be retested.

[0034] (III) Beneficial effects

[0035] The beneficial effects of the present application are:

[0036] The method provided by the present application for detecting 17 kinds of perfluorinated compounds and 25 kinds of antibiotics in marine biological samples has the advantages that the pretreatment method is simple to operate, does not need to be activated, eluted, and eluted through a solid phase extraction column, only needs to be frozen and centrifuged, has lower requirements for the experimental level of the operator, has smaller systematic errors, and can also achieve high recovery rate. The method reduces the use of extraction reagents, reduces raw material consumption, saves experimental links, and reduces experimental cost; at the same time, the internal standard correction and external standard quantification are used to jointly realize quality control of experimental data, and ensure the precision and accuracy of experimental results.

[0037] The results of the statistical data of 100 samples show that the internal standard recovery rate is in the interval of 61-117%, and the external standard recovery rate is shown in Tables 4 and 5. Compared with the previous research, the recovery rate is higher. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The chromatogram for perfluorinated compound detection;

[0039] Figure 2 The chromatogram for antibiotic detection. DETAILED DESCRIPTION

[0040] The application utilizes the physical and chemical properties of main impurities in the to-be-tested matrix, and realizes impurity removal by using the simplest operation method. For marine biological samples, the main matrix interference is derived from lipids and proteins. Traditional methods use various extractants to remove proteins and lipids. However, through a large number of experiments, the application first uses sodium chloride to precipitate proteins in the matrix system, uses the low-temperature solidification characteristics of lipids, and realizes lipid removal by multiple freezing and centrifugation, thereby reducing the use of various traditional dispersive solid-phase extractions. By utilizing the physical properties of the target matrix, efficient extraction and purification are realized. The method of the application is efficient and low in cost, and a simpler method is used for sample pretreatment, which reduces the addition of other substances to the to-be-tested system, reduces the experimental operation steps, and reduces experimental errors. On the other hand, the experimental cost is reduced, the experimental process is simplified, and the quality control standards required by the targeted quantitative method can also be achieved.

[0041] Secondly, the use of a suitable internal standard greatly improves the accuracy and precision of the analysis results and the reliability of the method. The use of isotopes of target compounds as internal standards can further reduce the interference of samples.

[0042] In order to better explain the application and facilitate understanding, the application is described in detail below through specific embodiments in combination with the accompanying drawings, wherein the internal standards of perfluorinated and bisphenol substances in the examples are purchased from Cambridge Isotope Laboratories, and the internal standards of antibiotic substances are purchased from Dr. Ehrenstorfer.

[0043] Example 1

[0044] S1, take 0.5g of freeze-dried marine organisms, wherein the marine organisms are different, and the sampling sites are different, such as fish taking the back of the skin and boneless muscle, shrimp taking the abdomen of the shell muscle, crab taking the chelicera muscle, and shellfish taking the closed shell muscle, etc. Sample powder is taken in a 50ml centrifuge tube, and 100ul of mixed internal standard is added to all samples. The specific internal standard is as follows: perfluoro-1-hexane [18O2] sodium sulfonate, perfluoro octane sulfonate-13C8, perfluoro-n-octanoic acid-13C8, perfluoro-n-nonanoic acid-13C9, roxithromycin-D7, azithromycin-D3, enoxacin-D5, sulfamethoxazole-D4, and sulfadimidine-D4. The concentration of each internal standard in the mixed internal standard is 100ng / ml.

[0045] S2. Take one sample from each of the two marine biological samples from S1. After adding 100 μl of the mixed internal standard as in step S1, add 100 μl of the mixed external standard to the newly taken sample as the spiked sample for quality control of the test. The mixed external standard is shown in Tables 1 and 2 below, specifically perfluorinated compounds: perfluorobutyric acid, perfluorovalerate, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluorooctadecanoic acid, potassium perfluorobutylsulfonate, sodium perfluorohexylsulfonate, sodium perfluorooctylsulfonate, sodium perfluorononylsulfonate, sodium perfluorodecylsulfonate; and macrolide antibiotics: azithromycin, clarithromycin, etc. Roxithromycin, quinolone antibiotics: enrofloxacin, flumethin, nalidixic acid, sinoxacin, diflufloxacin hydrochloride, sulfonamide antibiotics: sulfathiazole, sulfapyridine, sulfadiazine, sulfadimidine, sulfamethoxazole, trimethoprim, sulfadimethoxazole, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfadiazine, sulfamethoxypyrimidine, sulfadiazine, sulfachloropyrimidine, sulfabenzoyl, sulfadiazine, sulfachloropyrimidine. The concentration of each standard in the mixed external standard is 100 ng / ml; the sample obtained in step S1 is recorded as the unspiked sample.

[0046] S3. Place the spiked and unspiked samples into separate 50ml centrifuge tubes. Add 5ml of ultrapure water to each tube and vortex for one minute until homogeneous. Let the mixture stand at room temperature for 2 hours to equilibrate. Then add 5ml of acetonitrile, vortex for one minute, mix thoroughly, and sonicate for 10 minutes. Add 1g of NaCl (excess), vortex for one minute, mix thoroughly, and sonicate for 10 minutes. The addition of NaCl at this stage utilizes the principle of salting out; NaCl reduces the solubility of proteins, causing them to aggregate and precipitate from the solution.

[0047] S4. Then, centrifuge in a refrigerated centrifuge at 4°C and 9000 rpm for 10 minutes. Collect the supernatant into a new 15 ml centrifuge tube. Add 5 ml of acetonitrile to the remaining precipitate after collecting the supernatant, vortex for 1 minute, sonicate for 10 minutes, centrifuge at 9000 rpm for 10 minutes, and collect the supernatant. Combine the supernatants. Let the supernatant stand overnight in a -20°C refrigerator. Collect all the supernatant (10 ml) again and place it under a gentle nitrogen stream (the liquid level in the centrifuge tube will ripple slightly under nitrogen blowing, without violent disturbance) until nearly dry. Redissolve in 0.5 ml of methanol.

[0048] S5, After vortex mixing the reconstitution system, move it into a 1.5 ml centrifuge tube, centrifuge at 4℃, 15000 rpm for 10 minutes, take the supernatant into a new 1.5 ml centrifuge tube (discard the bottom part of salt and protein impurities) and stand at -20℃ for a night (a small part of the sample will have flocculent material). Centrifuge again at 4℃, 15000 rpm for 10 minutes, then take the supernatant into a sample injection vial for testing.

[0049] Table 1 Perfluorinated compounds

[0050]

[0051] Table 2 Antibiotics

[0052]

[0053]

[0054] S6, The separation and quantification of the above-mentioned 17 perfluorinated compounds (C4-C18) and 25 antibiotics of the three categories (macrolides, quinolones and sulfonamides) are detected by high performance liquid chromatography mass spectrometry (HPLC-Ms).

[0055] The LC-MS / MS platform of Agilent 1290 Infinity II ultra-high performance liquid chromatography (UHPLC) coupled with 6470 triple quadrupole mass spectrometer (QQQ-MS / MS) is used, equipped with an atmospheric pressure jet stream electrospray ion source (AJS-ESI) and an atmospheric pressure chemical ionization source (APCI). The chromatographic separation uses a Waters ACQUITY UPLC column (BEH C18 column, 2.1 x 100 mm, 1.7 μm).

[0056] The dynamic multiple reaction monitoring (dynamic MRM) in the negative ion mode of AJS-ESI is used for the qualitative and quantitative analysis of perfluorinated compounds, the mobile phases A and B are ultrapure water and methanol respectively, the injection volume is 5 μl, the flow rate is 0.2 ml / min, and the column temperature is 40℃. The elution gradient of the mobile phase is as follows: 0 min, 30% B; 2 min, 40% B; 5 min, 80% B; 7 min, 95% B; 9 min, 100% B; 10 min, 100% B; 11 min, 30% B; re-equilibrate for 2 min. The gas and gas temperature are 325℃ and 350℃ respectively, the flow rate is 10 and 11 ml / min respectively, and the nebulizer pressure is 20 psi.

[0057] The dynamic MRM in positive ion mode was used for qualitative and quantitative analysis of the antibiotics. The mobile phase A and B were ultrapure water (containing 0.1% formic acid) and methanol (containing 0.1% formic acid), respectively. The injection volume was 2 μl, the flow rate was 0.2 ml / min, and the column temperature was room temperature. The elution gradient of the mobile phase was as follows: 0 min, 10% B; 3.5 min, 40% B; 4 min, 48% B; 4.5 min, 30% B; 6 min, 100% B; 10 min, 100% B; 11 min, 10% B; and re-equilibration for 2 min. The gas and gas temperature were 325°C and 350°C, respectively, the flow rate was 10 and 11 ml / min, respectively, and the nebulizer pressure was 20 psi.

[0058] S7, The preparation of the standard can be as follows: 10 mg of single internal standard or external standard was weighed and dissolved in 100 ml of methanol to prepare a high-concentration single internal standard or external standard stock solution (100 μg / ml). A plurality of high-concentration single internal standard or external standard stock solutions were mixed and diluted to a mixed internal standard or mixed external standard with each standard at a concentration of 100 ng / ml (100 internal standard was taken from each single internal standard or external standard stock solution in a volumetric flask, and the volume was adjusted to 100 ml, i.e. diluted 1000 times. The original 100 μg / ml single standard solution was mixed and diluted to a mixed standard solution containing multiple standards, each at a concentration of 100 ng / ml). The prepared mixed internal standard and mixed external standard were used for sample spiking and calibration curve preparation in the experiment.

[0059] The standard sample of the standard curve is prepared by stepwise dilution, and the highest external standard concentration is 50 ng / ml, which is sequentially decreased to 20 ng / ml, 10 ng / ml, 5 ng / ml, 2 ng / ml, 1 ng / ml, 0.5 ng / ml, 0.2 ng / ml, and 0.1 ng / ml. The mixed external standard mother liquor concentration is 100 ng / ml. The preparation method of the 50 ng / ml standard sample is as follows: 250 μl of the 100 ng / ml mixed external standard mother liquor is taken into a sample injection vial, 100 μl of the mixed internal standard with a concentration of 100 ng / ml is added, and 150 μl of methanol is further added. The volume of the standard sample is adjusted to 500 μl, which is consistent with the volume of the resolubilized system after sample pretreatment. The concentration of each lower level external standard solution is diluted from the upper level, i.e., the 50 ng / ml mixed external standard solution is diluted 2 times from the mother liquor (100 ng / ml), the 20 ng / ml mixed external standard solution is diluted 2.5 times from the 50 ng / ml mixed external standard solution, and methanol is used as the diluent. Similar to the preparation of the 50 ng / ml standard sample, the 20 ng / ml standard sample is prepared by adding 200 μl of the 50 ng / ml mixed external standard, 100 μl of the mixed internal standard with a concentration of 100 ng / ml, and 200 μl of methanol. 100 μl of the mixed internal standard (with a concentration of 100 ng / ml) is added to each standard sample, and the volume difference between 500 μl is supplemented with methanol. The standard samples with concentrations of 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, 2 ng / ml, 1 ng / ml, 0.5 ng / ml, 0.2 ng / ml, and 0.1 ng / ml are sequentially prepared.

[0060] The different standard samples are detected by high performance liquid chromatography mass spectrometry (HPLC-Ms) in step S6 to obtain a standard curve.

[0061] In the qualitative process, the parent ion is bombarded into indefinite fragments in the mass spectrometer, two fragments with the highest abundance are selected as the qualitative sub-ion and the quantitative sub-ion, respectively, and the combination of the parent ion and the qualitative and quantitative sub-ion is specific to each substance. Each substance has a specific peak time because it has a certain and stable parent ion and sub-ion size and combination. Each peak in the chromatogram can be separated into a peak corresponding to each substance by spectrum analysis software, and each substance has a unique chromatogram. Therefore, the corresponding test substance is determined according to the specific peak time, and the peak time of each standard substance is shown in Tables 1, 4, and 5.

[0062] The quantitative process is realized on the quantitative analysis software, the peak corresponding to each substance of each sample in the mass spectrum is integrated, the response area is corresponding to the standard curve, and the concentration level corresponding thereto is determined.

[0063] The external standard recovery rate is the difference between the detection result of the sample with added standard and the detection result of the corresponding sample without added standard divided by the amount of the added external standard in the sample with added standard. The closer the external standard recovery rate is to 100%, the less the target substance is lost in the sample pretreatment process and the more likely the target substance can be detected.

[0064] Table 1: Internal standard list and peak time

[0065]

[0066] The perfluorinated compounds need to use negative ion scanning, and the chromatogram is as shown in Figure 1 , where the numbers represent the substances corresponding to the peaks, 1: perfluorobutyric acid; 2: perfluoropentanoic acid; 3: potassium perfluorobutyl sulfonate; 4: perfluorohexanoic acid; 5-6: perfluoroheptanoic acid, sodium perfluorohexyl sulfonate; 7: perfluorooctanoic acid; 8-9: perfluorononanoic acid, perfluorooctyl sulfonate; 10-11: sodium perfluorononyl sulfonate, perfluorodecanoic acid; 12-13: sodium perfluorodecyl sulfonate, perfluoroundecanoic acid; 14: perfluorododecanoic acid; 15: perfluorotetradecanoic acid; 16: perfluorohexadecanoic acid; 17: perfluorooctadecanoic acid; the antibiotics need to use positive ion scanning, and the chromatogram is as shown in Figure 2 , where the numbers represent the substances corresponding to the peaks, 1: sulfadiazine; 2: sulfathiazole; 3: sulfapyridine; 4: sulfamethazine; 5-7: trimethoprim, sulfisoxazole, sulfadimethoxine; 8: sulfamethoxazole; 9: enrofloxacin; 10: sulfachlorpyrazine; 11: difloxacin hydrochloride; 12-15: sulfamethoxazole, sulfisoxazole, sulfadimethoxine, sulfadimethoxazole; 16: sulfabenzamide; 17: sulfapyrazole; 18-19: sulfachlorpyrazine, cinoxacin; 20: sulfadimethoxine; 21: nalidixic acid; 22: flumequine, azithromycin, clarithromycin, roxithromycin.

[0067] The general external standard method refers to using a pure product of a component to be measured as a control substance, diluting the pure product to obtain a standard curve, each concentration of each substance has a different response on the mass spectrum, and a standard curve can be obtained according to the relationship between the concentration of each substance and the response of the mass spectrum. Under the same conditions, accurately sample and sample solution of the same volume as the control solution, according to the signal response of the component to be measured, the concentration thereof can be found from the standard curve to realize the quantification of the component to be measured.

[0068] The method additionally sets a sample group with added standard sample (as a control group), each group has two samples and is weighed from the same marine organism sample, and one of the two same samples is added with mixed external standard. Compared with the sample without added standard, the accuracy of detecting the measured substance is determined by the loss degree of the external standard. During the sample pretreatment process, the specific number of samples depends on the specific situation. In the case of adding internal standard to each sample to correct the signal loss of the measured substance, two groups are additionally set as sample standard groups. The added standard sample and the non-added standard sample in the sample standard group should be weighed from the same sample, and a known concentration and volume of mixed external standard is added to the added standard sample in the sample standard group to test whether the signal of the measured substance after internal standard correction reaches the expected recovery rate interval during the sample pretreatment process. In simple terms, we do not know the concentration level of the measured substance in the measured sample. A certain amount of external standard is added to the added standard sample in the sample standard group, and the difference between the measured concentration of the added standard sample and the measured concentration of the non-added standard sample should be the amount of added external standard without considering loss. However, after a series of pretreatment operations, the measured substance may be lost. At this time, the ratio of the aforementioned difference to the amount of added external standard is defined as the external standard recovery rate, which should be within an acceptable interval, preferably 60-130%. The data of the batch processing with the external standard recovery rate within this interval is considered as reliable data. The two samples in the sample standard group are weighed from the same sample, and the operations are consistent except for the difference whether to add mixed external standard. After the same sample pretreatment, the difference between the measured result of the added standard sample and the measured result of the non-added standard sample is the sample standard recovery rate, which is the ratio of the theoretical value of the added standard substance. Only when the sample standard recovery rate is within an acceptable space, the method for detecting the substance and the batch of substances is considered reliable. The sample standard group is set and the standard recovery rate is used to determine whether the target substance can be well detected after pretreatment (if 10 ng of external standard is added and only 1 ng is measured, it means that the target substance has a large degree of loss during sample pretreatment, and by analogy, it can be inferred that the method cannot detect the true content of the target substance in the sample). In addition, it can also be used to determine whether the detection results obtained by this batch of pretreatment are reliable (because experimental errors may be caused by experimental operations and other problems, and small changes or mistakes in the experimental process may affect the experimental results. The sample standard recovery rate can be used to determine whether the sample pretreatment is qualified and the results obtained are reliable), which plays a quality control role. If the recovery rate is not within the 60-130% interval, it means that an error has occurred during the experiment, and the results obtained are not reliable and need to be retested.

[0069] The internal standard is selected from the isotopes of the target analytes. In the present experiment, 1-2 isotopes of the target analytes of each class of substances (sulfonamide antibiotics, quinolone antibiotics, macrolide antibiotics, perfluorocarboxylic acids, and perfluorosulfonic acids) are selected as the internal standard of the class of substances. The target analyte and the isotope serving as the internal standard have similar chemical structures, and thus have similar behaviors in the pretreatment and instrument detection processes. The sample pretreatment process can cause loss of the signal of the target analyte. Due to the instability of the instrument, the response value also fluctuates when the same sample is continuously injected. In the present experiment, the internal standard is used to correct the loss of the signal of the target analyte in the pretreatment and the signal fluctuation caused by the instrument. The average of the measured response values of all samples in the standard curve sample is taken as the standard, and the measured response value of the internal standard in each sample is divided by the average to obtain the internal standard recovery rate of each sample. The internal standard recovery rate, like the external standard recovery rate, is used as a quality control index and needs to be in the range of 60-130%, indicating that the detection result is accurate and reliable. If it is not in the range, the experiment needs to be repeated.

[0070] Example 2

[0071] When the pretreatment method is established, first, the experimental conditions are adjusted to achieve good internal standard recovery rate, and then the sample spiked control group is set to screen the external standard with a recovery rate meeting the standard (60-130%) to complete the establishment of the method. When the method is optimized, part of the experimental operation and effect are as follows:

[0072] Scheme 1: Only acetonitrile is used as the extractant, the supernatant is taken for extraction, and nitrogen blowing is performed to dryness, and the operation of redissolving for instrument is performed without adding any other reagents and experimental steps. The specific operation is as follows: refer to Example 1 to weigh the sample and add the standard, add 5 ml of ultrapure water to the 50 ml centrifuge tube into which the sample and the standard have been added, vortex for 1 minute to mix uniformly, and stand at room temperature for 2 hours to balance the system. Then add 5 ml of acetonitrile, vortex for 1 minute to mix uniformly, and ultrasonic for 10 minutes. Then place it in a refrigerated centrifuge at 4℃ and 9000 rpm for 10 minutes, take the supernatant into a new 15 ml centrifuge tube. After taking the supernatant, add 5 ml of acetonitrile to the remaining precipitate, vortex for 1 minute, and ultrasonic for 10 minutes, and centrifuge at 9000 rpm for 10 minutes to take the supernatant, and combine the supernatants. The supernatant is placed in a -20℃ refrigerator overnight, and then the entire supernatant (about 10 ml) is taken again, and the supernatant is placed under a gentle nitrogen stream to blow to near dryness, and redissolved in 0.5 ml of methanol.

[0073] Scheme 2: refer to the experimental steps of Example 1. Scheme 3: 1 g of magnesium sulfate and 0.5 g of sodium chloride are added in the first extraction, and 70 mg of ethylenediamine-N-propylsilane (PSA, which can adsorb polar organic acids and some sugar and lipid impurities) is added in the second extraction. The supernatants of the two extractions are combined, nitrogen blowing is performed to dryness, and then redissolved for detection.

[0074] The specific operation is as follows: the sample is weighed and the standard sample is added according to the reference example 1, 5 ml of ultrapure water is added to the 50 ml centrifuge tube into which the sample and the standard sample have been added, and then vortexed for 1 minute to mix uniformly, and the system is balanced at room temperature for 2 hours. 5 ml of acetonitrile is added again, vortexed for 1 minute to mix uniformly, and then ultrasonicated for 10 minutes. 1 g of magnesium sulfate and 0.5 g of sodium chloride are added, vortexed for 1 minute to mix uniformly, and then ultrasonicated for 10 minutes. Then, it is placed in a refrigerated centrifuge and centrifuged at 4°C and 9000 rpm for 10 minutes, and the supernatant is taken into a new 15 ml centrifuge tube. After taking the supernatant, 5 ml of acetonitrile is added to the remaining precipitate, vortexed for 1 minute, and then ultrasonicated for 10 minutes. 70 mg of ethylenediamine-N-propylsilane is added, vortexed for 1 minute to mix uniformly, and then ultrasonicated for 10 minutes. It is centrifuged at 9000 rpm for 10 minutes, and the supernatant is taken. The supernatants are combined. The supernatant is left in the -20°C refrigerator overnight, and then the entire supernatant (about 10 ml) is taken again. The supernatant is blown to near dryness under a gentle stream of nitrogen, and then redissolved in 0.5 ml of methanol.

[0075] Scheme four, 1 g of magnesium sulfate and 0.5 g of sodium chloride are added during the first extraction, and 150 mg of magnesium sulfate and 100 mg of ethylenediamine-N-propylsilane (PSA, which can adsorb polar organic acids and some sugar and lipid impurities) are added during the second extraction. The supernatants obtained after the two extractions are combined, blown to dryness under nitrogen, and then redissolved for testing.

[0076] The specific operation is as follows: the sample is weighed and the standard sample is added according to the reference example 1, 5 ml of ultrapure water is added to the 50 ml centrifuge tube into which the sample and the standard sample have been added, and then vortexed for 1 minute to mix uniformly, and the system is balanced at room temperature for 2 hours. 5 ml of acetonitrile is added again, vortexed for 1 minute to mix uniformly, and then ultrasonicated for 10 minutes. 1 g of magnesium sulfate and 0.5 g of sodium chloride are added, vortexed for 1 minute to mix uniformly, and then ultrasonicated for 10 minutes. Then, it is placed in a refrigerated centrifuge and centrifuged at 4°C and 9000 rpm for 10 minutes, and the supernatant is taken into a new 15 ml centrifuge tube. After taking the supernatant, 5 ml of acetonitrile is added to the remaining precipitate, vortexed for 1 minute, and then ultrasonicated for 10 minutes. 70 mg of ethylenediamine-N-propylsilane is added, vortexed for 1 minute to mix uniformly, and then ultrasonicated for 10 minutes. It is centrifuged at 9000 rpm for 10 minutes, and the supernatant is taken. The supernatants are combined. The supernatant is left in the -20°C refrigerator overnight, and then the entire supernatant (about 10 ml) is taken again. The supernatant is blown to near dryness under a gentle stream of nitrogen, and then redissolved in 0.5 ml of methanol.

[0077] The subsequent operation is shown in example 1.

[0078] Except for the second scheme, there is no step of freezing the extraction liquid and the reconstitution system in the -20℃ refrigerator. The specific experimental results are shown in Table 3 by taking the recovery rate (%) of sulfamethoxazole-D4 internal standard as an example. The concentration and volume of the mixed internal standard added in all the markers and samples are the same. Due to the unstable response of the instrument, the response of the target substance will fluctuate a little when a sample is continuously injected. The average value of the internal standard response values of the nine standard samples of the standard curve is taken as the standard. The internal standard recovery rate is obtained by dividing the internal standard response value of each sample by the average response value of the standard curve.

[0079] Table 3

[0080]

[0081] The results show that the addition of the extraction agent in the extraction step can indeed improve the recovery rate, but the effect is limited. The experimental process is increased due to the addition of multiple substances, which makes the effect limited and unstable. The experimental results of the second scheme are better and stable.

[0082] Example 3

[0083] The preparation method and principle of the standard curve are shown in Example 1. The concentrations of the standard samples are sequentially reduced to 50, 20, 10, 5, 2, 1, 0.5, 0.2, and 0.1 ng / ml. The concentration of each external standard is 20 ng / ml. The standard samples of different concentrations are detected according to the detection conditions of the high performance liquid chromatography mass spectrometry (HPLC-Ms) in Example 1. Then the standard curve is drawn by software.

[0084] In the standard curve, the concentration of each test substance is linearly related to the response in the mass spectrometer. That is, as the concentration increases, the response value of the instrument increases. The standard curve is the functional relationship between the concentration of the standard substance and the response of the instrument. The linear fitting function of the relationship between the concentrations and responses of the nine standard samples is used to calculate the linear correlation coefficient R 2 As a correlation coefficient, it describes the correlation between the two. The larger the linear correlation coefficient, the stronger the linear relationship between the response and the concentration of the substance in the mass spectrometer. The limit of detection here is based on the standard curve. The limit of detection (LOD) = 3.3σ / S, where σ is the deviation of the response value (which can be obtained by measuring the standard deviation of the blank value or the remaining standard deviation of the standard curve or the standard deviation of the intercept), and S is the slope of the standard curve. The limit of detection can be calculated based on the standard curve. Results below the limit of detection are considered not detected and are marked as ND. The lower the limit of detection, the more sensitive the method is to the substance.

[0085]

[0086]

[0087] Table 4 perfluorinated compound quality control

[0088] Table 5 antibiotic quality control

[0089]

[0090]

[0091] All the detected substances have good linearity in the range of 0.1-50 ng / ml, and the correlation coefficients are all greater than 0.99, and the method detection limits of the substances range from 0.001 ng / g to 0.5 ng / g dry weight. The external standard combined with the internal standard method is used for quantification, the external standard recovery is 61.8-122.42%, the internal standard recovery is 61-117%, and the relative standard deviations are all less than 18%.

[0092] The application uses acetonitrile as an extractant, adopts ultrasonic-assisted extraction, removes protein by salting out with sodium chloride, and removes fat by freezing centrifugation to realize the two links of extraction and purification. Only acetonitrile and sodium chloride are added in the operation process, the addition of other substances is reduced, the matrix interference effect is reduced, the use of solid-phase extraction columns or dispersed extractants is omitted, and the experimental cost is reduced. If the Waters Oasis HLB solid-phase extraction column is used to realize concentration and purification, the cost of 30 yuan is increased for one sample; if the purification package in the QuEChERS method is used to remove impurities, the cost of 15 yuan is increased for one sample. For large batch sampling, a large amount of cost is saved.

[0093] The application adopts a pretreatment method, can simultaneously treat and detect a plurality of perfluorinated compounds and antibiotics in a complex biological matrix, and can ensure that both types of substances have high recovery rates (meet the standard range). Different chemical substances have different chemical properties and octanol-water partition coefficients due to differences in relative molecular mass, functional groups and structures, and have different distribution ratios in different extraction systems. Previous studies usually treat and detect one type of chemical substances with similar chemical properties. The method of the application simultaneously treats and detects two types of different chemical substances. Although perfluorinated compounds and antibiotics need to be scanned in different scanning modes of ESI ion source due to different ionization characteristics, and need to be sampled twice, one experimental operation can realize the pretreatment of the two types of substances, compared with the pretreatment of the two types of substances respectively, the workload is greatly reduced, and the experimental cost is also reduced.

[0094] The external standard recovery rate and the internal standard recovery rate are simultaneously used as the quality control index, the former is used for quality control of the target substance detection feasibility and batch processing quality, and the latter is used for quality control of batch processing quality and instrument fluctuation. In the experiment setting, the external standard recovery rate and the internal standard recovery rate are both in a reasonable and stable interval (both are 60-130%), and are considered as effective and reliable batch processing and can be repeated.

[0095] The above is only a preferred embodiment of the present application, and is not intended to limit the present application in other forms. Any person skilled in the art can modify or change the above disclosed technical content into equivalent embodiments with equivalent changes. However, any simple modification, equivalent change and modification of the above embodiments made according to the technical essence of the present application without departing from the technical solution content of the present application still belongs to the protection scope of the technical solution of the present application.

Claims

1. A method for detecting 17 perfluorinated compounds and 25 antibiotics in marine biological samples, characterized in that, It includes the following steps: S1. Weigh the marine biological sample powder and add internal standard to each sample; S2. Take one sample from each of any two marine biological samples from S1, add the internal standard as in step S1, and then add the mixed external standard to make a spiked sample. S3. Add the above samples to ultrapure water and vortex mix them evenly. Let them stand at room temperature for a period of time to balance the system. Add acetonitrile, vortex mix evenly, and then sonicate. Add NaCl, vortex mix evenly again, and then sonicate. S4. Then centrifuge and take the supernatant into a new centrifuge tube; Add acetonitrile to the remaining portion, vortex, sonicate, centrifuge, and take the supernatant. Combine the two supernatants, let the supernatant stand in a -20°C refrigerator to solidify, take the supernatant again, blow the supernatant to near dryness, and redissolve it in methanol. S5. After vortexing the reconstituted system until homogeneous, centrifuge, collect the supernatant and let it stand at -20℃ to solidify, then centrifuge again and collect the supernatant into a vial for testing. S6. Detection was performed using high performance liquid chromatography-mass spectrometry; S7. Prepare external standard standards of different concentrations for plotting standard curves. An internal standard is added to each standard, and the results are detected by high performance liquid chromatography-mass spectrometry. S8. Based on the chromatogram of the obtained standard, determine the corresponding chromatogram of the sample group to be tested, thereby obtaining the qualitative and quantitative results of perfluorinated compounds and antibiotics in the sample to be tested; The 17 perfluorinated compounds are: perfluorobutyric acid, perfluorovaleric acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluorostachyic acid, perfluorobutylsulfonate potassium, perfluorohexylsulfonate sodium, perfluorooctylsulfonate sodium, perfluorononylsulfonate sodium, and perfluorodecylsulfonate sodium; the 25 antibiotics are: macrolide antibiotics: azithromycin, clarithromycin, and roxithromycin; Quinolone antibiotics: enrofloxacin, flumethin, nalidixic acid, sinoxacin, diflufloxacin hydrochloride; Sulfonamide antibiotics: sulfathiazole, sulfapyridine, sulfadiazine, sulfadimethoxine, sulfamethoxazole, trimethoprim, sulfadimethoxine, sulfadimethoxazole, sulfamethazine, sulfamethoxypyrimidine, sulfamethoxypyrimidine, sulfadimethoxazole, sulfadiazine, sulfamethoxypyrimidine, sulfadiazine, sulfamethoxypyrimidine, sulfadiazine, sulfabenzoyl, sulfadimethoxazole, sulfachlorpyridazine.

2. The method as described in claim 1, characterized in that, The internal standards are sodium perfluoro-1-hexane[18O2]sulfonate, sodium perfluorooctanesulfonate-13C8, perfluorooctanoic acid-13C8, perfluorononanoic acid-13C9, roxithromycin-D7, azithromycin-D3, enrofloxacin-D5, sulfamethoxazole-D4, and sulfadiazine-D4.

3. The method as described in claim 1, characterized in that, In step S1, the amount of sample weighed is 0.1~1g, the concentration of each internal standard is 50~100ng / ml, and the total amount of internal standard added is 50~100μl; In step S2, the external standard includes perfluorinated compounds: perfluorobutyric acid, perfluorovalerate, perfluorohexanoic acid, perfluoroheptanoic acid, and perfluorooctanoic acid.

4. The method as described in claim 1, characterized in that, In step S2, the concentration of each external standard added to the spiked sample is 50~100 ng / ml, and the total amount added is 50~100 μl.

5. The method as described in claim 1, characterized in that, In step S3, the amount of ultrapure water and acetonitrile added is equal, and the amount of NaCl added is 10% by mass / volume percentage in the mixture, calculated as g / ml; the ultrasonication time is 5~15 min.

6. The method as described in claim 1, characterized in that, In step S4, the centrifugation conditions are 4°C and 9000~12000 rpm for 5~10 minutes. In step S5, the centrifugation conditions are 4°C and 12000~15000 rpm for 10~20 minutes.

7. The method as described in claim 1, characterized in that, In step S6, the conditions for qualitative and quantitative analysis of perfluorinated compounds by the high-performance liquid chromatography-mass spectrometry (HPLC-MS / MS) instrument are as follows: dynamic multiple reaction monitoring in AJS-ESI negative ion mode is used, mobile phases A and B are ultrapure water and methanol, respectively, the injection volume is 5 μl, the flow rate is 0.2 ml / min, and the column temperature is 40 ℃ constant temperature. The mobile phase elution gradient was as follows: 0 min, 30% B; 2 min, 40% B; 5 min, 80% B; 7 min, 95% B; 9 min, 100% B; 10 min, 100% B; 11 min, 30% B; reequilibration for 2 min. The gas and sheath gas temperatures were 325°C and 350°C, respectively, with flow rates of 10 and 11 ml / min, and the sprayer pressure was 20 psi.

8. The method as described in claim 1, characterized in that, In step S6, the conditions for qualitative and quantitative detection of antibiotics are as follows: dynamic multiple reaction monitoring in AJS-ESI positive ion mode is used, mobile phases A and B are ultrapure water containing 0.1% formic acid and methanol containing 0.1% formic acid, respectively, the injection volume is 2 μl, the flow rate is 0.2 ml / min, and the column temperature is room temperature. The mobile phase elution gradient was as follows: 0 min, 10% B; 3.5 min, 40% B; 4 min, 48% B; 4.5 min, 30% B; 6 min, 100% B; 10 min, 100% B; 11 min, 10% B; reequilibration for 2 min. The gas and sheath gas temperatures were 325°C and 350°C, respectively, with flow rates of 10 and 11 ml / min, and the sprayer pressure was 20 psi.

9. The method as described in claim 1, characterized in that, In step S8, the ratio of the measured concentration of the spiked sample minus the measured concentration of the corresponding sample without external standard to the theoretical value of the standard substance added to the spiked sample is the spike recovery rate. If the spike recovery rate is within the range of 60-130%, it indicates that the test results are accurate and reliable; if it is not within this range, the experiment needs to be repeated. The average of the measured response values ​​of the internal standard in all samples of the standard curve sample is used as the standard. The internal standard recovery rate of each sample is obtained by dividing the measured response value of the internal standard in each sample by the aforementioned average value. If the internal standard recovery rate is within the range of 60-130%, the test results are accurate and reliable. If it is not in this range, the experiment needs to be repeated.

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