CN100376893C - Simultaneous determination of sulfamethoxazole, acetylated sulfamethoxazole and trimethoprim in fish by liquid chromatography - Google Patents

Abstract

A liquid chromatography method for the simultaneous determination of sulfamethoxazole, acetylated sulfamethoxazole and trimethoprim in fish meat. Using hydrochloric acid and a 3:1 ratio of dichloromethane/acetone mixture as the extraction solution, the sulfamethoxazole, acetylated sulfamethoxazole and trimethoprim remaining in the fish tissue can be fully extracted, and the extracted impurities less, the purification process can be omitted; after adding the extract, let it stand in the refrigerator at 4°C for a period of time, which can fully denature the protein, thereby reducing the loss caused by protein adhesion on the high-speed homogenizer during extraction, and improving the recovery rate ; The extracted organic solvent is blown dry with nitrogen, filtered and can enter the high performance liquid chromatograph for determination. The method of the invention is easy and fast to operate, has low detection cost and high sensitivity, and can be used in scientific research institutes and import and export commodity inspection departments to determine the contents of sulfamethoxazole, acetylated sulfamethoxazole and trimethoprim in fish meat.

Description

Simultaneous determination of sulfamethoxazole, acetylated sulfamethoxazole and trimethoprim in fish by liquid chromatography

technical field

The present invention relates to the determination method of sulfonamide antibiotics; Specifically, adopt high-performance liquid chromatography to measure sulfamethoxazole (SMZ), acetylated metabolite (acetylated sulfamethoxazole, NSMZ) and methoxymethoxazole simultaneously in fish meat Benzidine (TMP) method.

Background technique

Sulfamethoxazole (also known as sulfamethoxazole) is a widely used drug in the prevention and control of aquaculture diseases. It is usually used in combination with the sulfa synergist trimethoprim; In addition to azoles, acetylated sulfamethoxazole is its main metabolite, which remains in the body for a long time. Therefore, it is very necessary to detect the content of acetylated metabolites and synergists when studying the residues of sulfonamides in aquatic products. At present, the detection of sulfa drugs such as sulfamethoxazole mostly uses high-performance liquid chromatography, and the relevant reports are as follows:

(1), related reports in the pharmaceutical industry: Yuan Mei et al. (2005, Guangzhou Huihua Animal Health Products Co., Ltd.) reported the simultaneous determination of sulfamethoxazole and methazoxazole in compound sulfamethoxazole water-soluble powder by reversed-phase high-performance liquid chromatography. Oxymethoprim; Chi Yumei et al. (2001, Nanjing University of Traditional Chinese Medicine) reported the simultaneous determination of the content of sulfamethoxazole and trimethoprim in compound sulfamethoxazole injection by high performance liquid chromatography. The sample pretreatment of the above method is relatively simple, and does not involve the sample pretreatment process of extracting drugs from tissues, and the method is not suitable for the content detection of drugs in aquatic products.

(2), related reports in the field of medicine and veterinary medicine: Cui Yanli et al. (2003, Guangxi Veterinary Drug Control Institute) reported the simultaneous determination of sulfamethoxine, sulfamethoxazole and trimethoprim in pig plasma by high performance liquid chromatography; Ding Chenguang et al. (1997, PLA Dalian Medical College) reported the determination of sulfamethoxazole and trimethoprim in human plasma by high performance liquid chromatography. Aquatic animals are different from mammals in that there are large differences in protein composition and fat content, and different sample matrices will affect the recovery rate of the analytical method and interfere with detection.

(3), relevant reports in the field of aquatic products: Xu Weihai et al. (2004, Institute of Oceanology, Chinese Academy of Sciences) reported the high performance liquid chromatography of 14 kinds of sulfonamide drug residues including sulfamethoxazole in aquatic products; Guo Gen and (2005, Fujian Academy of Agricultural Sciences) reported the determination of five sulfa drug residues in prawns by high performance liquid chromatography; Lin Haidan et al. Phase extraction HPLC assay; Zheng Zonglin et al. (2005, Shanghai Fisheries University) reported the determination of sulfamethoxazole in the hemolymph of Chinese mitten crab; Liu Changzheng et al. (2004, Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences) Report the determination of sulfamethoxazole and trimethoprim in aquatic products by high performance liquid chromatography. Most of these related reports only involve the detection of the sulfa drugs themselves, and do not involve the simultaneous detection of the synergist trimethoprim and acetylated metabolites, and the sample pretreatment methods used are also different from those of the present invention.

Contents of the invention

The invention provides a high-performance liquid chromatography method for simultaneously determining sulfamethoxazole, acetylated sulfamethoxazole and trimethoprim in fish meat, which has the characteristics of sensitivity, accuracy and reliability.

Realize the technical scheme of the present invention as follows:

Its technical methods include: sample preparation, sample pretreatment, standard solution preparation, mobile phase preparation and chromatographic conditions;

A. Sample preparation: collect fish muscle tissue samples, preliminarily cut them into pieces with scissors, homogenize them with a high-speed homogenizer, and store them frozen.

B. Sample pretreatment: After the sample is thawed, accurately weigh the fish tissue, add 0.3mol/L hydrochloric acid, let it stand for 1 hour, add anhydrous sodium sulfate, and then add dichloromethane/acetone mixed extract (3:1 ratio) , after standing in the refrigerator at 4°C for 8-12 hours, homogenize with a high-speed homogenizer, then clean the cutter head of the high-speed homogenizer with a dichloromethane-acetone mixed extract (homogenized), and combine the mixed extracts twice , oscillate fully, centrifuge (4000-6000r/min centrifuge 5-10min), take out all the supernatant, add dichloromethane/acetone mixed extract to extract the remaining residue, fully oscillate, centrifuge (4000-6000r/min centrifuge 5- 10min), take the supernatant and combine the first dichloromethane/acetone mixed extract, dry the mixed extract with nitrogen at 33-35°C, dissolve the residue with mobile phase, add n-hexane to fully shake, and let it stand for a while time, the n-hexane was discarded, and the lower liquid was filtered by a microporous membrane (0.22 μm or 0.45 μm) and then determined by a high-performance liquid chromatograph.

C. Preparation of standard solution

Accurately weigh 200mg sulfamethoxazole, 200mg acetylated sulfamethoxazole and 200mg trimethoprim standard and dissolve them in 100mL mobile phase together to make a mother solution (200μg/mL) containing three drugs at the same time, store in the refrigerator Store at 4°C, and then dilute into a series of gradient standard solutions before use.

D. Mobile phase preparation and chromatographic conditions

Take acetonitrile, redistilled water, and glacial acetic acid in the ratio of 20:80:0.5 (v/v/v) to prepare the mobile phase, and degas it by ultrasonic wave for 30 minutes before use.

Chromatographic conditions used:

a. The specification of chromatographic column is 4.6mm×250mm C ₁₈ ODS column;

b. Column temperature: 25-30°C;

c. Flow rate: 0.8-1.0mL/min; injection volume 10-20μL;

d. Ultraviolet detection: wavelength 272nm;

e. The mobile phase is redistilled water: acetonitrile: glacial acetic acid = 80:20:0.5.

The present invention has the following advantages:

1. The method of the present invention can simultaneously detect sulfamethoxazole, acetylated sulfamethoxazole and trimethoprim in fish meat, which can save a lot of manpower, material resources and time, and reduce the detection cost.

2. Adding hydrochloric acid helps to dissociate the drug from the fish muscle tissue protein and improve the recovery rate; dichloromethane-acetone is used as the extraction solution, which has a good extraction effect on the three drugs, and at the same time, there are few impurities leached and the matrix is easy to exclude Interference; the concentration temperature is lower than 35°C, which meets the characteristics of poor heat resistance of sulfa drugs; after adding dichloromethane/acetone and placing it in the refrigerator for 8-12 hours, it can not only fully extract the drug in the muscle tissue, but also fully denature the protein and reduce the extraction Process loss, extraction is ideal.

3. In this method, the drug peaks of sulfamethoxazole, trimethoprim and acetylated sulfamethoxazole can be well separated from miscellaneous peaks, and the minimum detection limit can reach 0.005, 0.01, 0.005 mg/kg, with accuracy and precision high degree.

Description of drawings

Figure 1: Liquid chromatogram of fish blank sample

Figure 2: LC chromatogram of a fish blank sample spiked with sulfamethoxazole, acetylated sulfamethoxazole, and trimethoprim standards

1-trimethoprim; 2-sulfamethoxazole; 3-acetylated sulfamethoxazole

Detailed ways

Describe technical content of the present invention in detail below in conjunction with accompanying drawing by embodiment:

Its technical methods include: sample preparation, sample pretreatment, standard solution preparation, mobile phase preparation and chromatographic conditions;

A. Sample preparation: Collect fish muscle tissue samples, cut them into pieces with scissors, homogenize them with a high-speed homogenizer, put them into plastic centrifuge tubes, seal them and store them in a freezer.

B. Sample pretreatment: Take 15 15ml plastic centrifuge tubes, add 1g of thawed blank muscle tissue respectively, put them into plastic centrifuge tubes, add standard solutions of different concentrations (0.5, 2.0, 10.0, 50.0, 200.0μg/mL ) each 0.1mL, each concentration set 3 in parallel, so that the concentrations of the three drugs in 1g muscle tissue reached 0.05, 0.20, 1.00, 5.00, 20.00μg/g respectively, fully mixed, let stand for 4 hours, added 0.3 mol/L hydrochloric acid 0.2mL, let it stand for 1 hour, add 0.5g of anhydrous sodium sulfate, add 3mL of dichloromethane/acetone (3:1 ratio) mixed solution, seal the centrifuge tube, put it in a refrigerator at 4°C for 8 hours, and use Homogenize with a high-speed homogenizer for 10s (16000r/min), then clean the knife head of the high-speed homogenizer with 3mL of dichloromethane/acetone mixed extract, combine the mixed extract twice, shake for 1min, and after standing for 5min, 5000r/ Centrifuge for 10 min, take out all the supernatant, put it into a 15mL glass centrifuge tube, add 4mL dichloromethane/acetone mixed extract to the remaining residue and extract again, shake for 1min, let stand for 5min, centrifuge at 5000r/min for 10min, and take the supernatant The solution was combined with the first dichloromethane/acetone mixed extract, dried under nitrogen at 35°C, the residue was dissolved in 1 mL of mobile phase, 1 mL of n-hexane was added, vortexed for 1 min, and allowed to stand for 10 min, and the n-hexane was discarded, and the lower layer The liquid was filtered with a 0.22 μm microporous membrane and then determined by a high-performance liquid chromatograph.

C. Preparation of standard solution

Accurately weigh 200mg sulfamethoxazole, 200mg acetylated sulfamethoxazole and 200mg trimethoprim standard and dissolve them in 100mL mobile phase together to make a mother solution (200μg/mL) containing three drugs at the same time, store in the refrigerator Store at 4°C, and dilute to 50.00, 20.00, 10.00, 5.00, 1.00, 0.50, 0.20, 0.10, 0.05, 0.02, 0.01μg/mL standard solution before use.

D. Mobile phase preparation and chromatographic conditions

Acetonitrile, redistilled water, and glacial acetic acid were used to prepare the mobile phase at a ratio of 20:80:0.5 (v/v/v), and degassed by ultrasonic wave for 30 minutes.

Chromatographic conditions: 4.6mm×250mm C ₁₈ ODS column; column temperature 25°C; flow rate 1.0mL/min; injection volume 20μL; UV detection wavelength 272nm; mobile phase redistilled water: acetonitrile: glacial acetic acid = 80: 20: 0.5 .

The method of the invention tests the recovery rate of fish muscle tissue in five concentration gradients of 0.05, 0.20, 1.00, 5.00 and 20.00 μg/mL. The results showed that after the pretreatment steps of this test, no obvious impurity peaks appeared in the chromatograms of the blank sample and the drug-added sample (Figure 1-2), and the samples were well separated. The addition of 5 concentration levels of SMZ, TMP and NSMZ in muscle The recoveries ranged from 82.12-90.87%, 79.64-88.67%, 78.21-86.08% (Table 1). The above 5 concentrations of samples were repeated 5 times within 1 day and measured in 5 days to measure the precision of the quantitative method (Table 2). The relative standards of the parallel samples of the three drugs at 5 different concentration levels in the test The deviation ranges are 1.44-3.13%, 2.01-4.23%, 1.09-4.56%, meeting the requirements for the analysis of sulfamethoxazole, trimethoprim and acetylated sulfamethoxazole residues.

Table 1: Recovery rates of SMZ, TMP and NSMZ in fish muscle tissue

Sample concentration (μg/mL) SMZ(%) TMP (%) NSMZ (%) 0.050.21.05.020.0 82.1290.8790.6290.2487.45 79.6484.4288.6785.5681.72 78.2183.418 6.0880.3278.75

Table 2: Intra-day and inter-day precision of SMZ, TMP and NSMZ in fish muscle tissue

Sample concentration (μg/mL) SMZ C.V(%) TMP C.V(%) NSMZ C.V(%) 0.050.21.05.020.0 1.06/2.131.54/3.130.80/1.441.55/2.160.67/2.33 0.87/2.011.07/2.110.96/3.421.87/4.230.66/3.76 2.44/4.561.43/3.722.07/2.170.69/1.092.11/3.16

Within the concentration range of 0.01-20mg/kg, SMZ, TMP, and NSMZ have a good linear relationship in fish muscle tissue, and the correlation coefficient can reach 0.9999; the method is defined by causing 2-3 times the signal-to-noise ratio (S/N) The minimum detection limit of SMZ, TMP, and NSMZ is 0.005, 0.01, and 0.005 mg/kg, respectively, fully meeting the requirements for the analysis of sulfamethoxazole, trimethoprim, and acetylated sulfamethoxazole residues.

Claims (1)

1. the liquid phase chromatography of Sulfamethoxazole, acetylation Sulfamethoxazole and Trimethoprim during a kind mensuration is oppressed simultaneously, the technical method that it is characterized in that it comprises preparation, the moving phase preparation of specimen preparation, sample pre-treatments, standard solution and measures used chromatographic condition;

A, specimen preparation: gather the musculature sample of fish, tentatively shred, with high speed homogenization device homogeneous, freezing preservation with scissors;

B, sample pre-treatments: take by weighing the structure of fish muscle that thaws, add hydrochloric acid, leave standstill, after adding anhydrous sodium sulfate, add methylene chloride/acetone mixed extract, after leaving standstill a period of time under 4 ℃ of conditions, with high speed homogenization device homogeneous, with the cutter head of methylene chloride/acetone mixed extract cleaning high speed homogenization device, mixed extract is centrifugal after fully vibrating, collect supernatant, residue extracts once more with methylene chloride/acetone mixed extract, merges 2 times mixed extract and dries up with nitrogen, and residue dissolves with moving phase, add the normal hexane degrease, subnatant is measured with the laggard high performance liquid chromatograph of filtering with microporous membrane;

Described sample pre-treatments, the concentration that adds hydrochloric acid is 0.3mol/L; The ratio that adds hydrochloric acid is a 0.2mL/g muscle; Time of repose behind the adding hydrochloric acid is 1 hour; The extract that adds is the methylene chloride/acetone mixed liquor of ratio mixing in 3: 1; Leaving standstill a period of time under 4 ℃ of conditions is after 4 ℃ of refrigerators leave standstill 8-12 hour;

The preparation of C, standard solution: accurately take by weighing 200mg Sulfamethoxazole, 200mg acetylation Sulfamethoxazole and 200mg Trimethoprim standard items respectively and be dissolved in together in the 100mL moving phase, be made into and contain the mother liquor that concentration is three kinds of medicines of 200 μ g/mL simultaneously, the standard solution that is diluted to serial gradient more successively with preceding is faced in 4 ℃ of preservations in refrigerator;

The preparation of D, moving phase: get acetonitrile, redistilled water, glacial acetic acid in 20: 80: 0.5 ratio preparation moving phase, standby behind the ultrasonic degas 30min;

E, measure used chromatographic condition: it is 4.6mm * 250mm C that chromatographic column is selected specification for use ₁₈The ODS post; Column temperature: 25-30 ℃; Flow velocity: 0.8-1.0mL/min; Sample size 10-20 μ L; Ultraviolet detection: wavelength 272nm; Moving phase is redistilled water: acetonitrile: glacial acetic acid=80: 20: 0.5.

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