A detection method for sulfonamide synergist

Abstract

The present invention provides a method for detecting sulfonamide synergists, and belongs to the technical field of chemical analysis. The present invention adopts a specific ratio of disodium hydrogen phosphate and phosphoric acid aqueous solution and methanol as extraction solvents, does not need to use a cation exchange column for enrichment and purification, and can achieve qualitative and quantitative detection of sulfonamide synergists (such as ormeprim) in drugs by optimizing the mobile phase and elution gradient, and has good accuracy, which is of great significance for the content detection of sulfonamide synergists.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and in particular relates to a detection method for sulfonamide synergists. Background Art

[0002] Sulfonamide synergists belong to the benzylaminopyrimidine class of compounds. They are synthetic broad-spectrum antibacterial agents that promote growth, prevent and treat diseases caused by microorganisms. They are effective against most Gram-positive and Gram-negative bacteria. Sulfonamide drugs used alone can easily cause bacteria to develop drug resistance. When used in combination with sulfonamide drugs, the bacterial folic acid metabolism can be doubly blocked. Therefore, sulfonamide synergists are often used in combination with sulfonamide drugs in clinical practice to enhance the antibacterial activity of sulfonamide drugs by several to dozens of times. Some of them have bactericidal effects, improve efficacy, shorten the course of treatment, and reduce the toxic and side effects of sulfonamide drugs. Therefore, they are often used to fight coccidia and prevent and treat digestive tract infections such as pullorum and fowl cholera. Since sulfonamide synergists have a long action time and metabolic time in the body, sulfonamide synergists ingested through any route may accumulate in the body. Long-term and large-scale use will accumulate excessive synergists, causing harm to animals and potential harm to human health through the food chain. They are restricted drugs.

[0003] There are many analytical detection methods for sulfonamide synergists residues, including titration, spectrophotometry, gas chromatography, liquid chromatography, capillary electrophoresis and bioanalysis methods, each of which has its advantages and disadvantages. The most commonly used is liquid chromatography, and commonly used detection methods for liquid chromatography include ultraviolet absorption, fluorescence, electrochemical detection and mass spectrometry. The detection wavelength of ultraviolet absorption is mostly selected between 226-360nm, and the sensitivity is usually 0.1mg / L. If a derivatization reaction is performed after the column, the detection sensitivity can reach 0.01mg / L. For the detection of sulfonamide synergists, liquid chromatography-tandem mass spectrometry has also been reported in the literature. It uses a mixed solution of triethylamine and acetonitrile for extraction, or a mixed solution of trichloroacetic acid and acetonitrile for extraction, a pretreatment technology of cation exchange solid phase extraction column purification and liquid chromatography-mass spectrometry / mass spectrometry detection, which can achieve the simultaneous detection of multiple sulfonamide synergists.

[0004] Omeprim is a common sulfonamide enhancer. The detection method reported in the literature is as follows:

[0005] Patent (CN112034054A) discloses a method for detecting the content of ormeprim in aquatic products, which includes extracting with ethyl acetate, concentrating the extract to dryness under reduced pressure, eluting with acetonitrile + 0.1 mol / L hydrochloric acid solution (1+1), purifying with a mixed cation exchange column (MCX), and dissolving with 10% acetonitrile water (containing 0.1% formic acid). Acetonitrile and 0.3% formic acid aqueous solution are used as mobile phases, separated by a C18 column, and determined by high performance liquid chromatography (including a diode array detector). This patent can quickly and accurately determine the residual amount of ormeprim in aquatic products through the characteristic spectrum of the diode array detector, with a detection limit of 0.006 mg / kg and a quantitative lower limit of 0.02 mg / kg, but the equipment requirements are relatively high, and the extraction and purification steps are complicated.

[0006] Journal article ("Determination of sulfonamide synergist residues in animal-derived foods by high performance liquid chromatography-tandem mass spectrometry", Jiang Yuan et al., Journal of Analysis and Testing, Issue 7, 2009) used high performance liquid chromatography-electrospray tandem mass spectrometry to determine three sulfonamide synergists, trimethoprim, dimethoprim and ormeprim, in honey, royal jelly, catfish, eel, pork, pig kidney, pig liver, chicken, beef and milk. Except for the royal jelly matrix, which was directly extracted with 10% trichloroacetic acid solution, the other matrices were extracted with a mixed solution of 10% trichloroacetic acid-acetonitrile (volume ratio 7:3), and the extract solution was enriched and purified by a cation exchange solid phase extraction column. A C18 column was used, and the mobile phase was methanol and 0.1% formic acid solution. One parent ion and two daughter ions were selected for reaction monitoring, and the three sulfonamide synergist residues were qualitatively determined, and the daughter ion with the strongest signal was selected for quantitative determination. However, this solution also has the problem of high equipment requirements and complicated extraction and purification steps.

[0007] Therefore, in view of the defects of the prior art sulfonamide enhancer detection method, the present invention optimizes the extraction solvent, eliminates the need for a cation exchange column for enrichment and purification, and optimizes the mobile phase and elution gradient, thereby achieving qualitative and quantitative detection of sulfonamide enhancers (such as ormeprim) in drugs with good accuracy, which is of great significance for the content detection of sulfonamide enhancers. Summary of the invention

[0008] The present invention aims at the problems existing in the prior art and provides a method for detecting sulfonamide synergists. The present invention adopts a specific ratio of disodium hydrogen phosphate and phosphoric acid aqueous solution and methanol as extraction solvents, does not need to use a cation exchange column for enrichment and purification, and can achieve qualitative and quantitative detection of sulfonamide synergists (such as ormeprim) in drugs by optimizing the mobile phase and elution gradient, and has good accuracy, which is of great significance for the content detection of sulfonamide synergists.

[0009] To achieve the above object, the present invention provides a method for detecting sulfonamide synergists, comprising the following steps:

[0010] 1) Sample pretreatment: dissolve the sample in methanol, sonicate, dilute with solution A, filter, and obtain a filtrate;

[0011] 2) dilute the filtrate with solution B to prepare the test solution; and detect the test solution by high performance liquid chromatography;

[0012] The elution conditions are: gradient elution using mobile phase A and mobile phase B; the mobile phase A is configured as follows: 3.12 g of sodium dihydrogen phosphate dihydrate is dissolved in 1000 mL of water, and the pH value is adjusted to 7.0 with 50% sodium hydroxide solution; the mobile phase B is methanol;

[0013] The elution gradient is:

[0014] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 70 30 10 30 70 10.1 70 30 15 70 30

[0015] In a preferred embodiment, the sulfonamide enhancer is ormeprim.

[0016] In a preferred embodiment, the chromatographic conditions are: using a C18 column or a chromatographic column with equivalent performance as the chromatographic column.

[0017] In a preferred embodiment, the solution A is prepared as follows: 2.5-4 g of sodium dihydrogen phosphate dihydrate is taken, 1000 mL of water is added to dissolve it, 0.5-5 mL of phosphoric acid solution is added, and mixed; the concentration of the phosphoric acid solution is 5-50 mg / mL.

[0018] In a preferred embodiment, the solution A is prepared as follows: 3.12 g of sodium dihydrogen phosphate dihydrate is taken, 1000 mL of water is added to dissolve it, 1.5 mL of phosphoric acid solution is added, and the mixture is mixed.

[0019] In a preferred embodiment, the solution B is a methanol aqueous solution with a volume concentration of 50-70%, more preferably a methanol aqueous solution with a volume concentration of 60%.

[0020] In a preferred embodiment, in step 1), the mass volume ratio of the sample to methanol is 2-5 mg:1 mL, the volume ratio of solution A to methanol is 0.4-1:1; the ultrasonic power is 200-400 W, and the ultrasonic time is 2-20 min.

[0021] In a preferred embodiment, in step 2), the volume ratio of the filtrate to solution B is 1:8-15.

[0022] In a preferred embodiment, the parameters of the chromatography column are: 4.6 mm×(150-250) mm, 3-5 μm, more preferably 4.6 mm×250 mm, 5 μm.

[0023] In a preferred embodiment, the flow rate is 0.5-1.5 mL / min, preferably 1.0 mL / min; the detection wavelength is 230-240 nm, preferably 235 nm; the column temperature is 30-40°C, preferably 35°C; and the injection volume is 10-20 μL, preferably 10 μL.

[0024] In a second aspect, the present invention provides the application of the aforementioned method for detecting sulfonamide enhancers in the detection of products containing ormeprim.

[0025] In a preferred embodiment, the product comprises a pharmaceutical product.

[0026] Compared with the prior art, the technical advantages of the present invention are:

[0027] 1. The present invention adopts an aqueous solution of disodium hydrogen phosphate and phosphoric acid and methanol in a specific ratio as extraction solvents, and does not require the use of a cation exchange column for enrichment and purification. By optimizing the mobile phase and elution gradient, the qualitative and quantitative detection of ometalate in the drug can be achieved with good accuracy, which is of great significance for the content detection of sulfonamide enhancers such as ometalate.

[0028] 2. The linear correlation coefficient of the detection method of the present invention is 0.9997; in the precision test, the RSD of 6 repeatability experiments is 0.52%, and the RSD of the intermediate precision experiment is 0.46%; in the accuracy test, the recovery rate is in the range of 98.7%-99.6%, and the RSD is 0.27%. The detection method of the present invention can accurately determine the content of ormeprim in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the blank solution chromatogram of Example 1;

[0030] Figure 2 It is the chromatogram of the reference substance solution of Example 1;

[0031] Figure 3 The chromatogram of the test solution of Example 1;

[0032] Figure 4 This is the linear relationship diagram of Ogilvy. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings of the specification. The test methods for which specific conditions are not specified in the preferred embodiments are usually carried out under conventional conditions. The examples are provided to better illustrate the contents of the present invention, but the contents of the present invention are not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above-mentioned invention contents, which still fall within the protection scope of the present invention.

[0034] Example 1

[0035] 1) Raw materials and equipment

[0036] Control substance: Omeprim, homemade;

[0037] Sample: model: primor, manufacturer: zoetis;

[0038] The remaining reagents were common commercial products, the solvent was of chromatographic grade, and the reagents other than the solvent were of analytical grade.

[0039] 2) Reagent configuration

[0040] Phosphoric acid solution: Dissolve 1 mL of phosphoric acid (AR) in water and make up to 100 mL.

[0041] Solution A: Take 3.12 g of sodium dihydrogen phosphate dihydrate, add 1000 mL of water to dissolve it, add 1.5 mL of phosphoric acid solution, and mix well.

[0042] Solution B: Take 60 mL of methanol and 40 mL of water and mix well.

[0043] Reference substance stock solution: Accurately weigh the ometalloprim reference substance and prepare the reference substance stock solution (ometalloprim 2000 μg / mL) with methanol.

[0044] Reference substance solution: Accurately measure the reference substance stock solution and quantitatively dilute it with solution B to prepare a solution containing 20 μg of ormeprim per 1 mL.

[0045] Test solution: Take 175 mg of sample, place it in a 100 mL volumetric flask, add 60 mL of methanol, ultrasonicate for 5 minutes (300 W), dilute to the scale with solution A, shake well, filter, accurately measure 1 mL of the filtrate, place it in a 10 mL volumetric flask, dilute to the scale with solution B, shake well, and use it as the test solution.

[0046] 3) Liquid chromatography conditions

[0047] Instrument: Agilent 1260 high performance liquid chromatograph (UV detector);

[0048] Chromatographic column: Themro C18 column, column parameters: 4.6mm×250mm, 5μm.

[0049] Mobile phase A: Take 3.12 g of sodium dihydrogen phosphate dihydrate, add 1000 mL of water to dissolve it, and adjust the pH value to 7.0 with 50 wt % sodium hydroxide solution.

[0050] Mobile phase B: methanol.

[0051] The elution gradient was set as follows:

[0052] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 70 30 10 30 70 10.1 70 30 15 70 30

[0053] Detector: UV; detection wavelength: 235 nm; flow rate: 1.0 mL / min; column temperature: 35 °C; injection volume: 10 μL.

[0054] 4) Detection

[0055] Take blank solution (solution B), reference solution and test solution for injection respectively, perform HPLC analysis, record the chromatogram, calculate the content of ormeprim in the sample. The chromatogram of blank solution is shown in the attached figure. Figure 1 The chromatogram of the reference solution is as follows: Figure 2 The chromatogram of the test solution is as follows: Figure 3 .

[0056] 5) Methodological validation

[0057] The above methods were validated by methodology, and the validation items included linearity, precision, and accuracy, as follows:

[0058] 5.1) Linearity test: Linear solutions (40%, 60%, 80%, 100% and 130%) were used for linearity verification. The results are shown in Table 1 and Figure 4 .

[0059] Table 1 Linearity verification results

[0060]

[0061] 5.2) Precision test: Different personnel prepared 6 test solutions on different dates and used different instruments to determine the content of omeprim. The results are shown in Table 2.

[0062] Table 2 Precision verification results

[0063]

[0064] 5.3) Accuracy test: Take 27.5 mg of blank auxiliary material, accurately weigh it, put it in a 50 ml volumetric flask, add the ormeprim reference substance according to the concentration level in Table 3, add 30 ml of methanol, ultrasonicate for 5 minutes, dilute to the scale with solution A, shake well, filter, accurately measure 1 ml of the filtrate, put it in a 10 ml volumetric flask, dilute to the scale with solution B, shake well, and use it as the recovery test solution. The recovery results of ormeprim are shown in Table 3.

[0065] Table 3 Accuracy verification results

[0066]

[0067] Finally, it should be noted that the above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. Application of a method for detecting sulfonamide synergists in the detection of products containing ormeprim, characterized in that: The product described also contains sulfadimethoxine; The detection method comprises the following steps: 1) Sample pretreatment: Take 175 mg of sample, place it in a 100 mL volumetric flask, add 60 mL of methanol, sonicate for 5 minutes, dilute to scale with solution A, shake well, filter, accurately measure 1 mL of the filtrate, place it in a 10 mL volumetric flask, dilute to scale with solution B, shake well, and use it as the test solution; Solution A: Take 3.12g of sodium dihydrogen phosphate dihydrate, add 1000mL of water to dissolve, add 1.5mL of phosphoric acid solution, and mix well; Solution B: Take 60 mL of methanol and 40 mL of water and mix well; 2) Detect the test solution by high performance liquid chromatography: Liquid chromatography conditions: Instrument: Agilent 1260 high performance liquid chromatograph; Chromatographic column: Themro C18 column, column parameters: 4.6 mm × 250 mm, 5 μm; Mobile phase A: Take 3.12 g of sodium dihydrogen phosphate dihydrate, add 1000 mL of water to dissolve, and adjust the pH to 7.0 with 50 wt% sodium hydroxide solution; Mobile phase B: methanol; The elution gradient was set as follows: Detector: UV; detection wavelength: 235 nm; flow rate: 1.0 mL / min; column temperature: 35 °C; injection volume: 10 μL.

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