A method for the simultaneous derivatization and extraction of biogenic amines for analytical detection

Abstract

The application discloses a kind of simultaneously derived and extracted biological amine analytical detection methods, it includes the following steps: sample solution, derivatization reagent, extraction material are mixed vortex reaction in the same centrifugal tube, complete derivatization extraction in this process, finally using suitable desorption solvent, and the derivative is desorbed from adsorption material, and the desorption solution containing derivative can be directly used for analysis and detection, compared with the pretreatment process in traditional method, avoid the problem that extraction column is easily blocked in solid-phase extraction, effectively simplify sample pretreatment process, shorten sample pretreatment time, operation step is more simple, fast and efficient.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology and relates to an analytical detection method for simultaneously derivatizing and extracting biogenic amines. Background Technology

[0002] Bioamines (BAs) are a class of small, amino-containing compounds with biological activity. They are primarily produced from amino acids via amino acid decarboxylases and serve as precursors for the synthesis of proteins, alkaloids, hormones, nucleotides, and other compounds. They play a crucial role in regulating nucleic acid and protein synthesis and stabilizing biological membranes, and are essential components of biologically active cells. BAs are widely found in various foods, especially those rich in protein and amino acids, including meat products, dairy products, seafood, fermented foods, fruits, and vegetables. Common BAs in food include histamine (His), tyramine (Tyr), putrescine (Put), cadaverine (Cad), 2-phenylethylamine (Phe), spermidine (Spd), and spermine (Spm). Ingesting appropriate amounts of biogenic amines can have a regulatory effect on the human body, promoting growth and metabolism. However, excessive intake can lead to poisoning, causing symptoms such as headache, difficulty breathing, and vomiting. Therefore, it is necessary to establish efficient analytical and detection technologies to ensure food quality and safety.

[0003] High-performance liquid chromatography (HPLC) is a commonly used method for detecting BAs. However, BAs are small-molecule active compounds that do not possess fluorescence or ultraviolet chromatin. Therefore, derivatization is usually required before HPLC detection of BAs. Before chromatographic analysis, due to the complexity of the sample matrix and the low concentration of the analyte, sample pretreatment is typically necessary to remove interfering drugs from the matrix and extract or enrich the target analyte from the sample. For BAs extraction methods, solid-phase extraction (SPE), solid-phase microextraction (SPE), liquid-liquid extraction (LC-LI), and dispersive liquid-liquid microextraction (DL-LI) are common. Traditional LC-LI consumes a lot of organic solvents and has poor selectivity, while traditional SPE is usually complex. Furthermore, these extraction techniques and derivatization methods are generally performed separately, either first extraction followed by derivatization or first derivatization followed by extraction. These methods complicate the sample pretreatment process, leading to sample loss and analytical errors. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the present invention aims to provide an analytical method for the simultaneous derivatization and extraction of biogenic amines. This method is characterized by its simple operation, rapid efficiency, and high accuracy and reliability. The present invention uses magnetic functionalized hydroxyl carbon nanotubes (MWCNTs-OH) as the extraction material, encapsulating the non-magnetic MWCNTs-OH with Fe3O4 to impart magnetism. The sample solution, extraction material, and derivatization reagent are mixed and vortexed together in a centrifuge tube, simultaneously performing derivatization and extraction. Fe3O4 / MWCNTs-OH selectively recognizes analyte derivatives primarily through hydrophobic interactions and π-π interactions. The derivatives are separated through washing and desorption steps under an applied magnetic field. The entire sample pretreatment process eliminates the need for centrifugation. Furthermore, this method is combined with a high-performance liquid chromatography-fluorescence detector (HPLC-FLD) for the determination of biogenic amines in samples such as wine, spirits, and beer.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] An analytical detection method for simultaneously derivatizing and extracting biogenic amines includes the following steps:

[0007] (1) Add the sample solution, derivatization reagent, extraction material and buffer salt into the same centrifuge tube and mix and vortex. During this process, derivatization and extraction are completed simultaneously. Under the action of an external magnetic field, the extraction material that has adsorbed biogenic amine derivatives can be separated from the sample solution.

[0008] (2) Add a certain amount of pure water, mix and vortex for a certain time, and separate and discard the supernatant under the action of an external magnetic field;

[0009] (3) Add a certain amount of desorption solvent, mix and vortex for a certain time to desorb the derivatized products from the material;

[0010] (4) The desorption solution containing the target analyte obtained in step (3) is detected and analyzed.

[0011] Furthermore, the sample solution is human urine, baijiu (Chinese white liquor), wine, red wine, beer, soy sauce, or huangjiu (yellow wine).

[0012] Furthermore, in step (1), the sample solution, derivatization reagent, and extraction material are added to the same centrifuge tube in any order for reaction.

[0013] Further, the derivatizing reagent in step (1) is 6-aminoquinolinyl-N-hydroxysuccinimide carbamate (AQC), the concentration of the derivatizing reagent is 1–2 mg / mL, preferably 2 mg / mL, and the volume of the derivatizing reagent is 50–400 μL, preferably 200 μL.

[0014] Further, the extraction material in step (1) is magnetic hydroxylated multi-walled carbon nanotubes (Fe3O4 / MWCNTs-OH), wherein the Fe3O4 / MWCNTs-OH is a mixture of Fe3O4 nanoparticles and MWCNTs-OH in a mass ratio of 4:1. The mixing method can be grinding and mixing until uniform. The amount of Fe3O4 / MWCNTs-OH material is 1–10 mg, preferably 4 mg.

[0015] Further, the buffer salt in step (1) is borax buffer salt, phosphate buffer salt or acetate buffer salt, and the pH of the buffer salt is 8.0-9.3, preferably 9.3.

[0016] Furthermore, the extraction and derivatization reactions in step (1) can be carried out by any one of the following methods: vortexing, oscillation, or ultrasound, with vortexing being preferred for extraction and derivatization.

[0017] Furthermore, the mixing vortex reaction time in step (1) is 1-10 min, preferably 1 min.

[0018] Furthermore, the desorption solvent in step (3) can be any one of acetone, acetonitrile, methanol, ethanol, or isopropanol, preferably acetone.

[0019] Furthermore, the desorption time in step (3) is 1-10 min, preferably 1 min.

[0020] Furthermore, the detection instrument in step (3) can be high performance liquid chromatography combined with a fluorescence detector or liquid chromatography-mass spectrometry.

[0021] Furthermore, the biogenic amine is any one of histamine, tyramine, putrescine, cadaverine, 2-phenylethylamine, spermine, or spermidine.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This invention provides an analytical detection method for the simultaneous derivatization and extraction of biogenic amines. The method involves mixing the sample solution, derivatization reagent solution, adsorbent material, and buffer salt in the same centrifuge tube and vortexing them to simultaneously carry out the derivatization and extraction processes. Following washing and desorption steps, an eluent containing the derivative of the analyte is obtained for detection. This method exhibits good extraction efficiency, meets the requirements for rapid analytical processing under certain conditions, and is simple, rapid, efficient, and demonstrates good sensitivity and selectivity. Attached Figure Description

[0024] Figure 1The HPLC-FLD chromatograms for the feasibility analysis of Comparative Examples 1-3 of this invention are shown, wherein A, blank solution, is subjected to simultaneous derivatization and extraction; B, analyte standard solution (5.0 μg / mL), is subjected to simultaneous derivatization and extraction; and C, analyte standard solution (5.0 μg / mL), is directly derivatized.

[0025] Figure 2 This is a graph showing the detection results corresponding to different amounts of extraction material in Example 2 of the present invention;

[0026] Figure 3 This is a graph showing the detection results corresponding to different amounts of the derivatization reagent in Example 3 of the present invention;

[0027] Figure 4 This is a graph showing the detection results for different pH values ​​in Example 4 of the present invention;

[0028] Figure 5 This is a graph showing the detection results at different times during the derivatization and extraction reactions in Example 5 of the present invention;

[0029] Figure 6 This is a graph showing the detection results for different types of desorption solvents in Example 6 of the present invention;

[0030] Figure 7 This is a graph showing the detection results corresponding to different amounts of desorption solvent in Example 7 of the present invention;

[0031] Figure 8 This is a graph showing the detection results corresponding to different desorption times in Example 8 of the present invention;

[0032] Figure 9 This is a schematic flowchart of the analytical detection method for simultaneously derivatizing and extracting biogenic amines according to the present invention. Detailed Implementation

[0033] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments.

[0034] Example 1

[0035] The analytical detection method for simultaneously derivatizing and extracting biogenic amines in this embodiment includes the following steps:

[0036] (1) Establishment of a model compound for the simultaneous derivatization and extraction analysis of biogenic amines

[0037] a. Preparation of analyte standard solutions: Accurately weigh appropriate amounts of histamine, tyramine, putrescine, cadaverine, 2-phenylethylamine, spermine, and spermidine using a 1 / 100,000 balance, and dissolve them separately in 60% acetonitrile water to prepare stock solutions of 2 mg / mL. These solutions can be diluted with water to provide concentrations for further use. All sample solutions should be stored in a refrigerator at 4°C protected from light.

[0038] b. Preparation of derivatization reagent solution: Accurately weigh an appropriate amount of 6-aminoquinolino-N-hydroxysuccinimide carbamate (AQC) using a 1 / 100,000 balance. Prepare a 2 mg / mL solution of AQC using chromatographic ACN as solvent and store it in a refrigerator at 4°C protected from light.

[0039] c. Preparation of buffer salt: Accurately weigh a certain amount of borax and prepare a 0.05M solution with pure water. The pH at this time is 9.3.

[0040] d. Preparation of Fe3O4 magnetic particles: Accurately weigh 15.0 g of FeCl3·6H2O and place it in a 1 L glass beaker. Add 300 mL of ethylene glycol and stir magnetically until the solid FeCl3·6H2O is completely dissolved in the ethylene glycol. Slowly add 45.0 g of sodium acetate to the above solution, then add 150 mL of 1,2-ethylenediamine and stir vigorously until the mixture becomes a clear solution. Transfer the solution to a polytetrafluoroethylene reaction liner and seal it in a stainless steel reactor. Place the reactor in a 200 °C oven and react for 10 h. After the reaction, the generated black precipitate (Fe3O4 magnetic particles) is repeatedly washed with ultrapure water and methanol under an external magnetic field until the supernatant is colorless and transparent. The final wash is with methanol to facilitate rapid drying in a vacuum drying oven. After washing, place the product in a vacuum drying oven at 60 °C for later use.

[0041] e. Preparation of the extraction material Fe3O4 / MWCNTs-OH: Accurately weigh 8.0g of Fe3O4 magnetic particles into a mortar, then add 2.0g of MWCNT-OH material. The two are mixed in a 4:1 ratio. Grind with a pestle until the mixture is uniform. Finally, pour it into a 100mL beaker. With the assistance of a magnet, wash the mixture repeatedly with ultrapure water and methanol, and finally wash with methanol. After washing, dry the product in a vacuum drying oven at 60℃ before use.

[0042] f. Preparation of sample solutions: Take 1 mL of wine, rice wine, and soy sauce into centrifuge tubes, dilute with pure water at a ratio of 1:5 (v / v), and filter through a 0.22 μm filter membrane. Take 30 mL of beer into centrifuge tubes, sonicate for 30 min to remove CO2, dilute with pure water at a ratio of 1:5 (v / v), and filter through a 0.22 μm filter membrane. Take 1 mL of baijiu into centrifuge tubes, dilute with pure water at a ratio of 1:10 (v / v), and filter through a 0.22 μm filter membrane.

[0043] (2) Simultaneous derivatization and extraction of biogenic amines: 2 mL of sample solution, 100 μL of borax buffer, 200 μL of AQC, and 4 mg of extraction material Fe3O4 / MWCNTs-OH were mixed and vortexed for 1 min. Then, an external magnetic field was applied. The magnetic material adsorbed with AQC-BAs derivatization products was separated from the sample solution under the action of the external magnetic field. The supernatant was discarded. Then, 0.3 mL of pure water was added, and the mixture was vortexed for 30 s. Under the action of the external magnetic field, the supernatant was discarded. Finally, 120 μL of acetone was added, and the mixture was vortexed for 1 min to desorb the derivatization products from the magnetic material. The magnetic material and the desorption solution were separated under the action of the external magnetic field. The obtained desorption solution can be directly used for HPLC-FLD analysis.

[0044] (3) The chromatographic column used was an Agilent 5TC-C18, model 4.6mm x 150mm, 5μm. The mobile phase adopted a gradient elution mode. Mobile phase A was pure water, and mobile phase B was methanol. The elution rates were: 0–2min, 75% A; 2–20min, 75–25% A; 20–25min, 30–70% A; 25–26min, 70–75% A; 26–30min, 75% A. The fluorescence detector conditions were set as follows: excitation wavelength 249nm, emission wavelength 398nm, column temperature 30℃, flow rate 1mL / min, and injection volume 20μL.

[0045] Comparative Example 1

[0046] The difference between Comparative Example 1 and Example 1 is that Comparative Example 1 is a blank extraction, specifically replacing the sample solution in Example 1 with pure water.

[0047] Comparative Example 2

[0048] The difference between Comparative Example 1 and Example 1 is that the sample solution in Example 1 is replaced with the analyte standard solution.

[0049] Comparative Example 3

[0050] The difference between Comparative Example 3 and Example 1 is that Comparative Example 3 is a direct solution derivatization experiment, that is, the analyte standard solution reacts directly with the derivatization reagent without going through the extraction method of the present invention, and the resulting derivatized solution is directly analyzed and detected. The specific steps are as follows: 200 μL of derivatization reagent solution and 100 μL of borax buffer salt are directly added to the analyte standard solution, and the mixture is vortexed for 1 min.

[0051] To evaluate the performance of the simultaneous derivatization and extraction method for biogenic amines, the methods were investigated for simultaneous derivatization and extraction of analyte standard solutions (Comparative Example 2), simultaneous derivatization and extraction of blank solutions (Comparative Example 1), and direct derivatization of analyte standard solutions (Comparative Example 3). Figure 1 As shown, Figure 1 The HPLC-FLD chromatograms for comparative examples 1-3 are shown. The results indicate that the detection method of this invention can detect all target analytes, and no interfering analyte peaks were observed in the blank control. Therefore, the simultaneous derivatization and extraction method provided by this invention can effectively analyze and detect biogenic amines.

[0052] Example 2

[0053] The difference between this embodiment and embodiment 1 is that the amount of extraction material used in step (2) of embodiment 1 is adjusted to 2mg, 4mg, 6mg, 8mg and 10mg respectively, and the remaining steps are the same as in embodiment 1.

[0054] Test results as follows Figure 2 As shown, as the amount of material increased from 2 mg to 4 mg, the peak area of ​​the biogenic amine derivative product increased significantly. When the amount of material continued to increase from 4 mg to 10 mg, the peak area of ​​the derivatized product did not change much, indicating that the amount of material of 4 mg was sufficient to adsorb the target analyte.

[0055] Example 3

[0056] The difference between this embodiment and Embodiment 1 is that the amount of derivatization reagent in step (2) of Embodiment 1 is adjusted to 50 μL, 100 μL, 150 μL, 200 μL, 300 μL, and 400 μL, respectively.

[0057] Test results as follows Figure 3 As shown, as the volume of the derivatization reagent increased to 200 μL, the peak area of ​​the derivatized product increased, and the derivatization extraction efficiency increased. However, with further increases in volume, the peak area of ​​the derivative decreased.

[0058] Example 4

[0059] The difference between this embodiment and embodiment 1 is that the pH of the buffer salt in step (2) of embodiment 1 is adjusted to 8.0, 8.3, 8.5, 8.8, 9.0, 9.3, 9.5 and 9.7 respectively, while the rest is the same as in embodiment 1.

[0060] The reaction between biogenic amines and AQC requires alkaline conditions; the pH value affects the derivatization efficiency of biogenic amines, as shown in the detection results. Figure 4 As shown, the peak area of ​​the derivatized product gradually increases with increasing pH. When the pH is greater than 9.3, the peak area of ​​the derivative does not change much, that is, the extraction derivatization efficiency reaches equilibrium.

[0061] Example 5

[0062] The difference between this embodiment and embodiment 1 is that the reaction time for simultaneous derivatization and extraction in step (2) of embodiment 1 is adjusted to 1 min, 2 min, 5 min, 8 min, and 10 min, while the rest is the same as in embodiment 1.

[0063] Test results as follows Figure 5 As shown, the peak area reaches its maximum value after 1 minute of mixed vortex reaction. As time increases, the peak area of ​​the derivative no longer increases, meaning that the extraction recovery rate does not increase further.

[0064] Example 6

[0065] The difference between this embodiment and Example 1 is that the desorption solvent in Example 1 is changed to acetone, acetonitrile, methanol, ethanol, and isopropanol, while the rest is the same as in Example 1.

[0066] The target analyte is polar and becomes hydrophobic after derivatization; therefore, the desorption solvent must have good solubility for the derivatization product of the target analyte. The detection results are as follows: Figure 6 As shown, the extraction effect is better when acetone is used as the desorption solvent.

[0067] Example 7

[0068] The difference between this embodiment and Example 1 is that the amount of methanol in acetone in step (2) of Example 1 is adjusted to 120μL, 150μL, 200μL, 250μL and 300μL respectively, while the rest is the same as in Example 1.

[0069] Test results as follows Figure 7 As shown, the extraction efficiency remains unchanged as the volume of desorption solvent increases, indicating that a desorption solvent volume of 120 μL is sufficient to desorb the derivative from the extraction material.

[0070] Example 8

[0071] The difference between this embodiment and embodiment 1 is that the desorption time in step (2) of embodiment 1 is adjusted to 1 min, 2 min, 5 min, 8 min and 10 min respectively, and the other steps are the same as in embodiment 1.

[0072] Test results as follows Figure 8 As shown, from 1 min to 2 min, the peak area of ​​the derivative increases significantly. If the desorption time is further increased, the peak area no longer increases, that is, the desorption efficiency reaches equilibrium.

[0073] Experimental Example 1

[0074] 1.1 Linearity and Detection Limit of the Detection Method of the Present Invention

[0075] A series of biogenic amine standard solutions of different concentrations were analyzed according to steps (2) and (3) in Example 1. Calibration curves were constructed by plotting the average peak area ratio of AQC-BAs derivatives to their corresponding analyte concentrations, yielding linear regression equations and correlation coefficients. Limits of detection (LODs) were calculated using a signal-to-noise ratio of 3:1, and limits of quantitation (LOQs) were calculated using a signal-to-noise ratio of 10:1. As shown in Table 1, within the range of 1–500 ng / mL, this method has a correlation coefficient (R0). 2 The linear relationship was good, with the detection limits of the seven biogenic amines ranging from 0.30 to 2.00 ng / mL and the quantitation limits ranging from 0.99 to 6.60 ng / mL.

[0076] Table 1

[0077]

[0078]

[0079] 1.2 Precision and accuracy of the detection method of the present invention

[0080] The accuracy and precision of the method were evaluated using intraday and interday relative recoveries and RSDs at three different concentrations (low, medium, and high). Analysis was performed according to steps (2) and (3) in Example 1, with the target analyte analyzed three times on the same day and over three consecutive days. Intraday and interday relative recoveries and RSDs were determined. Table 2 shows that the relative recoveries were 85.1–109.2%, and the intraday and RSDs were less than 9.7%.

[0081] Table 2

[0082]

[0083] 1.3 Detection Method for Simultaneous Derivatization and Extraction of Biogenic Amines in Food

[0084] Take 1 mL of each type of alcohol (baijiu, huangjiu, or wine) into a centrifuge tube, dilute with pure water at a ratio of 1:5 (v / v), and filter using a 0.22 μm filter membrane. Take 30 mL of beer into a centrifuge tube, sonicate for 30 min to remove CO2, dilute with pure water at a ratio of 1:5 (v / v), and filter using a 0.22 μm filter membrane. The detection method is the same as steps (2) and (3) in Example 1. The detection diagram is shown below. Figure 9 As shown.

[0085] The results are shown in Table 3. Histamine was detected in wine, rice wine, and baijiu. Tyramine was detected in all samples. Putrescine was detected in wine, rice wine, and beer. Cadaverine was detected in wine and beer. Spermine was detected in baijiu. Other biogenic amines were not detected. The results of these actual sample analyses indicate that the method in this study is suitable for the quantitative analysis of biogenic amines in different complex matrices.

[0086] Table 3

[0087]

[0088] The foregoing has shown and described the basic principles and main features of the present invention, as well as its advantages. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A rapid analytical detection method for simultaneously derivatizing and extracting biogenic amines, characterized in that, Includes the following steps: (1) Add the sample solution, derivatization reagent, extraction material and buffer salt solution into the same centrifuge tube and mix and vortex. During this process, derivatization and extraction are completed simultaneously. Under the action of an external magnetic field, the extraction material that has adsorbed biogenic amine derivatives can be separated from the sample solution. (2) Add a certain amount of pure water, mix and vortex for a certain time, separate and discard the supernatant under the action of an external magnetic field; (3) Add a certain amount of desorption solvent, mix and vortex for a certain time to desorb the derivatized product from the extraction material; (4) The desorption solution containing the target analyte obtained in step (3) is detected and analyzed; The derivatizing reagent in step (1) is 6-aminoquinolinyl-N-hydroxysuccinimide carbamate AQC, the concentration of the derivatizing reagent is 1-2 mg / mL, and the volume of the derivatizing reagent is 50-400 μL. The extraction material in step (1) is magnetic hydroxylated multi-walled carbon nanotubes Fe3O4 / MWCNTs-OH. The Fe3O4 / MWCNTs-OH is obtained by grinding and mixing Fe3O4 nanoparticles and MWCNTs-OH at a mass ratio of 4:1 until uniform. The amount of Fe3O4 / MWCNTs-OH material used is 1-10 mg. The mixing vortex reaction time in step (1) is 1 min; The mixing vortex reaction time in step (2) is 30s; The desorption solvent in step (3) is acetone; The desorption time in step (3) is 1 min.

2. The rapid analytical detection method for simultaneous derivatization and extraction of biogenic amines as described in claim 1, characterized in that, The sample solution in step (1) includes human urine, baijiu (Chinese white liquor), wine, red wine, beer, soy sauce, or huangjiu (yellow wine).

3. The rapid analytical detection method for simultaneous derivatization and extraction of biogenic amines as described in claim 1, characterized in that, The buffer salt solution in step (1) is a borax buffer salt solution, a phosphate buffer salt solution, or an acetate buffer salt solution, and the pH of the buffer salt solution is 8.0-9.

3.

4. The rapid analytical detection method for simultaneous derivatization and extraction of biogenic amines as described in claim 1, characterized in that, The detection instrument in step (3) is high performance liquid chromatography combined with a fluorescence detector or liquid chromatography-mass spectrometry.

5. The rapid analytical detection method for simultaneous derivatization and extraction of biogenic amines as described in claim 1, characterized in that, The biogenic amine is any one of histamine, tyramine, putrescine, cadaverine, 2-phenylethylamine, spermine, and spermidine.

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