CN109709227B - Method for measuring residual quantity of robenidine hydrochloride and metabolites thereof in aquatic product by high performance liquid chromatography-tandem mass spectrometry - Google Patents
Method for measuring residual quantity of robenidine hydrochloride and metabolites thereof in aquatic product by high performance liquid chromatography-tandem mass spectrometry Download PDFInfo
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Abstract
The invention discloses a method for determining residual quantity of robenidine hydrochloride and metabolites thereof in aquatic products based on time segmentation technology by high performance liquid chromatography-tandem mass spectrometry, which comprises the following steps: A. weighing aquatic product muscle tissues, adding acetonitrile containing 1% formic acid, anhydrous magnesium sulfate and ODS, extracting and purifying; B. adopting high performance liquid chromatography-tandem mass spectrometry to switch positive and negative ion modes and detect the residual amounts of robenidine hydrochloride, p-chlorobenzoic acid and p-chlorobenzamide acetic acid in the muscle of the aquatic product at different time periods; C. quantification by a standard curve method: the robenidine hydrochloride is quantified by an internal standard method, and the robenidine hydrochloride-d8As an internal standard, the p-chlorobenzoylglycine and p-chlorobenzoic acid are quantified by an external standard method. The method has the advantages of simple operation, low matrix effect, low detection limit, and high accuracy and precision.
Description
Technical Field
The invention belongs to the technical field of aquatic product safety detection, and particularly relates to a method for determining residual amounts of robenidine hydrochloride and metabolites thereof in aquatic products based on a time segmentation technology.
Background
Chlorobenzene guanidine hydrochloride (1, 3-bis (p-chlorobenzylideneamino) guanidine hydrochloride) belongs to a guanidyl derivative and is an achiral chemical synthetic drug. Chlorobenzeneguanidine hydrochloride-d8Is formed by replacing 8 hydrogen atoms on two benzene rings of robenidine hydrochloride by heavy hydrogen atoms. Robenidine hydrochloride is approved in europe as a feed additive for the control of coccidiosis in broiler chickens, turkeys and rabbits. Robenidine hydrochloride is used for treating fish sporulation diseases in China. Research shows that the preventive feeding of the robenidine hydrochloride can completely prevent enteritis of crucian caused by coccidium. The robenidine hydrochloride is widely used in animal-derived foods (including aquatic products), residues can be easily caused in the animal-derived foods (including aquatic products), and long-term intake of the robenidine hydrochloride-containing animal-derived foods (including aquatic products) by human bodies can easily cause the accumulation of drugs in the human bodies, thereby causing great harm to the health of human bodies. To avoid the need for the consumer to consume the productHealth risks caused by food of animal origin (including aquatic products) with chlorbenzoguandine hydrochloride residues, the european food safety agency has established a daily allowable intake (ADI) of chlorbenzoguandine hydrochloride of 0.11mg/kg body weight based on toxicological data. The European Union also established that the maximum residual limit value (MRLs) of robenidine hydrochloride in the liver, kidney, skin and fat (wet weight) of animal-derived foods other than broiler chickens, turkeys and rabbits was 50. mu.g/kg, and the maximum residual limit value in other foods (including aquatic products) was 5. mu.g/kg. Therefore, the problem of the residue of the robenidine hydrochloride in aquatic products is more and more concerned by people.
In order to more comprehensively evaluate the risk of aquatic products containing robenidine hydrochloride residue, guarantee the quality safety of the aquatic products and further protect the health of human beings, a reliable qualitative and quantitative method is provided for analyzing the robenidine hydrochloride and metabolite residue in the aquatic products, but only one ultra-high performance liquid chromatography literature report about the detection of robenidine hydrochloride and metabolite thereof in aquatic product muscles is found at home and abroad at present, but the detection Limit (LOD) and quantitative Limit (LOQ) of robenidine hydrochloride, p-chlorobenzoylglycine and p-chlorobenzoic acid in the method are respectively 25 mu g/kg and 50 mu g/kg, the requirement of European Union on the detection of robenidine hydrochloride residue in the aquatic products cannot be met, and the requirement of the maximum limit (MRLs) is 5 mu g/kg. Therefore, a detection method for rapidly determining the residual quantity of robenidine hydrochloride, metabolites thereof, p-chlorobenzoic acid and p-chlorobenzamide acetic acid in aquatic products, which has higher sensitivity and convenient operation, is needed to be developed.
At present, no report or patent application of a method for measuring residual amounts of parachlorobenzoic acid and parachlorobenzamide acetic acid of robenidine hydrochloride and metabolites thereof in aquatic products by high performance liquid chromatography-tandem mass spectrometry is found at home and abroad.
Disclosure of Invention
The invention aims to provide a method for measuring the residual quantity of robenidine hydrochloride, and metabolites thereof, namely p-chlorobenzoic acid and p-chlorobenzamide acetic acid in an aquatic product by using a high performance liquid chromatography-tandem mass spectrometry based on a time segmentation technology. The method for measuring the residual quantity of the robenidine hydrochloride, the metabolites thereof, namely, the p-chlorobenzoylglycine and the p-chlorobenzoic acid in the muscle tissue of the aquatic product is established by adopting a high performance liquid chromatography-heating electrospray ionization source-series quadrupole mass spectrometer and a method for presetting the segmentation time.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for measuring residual quantity of robenidine hydrochloride and metabolites thereof in aquatic products based on time segmentation technology by high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
A. and (3) processing a muscle sample: weighing homogeneous muscle sample, adding robenidine hydrochloride-d8Adding an extracting agent A and anhydrous magnesium sulfate as internal standards of robenidine hydrochloride in sequence, carrying out vortex oscillation, centrifuging, taking supernatant, repeatedly extracting residues, combining extracting solutions, adding the anhydrous magnesium sulfate and a purifying agent B, carrying out vortex oscillation and centrifuging, taking supernatant, carrying out nitrogen blow-drying, dissolving residues by using a mixed solution of methanol and water with a volume ratio of 70:30-80:20, filtering by using a filter membrane with the aperture not larger than 0.22 mu m, and detecting filtrate by using HPLC-HESI-MS/MS;
B. conditions for HPLC analysis: a chromatographic column: c18 column (100 mm. times.2.1 mm,3.5 μm); column temperature: 30 ℃, flow rate: 0.25 mL/min; mobile phase: the organic phase was methanol, the aqueous phase was 0.1% formic acid in water, gradient elution procedure: the initial gradient is methanol with the volume concentration of 25-35%, the methanol with the volume concentration of 85-95% is linearly increased within 4.5min, the initial gradient is kept for 2.5min, the methanol with the volume concentration of 25-35% is linearly decreased within 0.1min, and finally the initial gradient is maintained for 0.9min, and the sample injection volume is 20 mu L;
C. HESI-MS/MS analysis conditions: heating the electrospray ion source, and switching between a positive ion mode and a negative ion mode; the detection mode adopts a selective reaction monitoring mode; positive ion mode spray voltage: 3500V, negative ion mode spray voltage: -3000 v; temperature of the evaporated gas: 380 ℃; sheath gas pressure 50 arb; auxiliary gas pressure 15 arb; collision gas and pressure: the purity of argon is more than or equal to 99.999 and 1.5m Torr; ion transport capillary temperature: 350 ℃; the half-width of Q1 was set to 0.7, the half-width of Q3 was set to 0.7, and the detection parameters were as follows:
quantitative ions
D. Quantification by a standard curve method: the robenidine hydrochloride adopts an internal standard method to establish a standard curve, and the p-chlorobenzoic acid and the p-chlorobenzamide acetic acid adopt an external standard method to establish a standard curve, and the concentration of the sample is calculated according to the standard curve.
Furthermore, the time segment 1 is 0-5.3min, the time segment 2 is 5.3-6.0min, and the time segment 3 is 6.0-8.0 min.
Preferably, the extractant A is acetonitrile containing 1% formic acid.
Preferably, the scavenger B is octadecylsilane bonded silica (ODS).
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the invention adopts high performance liquid chromatography-tandem mass spectrometry to carry out ion mode switching and detection in different time periods, the method is simple and convenient to operate, the detection limit is low, the sensitivity, the accuracy and the precision are high, and the results are shown in tables 2 and 3.
(2) The method can be used for measuring the concentrations of the robenidine hydrochloride and metabolites thereof, namely p-chlorobenzoic acid and p-chlorobenzamide acetic acid in the muscle tissues of the aquatic animals after the robenidine hydrochloride is administrated, and further used for researching the pharmacokinetics and residue elimination rule of the robenidine hydrochloride in the aquatic animals.
Drawings
FIG. 1 shows different extractants of p-chlorobenzeneguanidine hydrochloride and chlorobenzeneguanidine hydrochloride-d8The effect on the recovery of p-chlorobenzoylamino acetic acid and p-chlorobenzoic acid.
FIG. 2 shows different purificant for chlorobenzeneguanidine hydrochloride and chlorobenzeneguanidine hydrochloride-d8The effect on the recovery of p-chlorobenzoylamino acetic acid and p-chlorobenzoic acid.
FIG. 3 is an HPLC-HESI-MS/MS chromatogram of spiked samples of robenidine hydrochloride, p-chlorobenzamide acetic acid and p-chlorobenzoic acid in muscle tissue of Chinese grass fish in example 2. Chromatogram of 1-p-chlorobenzoic acid, chromatogram of 2-p-chlorobenzamide acetic acid, chromatogram of 3-chlorobenzeneguanidine hydrochloride, chromatogram of 4-chlorobenzeneguanidine hydrochloride-d8And (4) chromatograms.
FIG. 4 is an HPLC-HESI-MS/MS chromatogram of robenidine hydrochloride, p-chlorobenzamide acetic acid and p-chlorobenzoic acid in muscles of grass carps after oral gavage of example 3. Chromatogram of 1-p-chlorobenzoic acid, chromatogram of 2-p-chlorobenzamide acetic acid, chromatogram of 3-chlorobenzeneguanidine hydrochloride, chromatogram of 4-chlorobenzeneguanidine hydrochloride-d8And (4) chromatograms.
FIG. 5 is the change of the concentration of robenidine hydrochloride and its metabolites in chlorobenzoic acid in the muscle samples of grass carp after oral gavage of example 3.
Detailed Description
Example 1 optimization of method for determining residual quantity of robenidine hydrochloride and metabolites thereof in aquatic products by high performance liquid chromatography-tandem mass spectrometry
1. Optimization of mass spectrometry conditions
The method comprises the steps of respectively injecting 10mg/L of robenidine hydrochloride, robenidine hydrochloride-d 8, p-chlorobenzamide acetic acid and p-chlorobenzoic acid into a mass spectrum through an injection pump, and selecting a proper detection mode according to the responsivity of the target in a positive ion mode and a negative ion mode, wherein the results show that the responsivity of the robenidine hydrochloride and the robenidine hydrochloride-d 8 in the positive ion mode is 5 times and 3 times that in the negative ion mode. And the response values of p-chlorobenzoylamino acetic acid and p-chlorobenzoic acid in the negative ion mode are 2 times and 1 time that in the positive ion mode. Thus, chlorobenzenediamine hydrochloride and chlorobenzenediamine hydrochloride-d8The analysis was performed in positive ion mode, while p-chlorobenzoylamino acetic acid and p-chlorobenzoic acid were analyzed in negative ion mode.
2. Determination of segment time
As the responsivity of the mass spectrometric detection of the robenidine hydrochloride and the robenidine hydrochloride-d 8 in the positive ion mode is high, and the responsivity of the mass spectrometric detection of the p-chlorobenzamide acetic acid and the p-chlorobenzoic acid in the negative ion mode is high. If positive and negative ions are adopted to switch, detect and analyze in real timeThe analyte can reduce the responsiveness of the analyte, and further the detection limit of the analyte can be increased, which is not beneficial to the qualitative and quantitative detection of low-content analyte in the sample. The present application tried different elution procedures, eventually the following were chosen: the initial gradient is methanol with the volume concentration of 25-35%, the methanol with the volume concentration of 85-95% is linearly increased within 4.5min, the methanol is kept for 2.5min, the methanol with the volume concentration of 25-35% is linearly reduced within 0.1min, the methanol with the volume concentration of 0.9min is finally maintained, the analyte can be detected by adopting a segment time method after being fully separated, the analyte is divided into 3 time segments for detection according to the peak-off time of the analyte, the p-chlorobenzamide acetic acid is detected in the first time segment, the peak-off time is 0-5.3min, the robenidine hydrochloride and the robenidine hydrochloride-d are detected in the second time segment8The peak-off time is 5.3-6.0min, and the peak-off time is 6.0-8.0min when p-chlorobenzoic acid is detected in the third time period.
3. Screening of the extractant
The extractants and purifiers suitable for extracting and purifying samples were evaluated and screened by muscle spiked samples (50 μ g/kg, n ═ 3) at spiked recoveries of robenidine hydrochloride, p-chlorobenzamide acetic acid, and p-chlorobenzoic acid.
The extraction agents acetonitrile, ethyl acetate, acetonitrile contain 0.1% formic acid, ethyl acetate contains 0.1% formic acid, acetonitrile contains 1% formic acid, ethyl acetate contains 1% formic acid, and the screening results are shown in figure 1. As can be seen from figure 1, the extraction recovery rate of acetonitrile containing 1% formic acid and ethyl acetate containing 1% formic acid, p-chlorobenzeneguanidine hydrochloride, p-chlorobenzamide acetic acid and p-chlorobenzoic acid is higher than that of other extracting agents. Since ethyl acetate containing 1% formic acid extracts more fat in the sample than acetonitrile containing 1% formic acid, and the fat in the sample has matrix effect on mass spectrometry of analytes, namely robenidine hydrochloride, p-chlorobenzamide acetic acid and p-chlorobenzoic acid, which can affect the accurate quantification of the analytes, particularly on some fishes with high fat, such as eel, the acetonitrile containing 1% formic acid is finally preferred as the extracting agent of the application. In addition, methanol, 0.1% formic acid in methanol and 1% formic acid in methanol are also selected as extracting agents, but the sample extracting solution is difficult to concentrate and is not adopted.
4. Screening of purification Agents
Three purifiers, octadecylsilane chemically bonded silica ODS (50mg), N-propylethylenediamine PSA (50mg) and multiwalled carbon nanotube MWCNT15(50mg), were used for purification of the extract of the spiked sample (50. mu.g/kg), with recovery as the evaluation criterion, and the results are shown in FIG. 2. The results showed that the recovery rates of robenidine hydrochloride, p-chlorobenzamide acetic acid and p-chlorobenzoic acid were more than 80% when ODS was used as a scavenger, and less than 75% when PSA or MWCNT15 was used as a scavenger. Therefore, ODS is preferred as a scavenger in the present application.
Example 2
A method for measuring residual quantity of robenidine hydrochloride and metabolites thereof in aquatic products based on time segmentation technology by high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
A. weighing 1g of a homogeneous grass carp muscle sample, adding robenidine hydrochloride, p-chlorobenzamide acetic acid and p-chlorobenzoic acid standard solution into a 15mL centrifuge tube to ensure that the concentrations of the robenidine hydrochloride, the p-chlorobenzamide acetic acid and the p-chlorobenzoic acid in the sample are respectively 1,3, 50, 100, 400 mu g/kg, 2.5, 5, 50, 100, 400 mu g/kg, 5, 10, 50 and 50 mu g/kg, and respectively adding 20 mu L of 1mg/L robenidine hydrochloride-d8Shaking the internal standard solution for 30s, adding 5mL of extractant acetonitrile containing 1% formic acid, shaking for 30s, ultrasonically extracting for 5min, adding 1g of anhydrous magnesium sulfate to remove water and part of fat in the sample, and vortexing. Centrifuging at 8000r/min for 5min, placing the supernatant in another 15mL centrifuge tube, re-extracting the residue once, mixing the two extractive solutions, adding 500mg anhydrous magnesium sulfate and 100mg purificant octadecylsilane chemically bonded silica (ODS) into the extractive solution, performing vortex oscillation for 30s, centrifuging at 8000r/min for 5min, transferring the supernatant into 15mL centrifuge tube, blowing nitrogen on a nitrogen blower at 50 deg.C, dissolving the residue with 1mL mixed solution of methanol and water solution (70:30, v/v), performing vortex oscillation for 30s, 10000r/min, centrifuging for 5min, filtering with 0.22 μm filter membrane, and analyzing the filtrate with HPLC-HESI-MS/MS.
B. Conditions for HPLC analysis: a chromatographic column: waters Symmetry C18 chromatography column (100 mm. times.2.1 mm,3.5 μm); column temperature: 30 ℃, flow rate: 0.25 mL/min; mobile phase: the organic phase was methanol, the aqueous phase was 0.1% formic acid in water, gradient elution procedure: the initial gradient was 30% methanol by volume, the linear increase to 90% methanol by volume within 4.5min, the hold time was 2.5min, the linear decrease to 30% methanol by volume within 0.1min, and the hold time was last 0.9 min. The injection volume was 20. mu.L.
C. HESI-MS/MS analysis conditions: heating an electrospray ion source (HESI), switching between a positive ion mode and a negative ion mode in time periods; the detection mode adopts a selective reaction monitoring mode (SRM); positive ion mode spray voltage: 3500V, negative ion mode spray voltage: -3000 v; temperature of the evaporated gas: 380 ℃; sheath gas pressure 50 arb; auxiliary gas pressure 15 arb; collision gas and pressure: the purity of argon is more than or equal to 99.999 and 1.5m Torr; ion transport capillary temperature: 350 ℃; the half-width of Q1 was 0.7, the half-width of Q3 was 0.7, and the specific detection parameters were as follows:
TABLE 1 specific test parameters
Quantitative ions
D. The quantification of the robenidine hydrochloride, the p-chlorobenzoic acid and the p-chlorobenzamide acetic acid in the aquatic products adopts a standard curve method. The standard curve of the robenidine hydrochloride is an internal standard method standard curve, and is prepared by the peak area of the quantitative ion of the robenidine hydrochloride in a standard solution and the robenidine hydrochloride-d with the concentration of 20 mu g/L in the standard solution8Quantitative ion peak area ratio Y of1As ordinate, chlorobenzene guanidine hydrochloride concentration value X1(concentration range is 1-500 mu g/L) as the abscissa, and a robenidine hydrochloride standard curve is drawn as Y1＝0.0271X1+0.0281，R 21 is ═ 1; the p-chlorobenzoic acid standard curve is an external standard method standard curve and is prepared by the peak area value Y of the quantitative ion of p-chlorobenzoic acid in a standard solution2As the ordinate and the concentration value X of p-chlorobenzoic acid2(5-500. mu.g/L) as the abscissa, and a p-chlorobenzoic acid standard curve is plotted as Y2＝47340X2+393833，R20.9991; p-chlorobenzamide acetic acid standard curveThe line is an external standard method standard curve and is prepared by the peak area value Y of the quantitative ion of p-chlorobenzamide acetic acid in a standard solution3As the ordinate and the concentration value X of p-chlorobenzoic acid3(2.5-500. mu.g/L) as the abscissa, and a p-chlorobenzoic acid standard curve is plotted as Y3＝74458X3+527833，R20.9990. Method for calculating residual quantity of robenidine hydrochloride in aquatic product muscle tissue, wherein robenidine hydrochloride and robenidine-d hydrochloride in sample are determined by HPLC-HESI-MS/MS8Quantitative determination of the ratio of ion peak areas as Y1Substituting into a robenidine hydrochloride standard curve to calculate the residual quantity X of the robenidine hydrochloride1(ii) a The method for calculating the residual quantity of p-chlorobenzoic acid and p-chlorobenzamide acetic acid in the muscle tissue of aquatic products respectively takes the peak area values of the p-chlorobenzoic acid and the p-chlorobenzamide acetic acid quantitative ions in a sample measured by HPLC-HESI-MS/MS as Y2And Y3Substituting into standard curves of p-chlorobenzoic acid and p-chlorobenzamide acetic acid to respectively calculate residual amounts X of chlorobenzoic acid and p-chlorobenzamide acetic acid2And X3。
Recovery (%). percent-standard sample assay concentration ÷ standard concentration × 100%
Accuracy (%) - (assay concentration for spiked samples-spiked concentration) ÷ spiked concentration × 100%
The precision degree comprises the intra-day precision degree and the inter-day precision degree, and the intra-day precision degree corresponds to the intra-day precision degree in the same adding concentration and the inter-measurement degree of different batches of standard adding samples. Day precision was calculated by continuously preparing and measuring the concentration of spiked samples over one week.
Precision ═ Standard Deviation (SD) ÷ mean value of the measured spiked sample concentration × 100%
The signal-to-noise ratio of more than or equal to 3 times is used as the detection limit of the method, and the signal-to-noise ratio of more than or equal to 10 times is used as the quantification limit of the method.
Obtaining robenidine hydrochloride, p-chlorobenzoic acid, p-chlorobenzamide acetic acid and robenidine hydrochloride-d in the muscle tissue of the grass carp8The labeled chromatogram of (2) is shown in FIG. 3. The standard recovery rate, accuracy and precision of robenidine hydrochloride, p-chlorobenzoic acid and p-chlorobenzamide acetic acid in grass carp muscle are shown in Table 2, and the detection limit and the determination are determinedThe amount limits are shown in Table 3. The results show that the recovery rate of 3 targets is 85.8-107.4%, the intra-day precision is 2.9-8.0%, and the inter-day precision is 3.5-11.2%. The detection limit and the quantification limit of the robenidine hydrochloride in the muscles of the grass carps are respectively 0.5 mug/kg and 1 mug/kg; the detection limit and the quantitative limit of p-chlorobenzoic acid are respectively 2.5 and 5 mu g/kg, and the detection limit and the quantitative limit of p-chlorobenzoylamino acetic acid are respectively 1 mu g/kg and 2.5 mu g/kg.
Table 2 accuracy, recovery and precision of robenidine hydrochloride, p-chlorobenzamide acetic acid and p-chlorobenzoic acid in muscle tissue of grass carp (n ═ 6)
TABLE 3 detection and quantitation limits for robenidine hydrochloride, parachlorobenzamide acetic acid and parachlorobenzoic acid in the muscle tissue of grass carp
Example 3
The method for determining the residual quantity of robenidine hydrochloride and metabolites thereof in aquatic products by high performance liquid chromatography-tandem mass spectrometry based on the time segmentation technology is applied as follows:
the grass carp is orally irrigated with robenidine hydrochloride in a dosage of 20mg/kg body weight, and the muscle tissue of the grass carp is collected at 0.5, 1, 2, 4, 6, 8, 10, 12, 24, 48, 72, 96, 120, 144 and 168 hours after administration. And (4) homogenizing the muscle tissue of the grass carp, freezing and storing at the temperature of 18 ℃ below zero, and waiting for analysis. Grass carp muscle samples were thawed before analysis. The grass carp muscle sample determination step comprises:
A. weighing 1g of homogeneous grass carp muscle sample into a 15mL centrifuge tube, adding 20 mu L of 1mg/L robenidine hydrochloride-d8Shaking the internal standard solution for 30s, adding 5mL of extractant acetonitrile containing 1% formic acid, shaking for 30s, ultrasonically extracting for 5min, adding 1g of anhydrous magnesium sulfate, and vortexing. Centrifuging at 8000r/min for 5min, placing the supernatant in another 15mL centrifuge tube, extracting the residue once again, mixing the two extractive solutions, and adding 500mg anhydrous magnesium sulfateAnd 100mg of octadecylsilane chemically bonded silica (ODS) is added into the extracting solution, the mixture is subjected to vortex oscillation for 30s and centrifugation for 5min at 8000r/min, the upper layer liquid is transferred into a 15mL centrifuge tube, a nitrogen blowing instrument with 50 ℃ is arranged in the centrifuge tube for nitrogen blowing, the residue is dissolved by using 1mL of mixed solution (70:30, v/v) of methanol and water solution, the vortex oscillation for 30s and the solution is 10000r/min and centrifugation for 5min, the mixture is filtered by a 0.22 mu m filter membrane, and the filtrate is subjected to HPLC-HESI-MS/MS analysis.
B. Conditions for HPLC analysis: a chromatographic column: waters Symmetry C18 chromatography column (100 mm. times.2.1 mm,3.5 μm); column temperature: 30 ℃, flow rate: 0.25 mL/min; mobile phase: the organic phase was methanol, the aqueous phase was 0.1% formic acid in water, gradient elution procedure: the initial gradient was 30% methanol by volume, the linear increase to 90% methanol by volume within 4.5min, the hold time was 2.5min, the linear decrease to 30% methanol by volume within 0.1min, and the hold time was last 0.9 min. The injection volume was 20. mu.L.
C. HESI-MS/MS analysis conditions: heating an electrospray ion source (HESI), switching between a positive ion mode and a negative ion mode in time periods; the detection mode adopts a selective reaction monitoring mode (SRM); positive ion mode spray voltage: 3500V, negative ion mode spray voltage: -3000 v; temperature of the evaporated gas: 380 ℃; sheath gas pressure 50 arb; auxiliary gas pressure 15 arb; collision gas and pressure: the purity of argon is more than or equal to 99.999 and 1.5m Torr; ion transport capillary temperature: 350 ℃; the half-width of Q1 was 0.7, the half-width of Q3 was 0.7, and the detection parameters were as in Table 1.
D. Quantification by a standard curve method: the standard curve drawn in the example 2 is adopted to calculate the residual quantity of the robenidine hydrochloride and the metabolites thereof in the muscle sample of the grass carp after oral administration of the robenidine hydrochloride.
By measuring the actual grass carp sample of the mouth-irrigated robenidine hydrochloride, the main existence of robenidine hydrochloride and p-chlorobenzoic acid as a metabolite thereof in the actual grass carp muscle sample is found, and the obtained chromatogram is shown in FIG. 4 and the time concentration data are shown in Table 4 and FIG. 5.
TABLE 4 concentration of chlorobenzeneguanidine hydrochloride and p-chlorobenzoic acid in grass carp muscle samples after oral gavage
| Time | Chlorobenzeneguanidine hydrochloride concentration (μ g/kg) | Concentration of p-chlorobenzoic acid (. mu.g/kg) |
| 0.5 | 295.80 | 69.92 |
| 1 | 382.40 | 69.65 |
| 2 | 874.11 | 168.52 |
| 4 | 604.53 | 112.50 |
| 6 | 1605.79 | 210.02 |
| 8 | 1374.64 | 321.75 |
| 10 | 1447.42 | 250.45 |
| 12 | 868.71 | 139.83 |
| 24 | 626.53 | 96.95 |
| 48 | 111.96 | 78.05 |
| 72 | 99.37 | 68.10 |
| 96 | 93.10 | 66.89 |
| 120 | 52.40 | 46.29 |
| 144 | 11.29 | 57.21 |
| 168 | 13.52 | 12.81 |
Claims (1)
1. A method for measuring residual quantity of robenidine hydrochloride and metabolites thereof in aquatic products based on time segmentation technology by high performance liquid chromatography-tandem mass spectrometry comprises the following steps:
A. and (3) processing a muscle sample: weighing homogeneous muscle sample, adding robenidine hydrochloride-d8As chlorobenzene hydrochlorideAdding an extracting agent A and anhydrous magnesium sulfate into an internal standard of guanidine in sequence, performing vortex oscillation, centrifuging, taking supernatant, repeatedly extracting residues, combining extracting solutions, adding the anhydrous magnesium sulfate and a purifying agent B, performing vortex oscillation and centrifuging, taking supernatant, drying by blowing nitrogen, dissolving residues by using a mixed solution of methanol and water with a volume ratio of 70:30-80:20, filtering by using a filter membrane with the aperture not larger than 0.22 mu m, and detecting filtrate by using HPLC-HESI-MS/MS; the extracting agent A is acetonitrile containing 1% formic acid, and the purifying agent B is octadecylsilane chemically bonded silica;
B. conditions for HPLC analysis: c18 chromatographic column, 100mm × 2.1mm,3.5 μm; column temperature: 30 ℃, flow rate: 0.25 mL/min; mobile phase: the organic phase was methanol, the aqueous phase was 0.1% formic acid in water, gradient elution procedure: the initial gradient is methanol with the volume concentration of 25-35%, the methanol with the volume concentration of 85-95% is linearly increased within 4.5min, the initial gradient is kept for 2.5min, the methanol with the volume concentration of 25-35% is linearly decreased within 0.1min, and finally the initial gradient is maintained for 0.9min, and the sample injection volume is 20 mu L;
C. HESI-MS/MS analysis conditions: heating the electrospray ion source, and switching between a positive ion mode and a negative ion mode; the detection mode adopts a selective reaction monitoring mode; positive ion mode spray voltage: 3500V, negative ion mode spray voltage: -3000 v; temperature of the evaporated gas: 380 ℃; sheath gas pressure 50 arb; auxiliary gas pressure 15 arb; collision gas and pressure: the purity of argon is more than or equal to 99.999 and 1.5m Torr; ion transport capillary temperature: 350 ℃; the half-width of Q1 was set to 0.7, the half-width of Q3 was set to 0.7, and the detection parameters were as follows:
quantitative ions
D. Quantification by a standard curve method: the robenidine hydrochloride adopts an internal standard method to establish a standard curve, and the p-chlorobenzoic acid and the p-chlorobenzamide acetic acid adopt an external standard method to establish a standard curve, and the concentration of the sample is calculated according to the standard curve.
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