CN115219614B - High performance liquid chromatography detection method for chlorbenzoguanide hydrochloride in chicken excreta - Google Patents

High performance liquid chromatography detection method for chlorbenzoguanide hydrochloride in chicken excreta Download PDF

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CN115219614B
CN115219614B CN202210707104.0A CN202210707104A CN115219614B CN 115219614 B CN115219614 B CN 115219614B CN 202210707104 A CN202210707104 A CN 202210707104A CN 115219614 B CN115219614 B CN 115219614B
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CN115219614A (en
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黄显会
张云晓
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South China Agricultural University
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Abstract

The application discloses a high performance liquid chromatography detection method of chlorbenzoguanamine hydrochloride in chicken excreta. The high performance liquid chromatography detection method of the chlorphenyl guanidine hydrochloride in chicken excreta comprises two steps of pretreatment and high performance liquid chromatography detection, the impurity peak and the main peak of the chlorphenyl guanidine hydrochloride of the sample treated by the pretreatment process have good separation effect, the main peak has good peak type, the detection limit and the quantitative limit of the detection method can meet the requirements of qualitative and quantitative analysis of medicines, and the detection sensitivity, the reproducibility, the accuracy, the recovery rate and the like can also meet the detection requirements. The method disclosed by the application not only can meet the content measurement of the chlorpheniramine hydrochloride in chicken excreta and the excretion research in chicken bodies, but also is beneficial to monitoring the influence of the chlorpheniramine hydrochloride on water sources, soil and the like after the chlorpheniramine hydrochloride is discharged into the environment along with the chicken excreta, and the reasonable application of the chlorpheniramine hydrochloride in the field of chicken coccidiosis prevention and treatment is monitored.

Description

High performance liquid chromatography detection method for chlorbenzoguanide hydrochloride in chicken excreta
Technical Field
The application belongs to the technical field of drug detection. More particularly relates to a high performance liquid chromatography detection method of the chlorbenzoguanamine hydrochloride in chicken excreta.
Background
The chlorbenzoguanamine hydrochloride belongs to guanidine derivatives, is an artificially synthesized anticoccidial drug, is mainly used for preventing and treating acute and chronic coccidian infection of chickens, and has good effects on Eimeria tenella, eimeria necatrix, eimeria acervulina, eimeria brunetti, eimeria mutans and the like. However, the excessive use or improper use of the chlorbenzoguanide hydrochloride, such as the illegal addition of high doses of chlorbenzoguanide hydrochloride in the feed, causes residues in the animal body and endangers the health of the human body; part of the animal excreta can be discharged in the form of original shape or metabolites along with the animal excreta, and the animal excreta can be scattered in farmlands as fertilizer, so that soil, water body and the like are polluted, the microbial ecological chain of the environment is influenced, and great risks exist for the environment and human health. Therefore, there is a need to establish a method for detecting the content of the chlorbenzoguanamine hydrochloride in the chicken excreta with high detection sensitivity and accurate detection result, and the method can be used for detecting the content of the chlorbenzoguanamine hydrochloride in the chicken excreta, so as to further judge whether the behavior of illegally adding the high-dose chlorbenzoguanamine hydrochloride into the feed exists.
At present, the separation and detection methods reported at home and abroad mainly comprise thin layer chromatography, high performance liquid chromatography and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although the thin layer chromatography has the advantages of simple and convenient operation, the qualitative and quantitative effects are poor, and the sensitivity is not high; although the liquid chromatography-tandem mass spectrometry has accurate qualitative and quantitative results and short peak time, the existing liquid quality detection method for the content of the chlorobenzoguanidine hydrochloride needs to use the chlorobenzoguanidine-D8 as an internal standard, and the pretreatment process needs to be subjected to column passing purification twice, so that the operation flow is complex and the time consumption is long. In addition, the existing separation and detection method of the chlorbenzoguanamine hydrochloride mainly aims at animal tissues such as muscles, livers, kidneys and the like, and at least the existing separation and detection method of the chlorbenzoguanamine hydrochloride in animal excreta is very few. Hu Shuangqing discloses a method for simultaneously detecting six types of 24 antibiotics in livestock and poultry manure by combining ultrasonic extraction-solid phase extraction pretreatment with liquid chromatography-tandem mass spectrometry, but the method utilizes liquid chromatography-tandem mass spectrometry and has the defects of complex pretreatment process operation, more reagents, long time consumption and the like.
Disclosure of Invention
The application aims to overcome the defects and the shortcomings of the prior art and provide a high performance liquid chromatography detection method for the chlorbenzoguanide hydrochloride in chicken excreta.
The above object of the present application is achieved by the following technical scheme:
the application provides a high performance liquid chromatography detection method of chlorbenzoguanamine hydrochloride in chicken excreta, which comprises the following steps:
s1, pretreatment: lyophilizing chicken excrement, adding pretreatment liquid, homogenizing, centrifuging to obtain supernatant, concentrating, redissolving, and filtering;
s2, high performance liquid chromatography detection.
The application can solve the problem of impurity interference caused by large sample sampling amount by freeze drying treatment of chicken excreta; secondly, the problems that the samples are difficult to homogenize and the samples are not uniform due to the fact that the water content of the chicken excrement is high can be solved.
Specifically, the preprocessing step in step S1 is as follows: freeze drying chicken excrement, adding acidified pretreatment liquid for homogenizing, centrifuging to obtain supernatant, concentrating, re-dissolving the residue with methanol, adding equal volume of n-hexane, mixing, centrifuging, and removing the lower layer liquid filtering membrane.
Specifically, the chicken excreta comprises urine and feces.
Specifically, the freeze-drying treatment is: quick freezing the sample in a refrigerator at-80 deg.c, and vacuum drying after freezing.
Specifically, the centrifugal temperature in the step S1 is 4 ℃, and the rotating speed is 5000-8000 r.min -1 The time is 5-10 min.
In order to remove impurities as much as possible, the application carries out two times of centrifugation, namely, the homogenization treatment is carried out at 4 ℃ and 8000 r.min -1 Centrifuging for 10min, sucking the upper layer extractive solution again at 4deg.C and 8000 r.min -1 And centrifuged again for 10min.
Preferably, the centrifugal rotational speed is 8000 r.min -1 The time was 10min, see example 1.
Specifically, the acidified pretreatment liquid is ethyl acetate containing formic acid or acetonitrile containing formic acid, and the volume percentage of the formic acid is 1-2%.
Preferably, the pretreatment liquid is acetonitrile containing 2% (v/v) formic acid, see example 1.
Specifically, the filter membrane is a 0.22 μm filter membrane.
Specifically, the chromatographic conditions of the high performance liquid chromatography detection in step S2 are as follows: chromatographic column: a C18 chromatographic column; mobile phase: the phase A (aqueous phase) is 0.1-0.2% formic acid aqueous solution or 0.025-0.01 moL/L phosphoric acid aqueous solution, and the phase B (organic phase) is methanol or acetonitrile according to the volume ratio A: b= (55 to 65): (35-45), gradient elution with the flow rate of 0.8-1.2 mL/min; sample injection volume: 15-25 mu L; column temperature: 38-42 ℃; detection wavelength: 300-330 nm.
Specifically, the column was a Agilent ZORBAX SB-C18 column (4.6mm.times.250mm, 5 μm), i.e., the inner diameter of the column was 4.6mm, the column length was 250mm, and the particle size of the packing was 5 μm.
Preferably, the A phase is 0.025moL/L aqueous phosphoric acid and the B phase is acetonitrile, see example 2.
Specifically, the gradient elution procedure is: 0-4 min,35% of phase B; 4-4.10 min,45% of phase B; 4.10-15 min,45% of phase B; 15-15.10 min,35% of phase B; 15.10-18 min,35% of phase B.
In particular, the detector used is a diode array detector; the flow rate is 1mL/min; the sample injection volume is 20 mu L; column temperature is 40 ℃; the PDA detection wavelength is 312nm.
After the key steps are optimized, the method for detecting the high performance liquid chromatography of the chlorbenzoguanamine hydrochloride in the chicken excrement comprises the following specific steps:
s1, pretreatment: lyophilizing chicken excreta, adding acetonitrile containing 2% (v/v) formic acid, homogenizing, i.e. extracting with strong shaking vortex for 2-5 min at 4deg.C and 8000r min -1 Centrifuging for 10min, sucking the upper layer extractive solution into a new centrifuge tube, and centrifuging at 4deg.C at 8000 r.min -1 Centrifuging for 10min again, sucking the upper layer extractive solution into another new centrifuge tube, and blowing up at 45deg.C with nitrogen blowerNear-drying; redissolving the residue with methanol, swirling for 30s, adding equal volume of n-hexane, swirling again for 30s at 4deg.C at 8000 r.min -1 Centrifuging for 10min; discarding the upper viscous liquid, transferring the lower solution into a centrifuge tube, and transferring at 4deg.C for 15000 r.min -1 And centrifuging again for 10min, and filtering the lower clarified liquid through a 0.22 mu m filter membrane to obtain a sample to be tested.
S2, high performance liquid chromatography detection, and a detector: a diode array detector; the chromatographic conditions are as follows: chromatographic column: agilent ZORBAX SB-C18 (4.6 mm. Times.250 mm,5 μm); mobile phase: phase A was 0.025moL/L phosphoric acid in water and phase B was acetonitrile, gradient elution procedure: 0-4 min,35% of phase B; 4-4.10 min,45% of phase B; 4.10-15 min,45% of phase B; 15-15.10 min,35% of phase B; 15.10-18 min,35% of phase B; flow rate: 1mL/min; sample injection volume: 20. Mu.L; column temperature: 40 ℃; PDA detection wavelength: 312nm.
The application has the following beneficial effects:
the application provides a high performance liquid chromatography detection method of chlorphenyl guanidine hydrochloride in chicken excreta, which comprises two steps of pretreatment of excreta samples and high performance liquid chromatography detection, wherein the separation effect of an interference peak and a main peak of the chlorphenyl guanidine hydrochloride of the samples treated by the pretreatment process is good, the main peak type is good, the detection limit and the quantitative limit of the detection method can meet the requirements of qualitative and quantitative analysis of medicines, and the detection sensitivity, the reproducibility, the accuracy, the recovery rate and the like can also meet the detection requirements.
The detection method disclosed by the application not only can meet the content measurement of the chlorphenidine hydrochloride in chicken excreta and the excretion research in chicken bodies, but also is beneficial to monitoring the influence of the chlorphenidine hydrochloride on water sources, soil and the like after the chlorphenidine hydrochloride is discharged into the environment along with the chicken excreta, and the reasonable application of the chlorphenidine hydrochloride in the field of chicken coccidiosis prevention and treatment.
Drawings
FIG. 1 is a standard graph obtained by examining the linear relationship in example 3 of the present application.
FIG. 2 is a chromatogram corresponding to the detection limit in example 3 of the present application.
FIG. 3 is a chromatogram corresponding to the limit of quantification described in example 3 of the present application.
FIG. 4 is a chromatogram of a blank fecal sample according to example 4 of the present application.
FIG. 5 is a chromatogram of a standard working solution (1. Mu.g/mL) of the blank stool added with progenitrile hydrochloride according to example 4 of the present application.
FIG. 6 is a chromatogram of an actual chicken excreta sample as described in example 4.
Detailed Description
The application is further illustrated in the following drawings and specific examples, which are not intended to limit the application in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 optimization of sample pretreatment conditions
1. Effect of chicken excreta freeze drying on homogenization treatment
The water content of the fresh chicken excreta (comprising urine and feces) is 50-60%, and the fresh chicken excreta is influenced by the water content of the fresh chicken excreta, if the sampling amount is small in the pretreatment process, the problem of uneven sampling can occur, and the water content cannot represent the change of the drug concentration in an actual sample; and excessive sampling amount is easy to cause excessive response of impurities in the matrix and influence the problem of low concentration quantification. Therefore, the chicken excreta is subjected to freeze-drying treatment, and the influence on homogenization treatment after the chicken excreta freeze-drying treatment is explored; the freeze drying treatment comprises the following steps: quick freezing the sample in a refrigerator at-80 deg.c, and vacuum drying after freezing.
Taking an equal amount of fresh chicken excreta, freeze-drying to semi-dry and completely dry chicken excreta, adding the same solvent, strongly shaking, vortex-extracting for 5min, and observing the homogenizing effect, wherein the result is shown in Table 1:
TABLE 1 Effect of different moisture content of chicken excreta on homogenization treatment effect
2. Centrifugal condition optimization
Although the method of freeze drying chicken excreta and then homogenizing is adopted, the problems of excessive impurity response caused by excessive sampling amount, influence on quantification and uneven sampling can be avoided. However, after adding the extraction reagent for homogenization, the extraction reagent system becomes very turbid, which is unfavorable for subsequent detection. In order to obtain a clear extraction reagent system, the application performs an influence test of the centrifugal speed and the centrifugal time on the extraction effect, and the results are shown in table 2. As is clear from the results shown in Table 2, when the rotational speed is 8000 rmin -1 When the centrifugation time is 10min, the system state is clear and transparent, so that impurities in chicken manure samples can be well removed, and interference is reduced.
TABLE 2 influence of centrifuge time and speed on System State
3. Optimization of drug extraction reagents
The results of the preliminary test show that the application has better effect by using ethyl acetate or acetonitrile as an extraction reagent, and in order to further optimize the application, after the chicken excreta are freeze-dried, the application extracts and recovers the chlorbenzoguanamine hydrochloride in the chicken excreta by respectively using 1% (v/v) formic acid to acidify the ethyl acetate, 1% (v/v) formic acid to acidify the acetonitrile, 0.1% (v/v) formic acid to acidify the acetonitrile and 2% (v/v) formic acid to acidify the acetonitrile.
Adding lyophilized chicken excreta into 1% (v/v) formic acid ethyl acetate, 1% (v/v) formic acid acetonitrile, 0.1% (v/v) formic acid acetonitrile and 2% (v/v) formic acid acetonitrile, and extracting with strong shaking vortex for 3min at 4deg.C and 8000 r.min -1 Centrifuging for 10min, sucking the upper layer extract into a new centrifuge tube, and blowing up to near dryness with a nitrogen blower at 45deg.C; redissolving the residue with methanol, swirling for 30s, adding equal volume of n-hexane, swirling again for 30s at 4deg.C at 8000 r.min -1 Centrifuging for 10min; the upper viscous liquid was discarded, the lower clarified liquid was filtered through a 0.22 μm filter to obtain the sample to be tested, and the recovery test results are shown in Table 3. As is clear from the results shown in Table 3, when 2% (v/v) formic acid-acidified acetonitrile was used as the extraction reagent, the recovery rate of the chlorobenzoguanidine hydrochloride was high, and the parallelism was good, so that 2% (v/v) formic acid-acidified acetonitrile was subsequently selected as the extraction reagent.
TABLE 3 influence of different extraction reagents on recovery
4. Determination of pretreatment method
After a series of optimization, the pretreatment method of the chicken excrement is determined as follows: weighing 0.20 g+ -0.01 g of freeze-dried chicken manure in a 50mL centrifuge tube, adding 10mL acetonitrile containing 2% (v/v) formic acid, extracting with strong shaking vortex for 2min, extracting at 4deg.C for 8000 r.min -1 Centrifuging for 10min, collecting supernatant, and centrifuging at 4deg.C and 8000 r.min -1 Centrifuging again for 10min, collecting supernatant, and blow-drying on nitrogen blower at 45deg.C; the residue was redissolved in 1mL of methanol, vortexed for 30s, then added with 1mL of n-hexane, vortexed again for 30s, and at 4℃8000 r.min -1 Centrifuging for 10min; discarding the upper viscous liquid, transferring the lower solution into a 2mL centrifuge tube, and transferring the lower solution into 15000 r.min at 4deg.C -1 Centrifuging again for 10min, taking the clarified liquid at the lower layer, and filtering with a 0.22 μm filter membrane to obtain a sample to be tested, and performing HPLC analysis.
EXAMPLE 2 optimization of chromatographic conditions
1. Determination of detection wavelength
When the high performance liquid chromatography detection is carried out on the obtained sample to be detected, the detector is a diode array, chromatographic conditions of the high performance liquid chromatography detection are further optimized to improve detection sensitivity, accuracy and the like, the detector carries out full-wavelength detection on the chlorphenyl guanidine hydrochloride standard solution (1 mug/mL) through the diode array detector of the high performance liquid chromatograph, the response value and the peak area of the chlorphenyl guanidine hydrochloride in the wavelength range of 200-400 nm are compared, and the result shows that the chlorphenyl guanidine hydrochloride has higher response intensity in the wavelength range of 300-330 nm, has the highest response intensity at 312nm and can meet the detection requirement. Therefore, 312nm is selected as the final detection wavelength in the method.
2. Selection of mobile phases in chromatographic conditions
The application optimizes the chromatographic conditions by adopting different organic phases and water phases respectively. According to the application, methanol and acetonitrile are respectively used as organic phases, high performance liquid chromatography detection is carried out on a standard solution (1 mug/mL) of the chlorbenzoguanamine hydrochloride, and as a result, the acetonitrile has stronger eluting capability on the chlorbenzoguanamine hydrochloride, better peak type and smaller viscosity, and under the premise of the same other chromatographic conditions, the column efficiency is higher and the column pressure is lower, so that the acetonitrile is selected as the organic phase for HPLC analysis.
According to the application, 0.1% formic acid water and 0.025moL/L phosphoric acid water are respectively used as water phases, high performance liquid chromatography detection is carried out on a standard solution of the chlorobenzoguanidine hydrochloride (1 mu g/mL), and as a result, when 0.025moL/L phosphoric acid water is used as a water phase mobile phase, the obtained chromatographic peak of the chlorobenzoguanidine hydrochloride is sharper and more symmetrical, the tailing factor (T) is between 0.95 and 1.05, the separation degree is more than 1.5, the theoretical plate number is more than 2000, and therefore, 0.025moL/L phosphoric acid water is selected as the water phase for HPLC analysis.
After determining the reagents used for the mobile phase, the present application detects the presence of the guanidine hydrochloride in chicken excreta using different phase ratios of the same flow, i.e., 20%, 25%, 30%, 35%, 40%, 45%, 50% and 55% (v/v). As a result, it was found that the effect of the off-peak of the clomazone hydrochloride was good in the 45% organic phase state, the peak was sharp and symmetrical, and the effect of separating the impurity peaks in the matrix was good. In combination with consideration of peak time and separation degree of main peak and impurity peak, the application finally determines to use an organic phase gradient elution mode from 35% to 45%. The gradient procedure is detailed in table 4 below:
TABLE 4 gradient elution procedure
3. Determination of chromatographic conditions
After a series of optimization, the application determines chromatographic conditions of the high performance liquid chromatography detection, and the chromatographic conditions are specifically as follows: a detector: a diode array detector; detection wavelength: 312nm; chromatographic column: agilent ZORBAX SB-C18 (4.6 mm. Times.500 mm,5 μm); mobile phase: phase A was 0.025moL/L phosphoric acid in water and phase B was acetonitrile, gradient elution procedure: 0-4 min,35% of phase B; 4-4.10 min,45% of phase B; 4.10-15 min,45% of phase B; 15-15.10 min,35% of phase B; 15.10-18 min,35% of phase B; flow rate: 1mL/min; sample injection volume: 20. Mu.L; column temperature: 40 ℃; PDA detection wavelength: 312nm.
EXAMPLE 3 methodology investigation
1. Linear relationship investigation
A plurality of parts of 0.20g of freeze-dried chicken manure are accurately weighed, 100 mu L of standard working solution of the chlorobenzoguanidine hydrochloride is sequentially added, so that the mass concentration of the added chlorobenzoguanidine hydrochloride is 0.02mg/kg, 0.5mg/kg, 2mg/kg, 5mg/kg, 10 mg/kg, 20 mg/kg, 40mg/kg and 50mg/kg respectively, the added chlorobenzoguanidine hydrochloride is processed according to the pretreatment method determined in the embodiment 1 of the application, the measurement is carried out according to the chromatographic conditions determined in the embodiment 2, and the HPLC analysis is carried out by sequentially sampling according to the sequence from low concentration to high concentration.
Drawing a standard curve by taking the measured chromatographic peak area (A) of the chlorbenzoguanamine hydrochloride as an abscissa and the drug concentration (C) as an ordinate to obtain a linear regression equation and a correlation coefficient; as shown in fig. 1, the standard curve shows a good linear relationship in the detection method as shown in fig. 1. The linear regression equation was c= 8.6441 ×10 -5 A+0.1174, linear correlation coefficient R 2 ＝0.9997。
2. Detection and quantification limit
0.20g of blank lyophilized chicken manure is accurately weighed, and standard solutions of chlorobenzoguanidine hydrochloride with mass concentrations of 0.01mg/kg, 0.02mg/kg, 0.05mg/kg, 0.1mg/kg, 0.2mg/kg and 0.5mg/kg are respectively added, treated according to the pretreatment method described in example 1, and measured according to the chromatographic conditions described in example 2, wherein the detection Limit (LOD) is calculated with S/N not less than 3, and the quantitative Limit (LOQ) is calculated with S/N not less than 10.
The detection limit of the detection method for the chlorbenzoguanamine hydrochloride in the chicken excreta is 0.01mg/kg, and a chromatogram corresponding to the detection limit is shown in figure 2; the quantitative limit of the detection method on the chlorbenzoguanamine hydrochloride in the chicken excreta is 0.02mg/kg, and a chromatogram corresponding to the quantitative limit is shown in figure 3.
3. Recovery rate and precision
Accurately weighing 0.20g of blank freeze-dried chicken manure, adding a standard working solution of the chlorbenzoguanamine hydrochloride, so that the mass concentration of the chlorbenzoguanamine hydrochloride after the addition is respectively 0.5, 5 and 40mg/kg, wherein 5 mass concentrations are parallel, processing according to the pretreatment method described in the embodiment 1, measuring according to the chromatographic conditions described in the embodiment 2, comparing the peak area of the measured chlorbenzoguanamine hydrochloride with the peak area of a standard product with the corresponding concentration, quantifying by a single-point method, calculating the absolute recovery rate of the chlorbenzoguanamine hydrochloride in a manure sample, and performing accuracy analysis; and 3 analytical batches were prepared and examined consecutively on different days, and the method was evaluated for intra-and inter-batch precision with relative standard deviation, and the results are shown in Table 5:
TABLE 5 absolute recovery of Chlorobenzguanide hydrochloride and relative Standard deviation between batches
As can be seen from the results shown in Table 5, the average recovery rate of the chlorobenzoguanidine hydrochloride in the chicken manure sample at 3 addition concentrations (0.5, 5, 40 mg/kg) was 91.26% -103.46%, the relative standard deviation between batches was 1.44% -4.49%, the relative standard deviation between batches was 4.12% -5.97%, and the recovery rate and precision were both at high levels.
The result shows that the detection limit and the quantitative limit of the high performance liquid chromatography detection method of the chlorbenzoguanamine hydrochloride in the chicken excrement meet the requirements of qualitative and quantitative analysis of medicines, and the detection sensitivity is high, and the method has the advantages of high recovery rate, high precision, good repeatability and high accuracy.
Example 4 actual sample detection
1. Experimental animal and sample collection
10 healthy Yue-bird king hens with the age of 30 weeks are placed in a metabolism cage for independent feeding, the average weight is 1.42+/-0.14 kg, animal diet, behavior, physical characteristics and the like are observed before test development, and subsequent experiments are carried out after clinical manifestations are healthy; suspending the chlorbenzoguanamine hydrochloride in 0.1% sodium carboxymethylcellulose aqueous solution to prepare an oral liquid with the concentration of 5mg/mL, preparing the oral liquid by pouring in a 15 mg/kg.bw dosage currently, respectively 4h, 8h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h, 120h, 132h, 144h, 156h and 168h before and after the administration, collecting all fecal-urine mixtures, weighing and recording wet weights; quickly freezing the sample in a refrigerator at-80 ℃, carrying out vacuum treatment after the sample is completely frozen, weighing again after the sample is completely freeze-dried (the water content is less than 2%), and recording the dry weight of the sample; the resulting chicken excreta samples were sealed and stored in a foam box with desiccant for testing.
2. Sample detection and results
The resulting chicken excreta samples were each treated according to the pretreatment method described in example 1 and were assayed according to the chromatographic conditions described in example 2; in addition, after addition of a standard solution of chlorobenzoguanidine hydrochloride (1. Mu.g/mL) to the blank chicken manure, the resulting chicken manure samples were treated separately in the same manner as in the pretreatment described in example 1 and were assayed according to the chromatographic conditions described in example 2.
The chromatogram of the blank fecal sample is shown in fig. 4; the chromatogram of the standard working solution (1 mug/mL) with the addition of the chlorbenzoguanamine hydrochloride to the blank feces is shown in FIG. 5; the chromatogram of the actual chicken faeces sample is shown in figure 6. The result shown in fig. 5 shows that the retention time of the chlorbenzoguanamine hydrochloride is 11.567min, which is consistent with the retention time in the chromatogram (fig. 6) of the actual chicken excrement sample, and meanwhile, the blank excrement sample has no impurity peak at the corresponding time point (fig. 4), which shows that no other impurity is interfered near the retention time of the chlorbenzoguanamine hydrochloride, and the endogenous impurities of the sample can not influence the detection of the drug chlorbenzoguanamine hydrochloride by the detection method provided by the application, so that the separation effect is good, the quantification is accurate, and the detection requirement can be met. In addition, according to the detection result of an actual sample, after the chicken takes the chlorbenzoguanamine hydrochloride orally, the chlorbenzoguanamine hydrochloride can be discharged out of the body along with excrement quickly, the period of 0-4 hours is a excretion peak period, the average excretion amount is 6514.88 +/-1714.44 mug, and the chicken accounts for 75.56 percent of the accumulated excretion amount and accounts for 29.84 percent of the administration dosage; the main excretion time is within 24 hours after administration, and the excretion amount accounts for 93.50% of the accumulated excretion amount; the drug administration is carried out for 7 days, and the excretion is basically completed.
The above examples are preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present application should be made in the equivalent manner, and the embodiments are included in the protection scope of the present application.

Claims (4)

1. The high performance liquid chromatography detection method of the clomazone hydrochloride in chicken excreta is characterized by comprising the following steps of:
s1, pretreatment: freeze drying chicken excrement, adding acidified pretreatment liquid for homogenization treatment, centrifuging to obtain supernatant, concentrating, re-dissolving the obtained residue with methanol, adding equal volume of n-hexane, mixing, centrifuging, and collecting lower layer liquid filtering membrane; the acidified pretreatment liquid is ethyl acetate containing formic acid or acetonitrile containing formic acid, and the volume percentage of the formic acid is 1% -2%; the centrifugal temperature is 4 ℃, and the rotating speed is 5000-8000 r.min -1 The time is 5-10 min;
s2, high performance liquid chromatography detection; the chromatographic conditions of the high performance liquid chromatography detection are as follows: chromatographic column: a C18 chromatographic column; mobile phase: the phase A is 0.1 to 0.2 percent formic acid aqueous solution or 0.025 to 0.01moL/L phosphoric acid aqueous solution, and the phase B is methanol or acetonitrile; the gradient elution procedure was: 0-4 min,35% of phase B; 4-4.10 min,45% of phase B; 4.10-15 min,45% of phase B; 15-15.10 min,35% of phase B; 15.10-18 min,35% of phase B, and gradient elution at the flow rate of 0.8-1.2 mL/min; sample injection volume: 15-25 mu L; column temperature: 38-42 ℃; detection wavelength: 300-330 and nm.
2. The method according to claim 1, wherein the chromatographic column is a Agilent ZORBAX SB-C18 chromatographic column, the inner diameter of the column is 4.6mm, the column length is 250mm, and the particle size of the packing is 5 μm.
3. The method according to claim 1, wherein the phase A is 0.025moL/L phosphoric acid aqueous solution and the phase B is acetonitrile.
4. The method of claim 1, wherein the detector used is a diode array detector; the flow rate is 1mL/min; the sample injection volume is 20 mu L; column temperature is 40 ℃; the PDA detection wavelength was 312nm.
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