A rapid detection method for synchronously deriving and extracting sulfonamide residues

Abstract

The application provides a rapid detection method for synchronously deriving and extracting sulfonamide drug residues, comprising the following steps: (1) sample preparation; (2) synchronous derivation and extraction; (3) elution of the derivative product; (4) detection and analysis by a liquid phase fluorescence chromatograph. The method synchronously performs the extraction and derivation steps which are usually performed separately, and accelerates the completion of sample pretreatment by a magnetic dispersion solid phase extraction mode. Compared with a traditional detection method, the method effectively simplifies the sample pretreatment process, greatly shortens the sample pretreatment time, reduces possible human-induced errors, improves the sample pretreatment efficiency and realizes accurate detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of drug detection, and more particularly relates to a rapid detection method for synchronously derivatizing and extracting sulfonamide drug residues. BACKGROUND

[0002] Sulfonamides (SAs) are a class of synthetic antibacterial drugs, which are widely used in human and animal medicine due to their low cost, high efficiency, and broad-spectrum antibacterial activity. However, humans and animals cannot completely degrade SAs, and a part of the original drugs will enter the environment through urine and feces, causing soil and water pollution. The widespread use of SAs can lead to the residues of SAs in the environment and animal-derived products, and accumulate in the human body through the food chain, causing a series of adverse reactions such as drug resistance, allergy, and urinary system damage. Therefore, it is necessary to establish an analysis method for accurately detecting SAs residues.

[0003] Sulfonamides can be detected by electrochemistry, digital image measurement, spectroscopic techniques, and chromatographic techniques. Among these methods, high-performance liquid chromatography (HPLC) is widely used due to its high selectivity and good reproducibility. However, due to the complexity of food and environmental sample matrices and the extremely low concentration of SAs, direct and accurate analysis by HPLC is not possible. Therefore, effective sample pretreatment such as extraction, concentration, and derivatization must be performed before HPLC analysis.

[0004] In recent years, the combination of chemical derivatization and extraction technology has been widely used in environmental, food, and pharmaceutical research fields. This new type of sample pretreatment technology not only can achieve the enrichment and impurity removal of analytes, but also can improve the selectivity and sensitivity of the method, which will be very beneficial to the HPLC detection of SAs. However, for the determination of SAs, extraction and derivatization are usually performed separately or sequentially, which introduces additional derivatization steps, and the operation is relatively complicated and time-consuming; at the same time, it may increase the sample transfer steps, introduce human errors, and reduce the accuracy of the detection results. In-situ derivatization can combine the extraction and derivatization steps of the target analyte into one, simplifying the sample pretreatment process and shortening the pretreatment time. However, the sample preparation method can be further simplified.

[0005] Carbon nanomaterials are a new type of solid-phase adsorbents, including activated carbon, graphene, carbon nanotubes (CNTs) and multi-walled carbon nanotubes (MWCNTs) and so on. Among them, MWCNTs are widely concerned by analysts due to its good stability, porous structure and large specific surface area. For chemical derivatization of SAs, fluorescent amine (FA) is the most commonly used fluorescent derivatization reagent, which is frequently used in HPLC analysis and detection. The reaction product of SAs and FA has certain hydrophobicity, and MWCNT is a hydrophobic material with high surface activity, which can effectively adsorb SAs-FA product through hydrogen bonding or π-π interaction. Therefore, the sample preparation method of synchronous derivatization and extraction of SAs is of practical significance. SUMMARY

[0006] In view of the deficiencies of the prior art, the present application provides a rapid detection method for synchronous derivatization and extraction of sulfonamide drugs. The derivatization and extraction steps are completed synchronously by mixing sample solution, derivatization reagent and adsorbent; and the separation and purification of analytes are completed by magnetic dispersion solid-phase extraction and external magnet, which solves the problems of the prior SAs detection method, such as complicated sample pretreatment steps, time-consuming, easy to introduce human error and unsuitable for batch processing of samples.

[0007] To solve the above technical problems, the present application adopts the following technical solutions:

[0008] A rapid detection method for synchronous derivatization and extraction of sulfonamide drugs, comprising the following steps:

[0009] (1) sample preparation, (2) synchronous derivatization and extraction, (3) elution of derivatization product, (4) detection and analysis by liquid phase fluorescence chromatograph.

[0010] Further, the sample in step (1) is a liquid, semi-solid or solid sample, such as environmental water, honey, meat products, liver and the like.

[0011] Preferably, the sample in step (1) is prepared into a sample to be measured with water as the matrix.

[0012] Preferably, the derivatization reagent in step (2) is an acetonitrile solution of fluorescent amine, wherein the concentration of fluorescent amine in the derivatization reagent is 1-15 g / L; and the concentration of sulfonamide in the sample to be measured is 0.01-10 μg / mL.

[0013] Preferably, the buffer salt solution in step (2) can be acetic acid buffer salt, phosphoric acid buffer salt and the like; and the pH of the buffer salt solution is 3.0-6.0.

[0014] Preferably, the extraction medium in step (2) is magnetic hydroxylated carbon nanotubes, which are prepared from ferroferric oxide and commercial hydroxylated carbon nanotubes by a conventional synthesis method, wherein the magnetic hydroxylated carbon nanotubes are used in an amount of 1-10 mg.

[0015] Preferably, step (2) mixes the sample to be tested, a buffer salt solution, a derivatization reagent and an extraction medium, and completes the derivatization and magnetic dispersive solid-phase extraction under the condition of vortex assistance in one step, and the simultaneous derivatization and extraction time is 1-10 min.

[0016] Preferably, step (3) separates the sample solution after derivatization in step (2) by an external magnet, and adds acetone to the solid phase for elution.

[0017] Preferably, the elution step in step (3) is performed by vortex, and the elution time is 1-10 min.

[0018] Preferably, step (4) takes out the eluent in step (3) by an external magnet to obtain a purified acetone extract.

[0019] Preferably, step (4) detects and analyzes the obtained purified acetone extract by a liquid chromatograph with fluorescence detection.

[0020] Compared with the prior art, the method has the following beneficial effects:

[0021] The application provides a rapid detection method for simultaneously derivatizing and extracting sulfonamide drugs. First, a sample to be tested is simply treated, such as filtration and organic solvent extraction, to prepare a sample to be tested with pure water as a matrix. The prepared sample to be tested, a buffer salt solution, a derivatization reagent and a solid-phase adsorbent are mixed and vortexed to simultaneously complete derivatization and extraction. Compared with a traditional method, the original separate or continuous derivatization and extraction steps are integrated into one step, the sample pretreatment process is simplified, possible human errors are reduced, and the accuracy of sample detection is improved. In addition, the extraction technology used is magnetic dispersive solid-phase extraction technology, which can complete the derivatization, extraction and elution steps by simple vortexing, avoids the use of organic solvents in liquid-liquid extraction and the cumbersome steps such as activation and washing in solid-phase extraction, and separates solid and liquid by an external magnet, greatly shortening the sample pretreatment time and being helpful to realize batch preparation of samples. In summary, the application effectively simplifies the sample pretreatment process, reduces the operation complexity, reduces possible human errors, and greatly shortens the sample pretreatment time, improves the sample preparation efficiency, and also ensures accurate analysis of sulfonamide drugs. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1Flow chart of the method for determination of sulfonamides in the present application.

[0023] Figure 2 Chromatograms of the derivatized products of Example 1 sulfonamides passed or failed magnetic dispersive solid phase extraction. (A) Blank water solution passed magnetic dispersive solid phase extraction after derivatization, (B) spiked water solution (20 ng / mL) failed magnetic dispersive solid phase extraction after derivatization, (C) spiked water solution (20 ng / mL) passed magnetic dispersive solid phase extraction after derivatization.

[0024] Figure 3 Effect of derivatization reagent concentration on recovery of simultaneous derivatization and extraction of sulfonamides in Example 1.

[0025] Figure 4 Effect of buffer salt pH on recovery of simultaneous derivatization and extraction of sulfonamides in Example 1.

[0026] Figure 5 Effect of simultaneous derivatization and extraction time on recovery of simultaneous derivatization and extraction of sulfonamides in Example 1.

[0027] Figure 6 Effect of solid phase adsorbent amount on recovery of simultaneous derivatization and extraction of sulfonamides in Example 1.

[0028] Figure 7 Effect of elution solvent type on recovery of simultaneous derivatization and extraction of sulfonamides in Example 1.

[0029] Figure 8 Effect of elution solvent volume on recovery of simultaneous derivatization and extraction of sulfonamides in Example 1.

[0030] Figure 9 Effect of elution time on recovery of simultaneous derivatization and extraction of sulfonamides in Example 1. DETAILED DESCRIPTION

[0031] The present application will be further described below in conjunction with the accompanying drawings and specific embodiments, it should be noted that the following described embodiments are only used to explain the present application, and are not used to limit the present application.

[0032] Example 1

[0033] The present application provides a rapid detection method for simultaneous derivatization and extraction of sulfonamides in honey, comprising the following steps:

[0034] (1) Sample preparation: 9 sulfonamides were selected as the analytes, i.e. sulfaguanidine (SGN), sulfanilamide (SA), sulfadiazine (SDZ), sulfamethoxazole (SMZ), sulfisoxazole (SIS), sulfamonomethoxine (SMM), sulfadoxine (SDM), sulfabenzamide (SB). 5 g of honey sample was taken in a 15 mL centrifuge tube, 10 mL of acetonitrile was added, and the sample was ultrasonically extracted for 20 min. After filtration through a 0.22 μm filter membrane, 3 mL of the crude extract was taken and blown to near dryness under N2 at room temperature. 1.5 mL of a mixed standard solution of a certain concentration was added to reconstitute the sample to the desired concentration.

[0035] (2) Simultaneous derivatization and extraction: 1.5 mL of the simply prepared sample solution, 200 μL of sodium acetate buffer salt solution (0.25 M, pH 3.5), 50 μL of 12 mg / mL fluorescent amine acetonitrile solution and 5 mg of magnetic MWCNTs-OH material were mixed in a 2 mL centrifuge tube, and vortexed for 3 min for simultaneous derivatization and dispersive solid phase extraction.

[0036] (3) Elution of the derivatized product: the above sample solution after derivatization was discarded, and 100 μL of acetone was used to vortex for 2 min for elution.

[0037] (4) High performance liquid chromatography fluorescence detector analysis: the acetone extract was separated by solid-liquid separation using an external magnet, and the separated extract was detected and analyzed by high performance liquid chromatography fluorescence detector. The above liquid chromatography conditions are as follows: Agilent 5 TC-C18 (4.6 mm x 150 mm, 5 μm) was used as the sulfonamide separation chromatographic column; the mobile phase composition was: A (0.1% formic acid aqueous solution), B (0.1% formic acid acetonitrile solution); the gradient elution program was: 0-15 min 70% A, 15-20 min 68% A, 20-40 min 58% A, 40-45 min 70% A; 45-50 min 70% A; the flow rate was set to 1 mL / min, the column temperature was set to 35℃, the injection volume was set to 20 μL, and the fluorescence detector conditions were: excitation wavelength 395 nm, emission wavelength 491 nm.

[0038] Comparative Example 1

[0039] The difference between Comparative Example 1 and Example 1 is that the sample in Comparative Example 1 is a blank control sample, specifically, the 1.5 mL sample solution in step (1) of Example 1 is replaced with 1.5 mL of deionized water.

[0040] Comparative Example 2

[0041] The difference between Comparative Example 2 and Example 1 is that Comparative Example 2 is directly derivatized from a sulfanilamide standard aqueous solution, specifically 1.5 mL of a sulfanilamide standard solution, 200 μL of a sodium acetate buffer salt solution (0.25 M, pH 3.5), 50 μL of a 12 mg / mL fluorescamine acetonitrile solution are mixed in a 2 mL centrifuge tube, vortexed for 3 min to perform derivatization, and the obtained derivatized product solution is directly subjected to instrument detection.

[0042] To verify the feasibility of the experimental scheme, each experiment is repeated 3 times, the same as below.

[0043] To investigate the specificity of the method, the samples with added standard are subjected to synchronous derivatization and extraction (Example 1), the blank control samples are subjected to synchronous derivatization and extraction (Comparative Example 1), and the added standard aqueous solution is directly derivatized without synchronous derivatization and extraction (Comparative Example 2), as shown in Figure 2 , and Figure 2 are liquid chromatograms of Example 1, Comparative Examples 1 and 2. The results show that the detection method of the present application can detect all the analytes to be detected, and compared with the blank control sample, no signal interfering with the derivatized product is generated, at the same time, three groups of parallel experiments are performed to investigate the repeatability, the relative standard deviation is within 15%, indicating that the method has specificity and repeatability.

[0044] To obtain higher derivatization and extraction efficiency, the main influencing factors are investigated, including the concentration of derivatization reagent, the pH of buffer salt, the synchronous derivatization and extraction time, the amount of solid-phase adsorbent, the type of elution solvent, the volume of elution solvent and the elution time. At the same time, in order to obtain more reliable experimental conditions, we use a higher concentration of added standard water sample (20 ng / mL) to perform optimization experiments, and three groups of parallel experiments are set up for each condition.

[0045] 1.1 Influence of Derivatization Reagent Concentration on Derivatized Product Recovery

[0046] In step (2) of Example 1, 50 μL of 1, 2, 5, 8, 10, 12 and 15 mg / mL fluorescamine acetonitrile solution concentrations are used for experiments, and the remaining steps are the same as Example 1. Each group of experiments is repeated three times. The experimental results are shown in Figure 3 , when the concentration of fluorescamine is 10 mg / mL, the recovery rate of the derivatized product is the highest, and considering the stability of the experimental results, the concentration of 12 mg / mL fluorescamine acetonitrile solution is finally selected.

[0047] 1.2 Influence of Acetic Acid Buffer Solution pH on Recovery

[0048] In step (2) of Example 1, 200 μL of sodium acetate buffer solution (0.25 M) with pH 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0, respectively, were used, and the rest of the steps were the same as in Example 1. Each group of experiments was repeated three times. The experimental results are shown in Table 1. Figure 4 As shown in Table 1, the recovery rate was higher when the pH of the acetate buffer solution was 3.5.

[0049] 1.3 Influence of simultaneous derivatization and extraction time on recovery rate

[0050] In step (2) of Example 1, 1.5 mL of the simply prepared sample solution, 200 μL of sodium acetate buffer solution (0.25 M, pH 3.5), 50 μL of 12 mg / mL fluorescent amine acetonitrile solution and 5 mg of magnetic MWCNTs-OH material were mixed, and the vortex was performed for 1, 2, 3, 5, 8 and 10 min, respectively, for simultaneous derivatization and extraction, and the rest of the steps were the same as in Example 1. Each group of experiments was repeated three times. The experimental results are shown in Table 2. Figure 5 As shown in Table 2, the recovery rate and repeatability were better when the vortex was performed for 3 min.

[0051] 1.4 Influence of solid-phase adsorbent dosage on recovery rate

[0052] In step 4) of Example 1, 2, 4, 5, 6, 8 and 10 mg of magnetic MWCNTs-OH material were used, respectively, and the rest of the steps were the same as in Example 1. Each group of experiments was repeated three times. The experimental results are shown in Table 3. Figure 6 As shown in Table 3, the recovery rate was higher and the results were less likely to fluctuate when the adsorbent dosage was 5 mg.

[0053] 1.5 Influence of elution solvent type on recovery rate

[0054] In step (3) of Example 1, methanol, acetonitrile and acetone were used to elute the derivatized product, respectively, and the rest of the steps were the same as in Example 1. Each group of experiments was repeated three times. The experimental results are shown in Table 4. Figure 7 As shown in Table 4, the extraction efficiency was higher when acetone was used as the elution solvent.

[0055] 1.6 Influence of elution solvent volume on recovery rate

[0056] In step (3) of Example 1, 100, 150, 200, 250 and 300 μL of acetone were used to elute the derivatized product, respectively, and the rest of the steps were the same as in Example 1. Each group of experiments was repeated three times. The experimental results are shown in Table 5. Figure 8 As shown in Table 5, the response of the derivatized product was the highest and the detection method was the most sensitive when the volume of acetone was 100 μL.

[0057] 1.7 Influence of elution time on recovery rate

[0058] In step (3) of Example 1, the elution of the derivatized product was performed by vortexing for 1, 2, 5, 8 and 10 min, respectively, and the remaining steps were the same as in Example 1. Each set of experiments was repeated three times. The experimental results are shown in Table 1. Figure 9 As shown in Table 1, almost complete elution was achieved at an elution time of 2 min, and the results were reproducible.

[0059] The quantitative analysis method used a matrix-matched calibration curve method. The sulfonamides were diluted with pure water (prepared with methanol) to prepare a series of standard solutions with concentrations of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2, 5 and 10 ng / mL. The honey without the analyte was used as a blank matrix. The simple processing according to step (1) of Example 1 was performed, and 1.5 mL of the standard aqueous solution with the above-mentioned series of concentrations was added for reconstitution to prepare honey samples with different concentrations. The remaining steps were performed according to the optimized method of Example 1, and the peak area of the derivatized product (3 sets of parallel samples for each concentration) was recorded to obtain the linear regression equation and the correlation coefficient by linear regression of the average peak area with respect to the corresponding spiked concentration. The detection limit (LOD) and the quantification limit (LOQ) were calculated at signal-to-noise ratios of 3:1 and 10:1, respectively, as shown in Table 1, indicating that the sulfonamides had a good linear relationship in the concentration range of 0.01-10.0 ng / g, with R 2 > 0.9989.

[0060] Table 1 Linear range, detection limit and quantification limit

[0061]

[0062] The precision (reproducibility) and accuracy of the method were measured by the relative standard deviation (RSD) and the relative recovery, respectively. The target analyte was added to the blank honey at low, medium and high concentrations. At these three concentrations, 3 sets of parallel samples were prepared and tested within a day, and the peak area was recorded to calculate the intraday relative standard deviation. The interday relative standard deviation was calculated by recording the peak area from 3 sets of parallel samples prepared and tested on 3 consecutive days. The results are shown in Table 2, and the accuracy was in the range of 86.2-111.2%, with RSD≤10.5%.

[0063] Table 2 Precision and accuracy

[0064]

[0065] Continued Table 2

[0066]

[0067] From the above data, it can be seen that the quantitative detection method of the application has good linearity, accuracy and detection sensitivity, and can meet general detection requirements.

[0068] In order to verify whether the method of the application has application value, 8 kinds of commercialized honey (1, linden tree honey; 2, willow honey; 3, jujube honey; 4, buckwheat honey; 5, honeysuckle honey; 6, medlar honey; 7, locust honey; 8, hundred flower honey) were detected and analyzed, and no signal of 9 kinds of sulfonamides was detected. In order to further verify the quantitative analysis ability, the above 8 kinds of honey samples were randomly added with standard. The results are shown in Table 3, the detection level is equivalent to the standard addition level, and the relative standard deviation is within 20%.

[0069] Table 3 Detection of 8 kinds of commercialized honey samples

[0070]

[0071] The above embodiments are only preferred embodiments of the application, and cannot be used to limit the protection scope of the application. Any non-substantial changes and replacements made by those skilled in the art on the basis of the application shall fall within the protection scope of the application.

Claims

1. A rapid detection method for simultaneous derivatization and extraction of sulfonamides, characterized in that, It comprises the following steps: (1) preparing the sample into a sample to be tested; (2) simultaneous derivatization and extraction; (3) elution of the derivatization product; (4) detection and analysis by liquid phase fluorescence chromatograph; The method of step (2) is as follows: the sample to be tested, a buffer salt solution, a derivatization reagent and an extraction medium are mixed in the same test tube, and simultaneous derivatization and extraction are carried out under the condition of shaking or vortexing; The buffer salt solution is a phosphoric acid buffer salt solution, an acetic acid buffer salt solution or a boric acid buffer salt solution, and the pH of the buffer salt solution is 3.0-6.0; The derivatization reagent is an acetonitrile solution of fluorescent amine, and the concentration of fluorescent amine in the derivatization reagent is 1-15 g / L; the concentration of sulfonamides in the sample to be tested is 0.01-10 μg / mL; and the extraction medium is magnetic hydroxylated carbon nanotubes, wherein the magnetic hydroxylated carbon nanotubes are prepared from ferroferric oxide and commercial hydroxylated carbon nanotubes by a conventional synthesis method, and the amount of the magnetic hydroxylated carbon nanotubes is 1-10 mg; The time for simultaneous derivatization and extraction under the condition of vortexing in step (2) is 1-10 min; The sample in step (1) is honey; The elution time is 1-10 min; The sulfonamides are sulfaguanidine SGN, sulfanilamide SA, sulfadiazine SDZ, sulfadimethoxine SMZ, sulfisoxazole SMM, sulfamonomethoxine SMD, sulfamethoxazole SMX, sulfadimethoxine SDM and sulfabenzamide SB; The liquid chromatography conditions in the detection and analysis by liquid phase fluorescence chromatograph in step (4) are as follows: Agilent 5 TC-C18 is used as a sulfonamide separation chromatographic column; the mobile phase composition is as follows: A: 0.1% formic acid aqueous solution, B: 0.1% formic acid acetonitrile solution; the gradient elution program is as follows: 0-15 min 70% A, 15-20 min 68% A, 20-40 min 58% A, 40-45 min 70% A; 45-50 min 70% A; the flow rate is set to be 1 mL / min, the column temperature is set to be 35℃, the injection volume is set to be 20 μL, and the fluorescence detector conditions are as follows: excitation wavelength 395 nm, emission wavelength 491 nm.

2. The rapid detection method according to claim 1, characterized in that, In step (1), the sample is prepared into a sample to be tested with water as the matrix, and the treatment mode adopts centrifugation, filtration or organic solvent extraction and blowing dry.

3. The rapid detection method according to claim 1, characterized in that, In step (3), the sample solution after simultaneous derivatization and extraction in step (2) is removed, then an elution solvent is added, and elution is carried out under the condition of vortexing or shaking.

4. The rapid detection method according to claim 3, characterized in that, In step (3), the removal is carried out by using an external magnet to separate the sample solution of magnetic hydroxylated carbon nanotubes; and the elution solvent is acetone.

5. The rapid detection method of claim 1, wherein, In step (4), the eluate in step (3) is transferred out by using an external magnet, and detection and analysis are carried out by using a liquid phase fluorescence chromatograph.

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