A method for selective and rapid measurement of volatile biogenic amines

Abstract

The present invention mainly provides a highly selective and highly sensitive ion migration spectrometry method for rapid determination of volatile biogenic amines. The present invention utilizes electrospray ionization to generate a single gaseous ammonium ion from an ammonia solution, and uses it as a reagent ion to react with volatile biogenic amine molecules through a proton transfer reaction to generate protonated biogenic amine ions, and then introduces the protonated biogenic amine ions into an ion migration spectrometer under atmospheric pressure, enters the migration zone through a periodically opened ion gate, and collides with the migration gas molecules under the action of the electric field in the migration zone, so that the reagent ions and the biogenic amine ions are separated and arrive at the detector at different times, thereby achieving qualitative and quantitative determination of trimethylamine and other volatile biogenic amines.

Description

Technical Field

[0001] The present invention relates to a highly selective and highly sensitive separation and detection method for volatile biogenic amines. Specifically, an ammonia-containing solution is used to generate gaseous ammonium ions by electrospray ionization, and the gaseous ammonium ions and volatile biogenic amines are subjected to proton transfer to generate gaseous protonated biogenic amine ions, and then ion migration spectrometry is used to rapidly identify and quantify the volatile biogenic amines with high sensitivity and selectivity, which belongs to the field of food safety. Background Art

[0002] Volatile biogenic amines are a class of low molecular weight organic substances containing nitrogen. As a representative of volatile biogenic amines, trimethylamine is a volatile toxic gas with a strong ammonia smell. Aquatic products are rich in nutrients such as protein. After their death, a series of physical and chemical changes will occur, such as protein decomposition, the production of biogenic amines, fat oxidation, etc. The oxidized trimethylamine in the aquatic product is converted into dimethylamine and trimethylamine under the action of enzymes and microorganisms, accompanied by the generation of other volatile biogenic amines and the generation of fishy odors. As the storage time of aquatic products increases, trimethylamine accumulates more and more, and the fishy smell becomes stronger, so trimethylamine becomes an important indicator for evaluating the freshness of aquatic products. Biogenic amines are normal components of the human body, but excessive amounts can cause some problems to human health, such as headaches, low or high blood pressure, renal failure, anaphylactic shock, and even death. Trimethylamine can be absorbed by the human body. Part of it can be converted into carcinogenic N-nitrosoamine compounds in the body, and the other part is activated into trimethylamine oxide. Studies have shown that there is a connection between trimethylamine oxide levels and cardiovascular disease, diabetes, chronic kidney disease and metabolic syndrome. It can be seen that whether from the perspective of the safety of aquatic products or from the perspective of human health, it is imperative to monitor the concentration changes of trimethylamine and volatile biogenic amines and control their content.

[0003] At present, analytical methods have been developed at home and abroad to detect trimethylamine. The main analytical methods are gas chromatography, mass spectrometry, high performance liquid chromatography, ion chromatography, capillary electrophoresis and ion mobility spectrometry. Among them, ion mobility spectrometry can work under atmospheric pressure. It is an analytical detection technology that characterizes chemical ion substances based on the difference in the migration speed of ions in an electric field. Under atmospheric pressure conditions, solid, liquid or gas sample molecules are ionized to form ions by applying high voltage to them. Under the action of the electric field, the ion gate switching time is periodically controlled, and the ions enter the migration zone from the ionization zone and continuously collide with the migrating gas molecules in the countercurrent. Due to the different migration rates of each ion in the drift zone, it is separated and arrives at the detector for detection one after another, thus forming an ion migration spectrum, which has the advantages of small size, light weight and short response time.

[0004] When using ion mobility spectrometry to detect trimethylamine, the trimethylamine product ion peak overlaps with the hydrated proton peak and cannot be separated. Usually, a dopant is required to first react the hydrated proton with the dopant to generate a protonated dopant, and then trimethylamine reacts with the protonated dopant to form a protonated trimethylamine ion, thereby performing ion mobility spectrometry separation and detection of trimethylamine. However, this method requires strict control of the flow rate and concentration of the dopant, and optimization of the airflow field, and the detection performance is not stable enough. At the same time, dopants pollute the environment and have a great impact on the performance of the ion mobility spectrometry.

[0005] Electrospray ionization-ion mobility spectrometry can be used to analyze some biogenic amines, but the electrospray ion source mainly analyzes non-volatile substances and has weak analytical performance for volatile substances. An electrospray ion source can also be combined with a gas sampling device to use the hydrated protons generated by electrospray to react with biogenic amines to achieve ionization of biogenic amines, forming a so-called secondary electrospray ionization-ion mobility spectrometry analysis method, which mainly detects some illegal drugs (such as heroin, morphine and cocaine), chemical warfare agent simulants and some explosives, such as pentaerythritol tetranitrate, nitroglycerin, etc. In the detection of trimethylamine, when using electrospray ionization-ion mobility spectrometry alone for detection, its peak position is connected to the reaction ion peak and cannot be separated.

[0006] The present invention is based on the improvement of the electrospray ion source, and can quickly and conveniently determine trimethylamine and volatile biogenic amines without adding dopants. Under atmospheric pressure, an ammonia solution is used for electrospray ionization to specifically generate (NH3)H + As the reactive ions, the sample gas and the reactive ions undergo a gaseous proton transfer reaction in the reaction zone to form sample ions, which then enter the migration zone through the ion gate to form a spectrum of ion current intensity corresponding to ion migration time, ultimately achieving the purpose of identifying and quantifying trimethylamine and volatile biogenic amines. This method does not require dopants, and the generated (NH3)H + The concentration of reactive ions is high, no other ions are contained, and the ionization process is very stable, which can separate the characteristic peak of trimethylamine from the original reactive ion peak, which greatly improves the detection efficiency and makes the detection process faster and faster. At the same time, since the proton affinity of most volatile compounds is smaller than that of ammonia, common volatile substances such as ketones, aldehydes, esters, alcohols, and aromatic hydrocarbons cannot be ionized, and most interference peaks can be excluded. Summary of the invention

[0007] The present invention performs specific recognition and sensitive quantitative detection of trimethylamine and volatile biogenic amines in a positive ion mode.

[0008] In order to achieve the above object, the technical solution of the present invention is as follows:

[0009] A method for selectively and rapidly measuring volatile biogenic amines, wherein the device is composed of an electrospray ionization source, a gas sampling device, and an ion mobility spectrometer. The ion mobility spectrometer is composed of four parts: an ionization region 9, an ion gate 6, a migration region 8, and a detector 7. An ion gate 6 is arranged between the ionization region 9 and the migration region 8, and the ions are allowed to enter the migration region from the ionization region by periodically controlling the switch of the ion gate. The region between the ion gate 6 and the ion source is the ionization region 9, and the region between the ion gate 6 and the detector 7 is the migration region 8.

[0010] In the migration region 8 near the detector 7, a migration gas inlet 3 is provided on the outer wall of the ion migration tube, and in the ionization region 9 near the outer wall of the ionization source, an exhaust gas outlet 2 is provided.

[0011] The secondary electrospray ionization source comprises an electrospray device and a sample gas channel. The electrospray device is composed of an electrospray ionization source 4 and a microinjection pump 1.

[0012] The sample gas channel includes a sample inlet pipeline 5 and a gas generating device 12 .

[0013] The sample gas inlet is located directly above the electrospray ion source.

[0014] The trimethylamine and volatile biogenic amine gases to be detected enter the ionization region 9 through the sample gas channel.

[0015] The floating gas is one of air and nitrogen.

[0016] In the positive ion mode, the electrospray solution is formed in the ionization region 9 under the action of the electric field.

[0017] Single gaseous ammonium ion, sample gas (trimethylamine, etc.) enters ionization zone 9 and reacts with electrospray ionization to form charged droplets ((NH3)H + ) and ions interact to form protonated biogenic amine ions, which then enter the migration zone through the periodically opened ion gate 6 and collide with the migrating gas molecules under the action of the electric field in the migration zone 8, so that the ammonium ions and the biogenic amine ions are separated and arrive at the detector 7 at different times, and a spectrum of the ion current intensity corresponding to the ion migration time is formed, thereby realizing the qualitative and quantitative analysis of trimethylamine and volatile biogenic amines.

[0018] The ionization source is an electrospray ionization source; the ion gate is a Bradbury-Nielsen type ion gate or a TP type ion gate.

[0019] The migration gas flow rate is 200-600 ml / min; the sample gas flow rate is 20-60 ml / min.

[0020] The advantages of the present invention are:

[0021] When trimethylamine is detected by electrospray ionization-ion mobility spectrometry alone, the trimethylamine peak is connected to the reaction ion peak, and it is impossible to identify and quantitatively detect it. Therefore, it is often necessary to use the assistance of a dopant to transfer the reaction ion peak, and trimethylamine and the transferred ion peak form the ion peak of trimethylamine, so as to detect trimethylamine. The present invention uses an ammonia-containing solution to generate gaseous ammonium ions singly, and a gaseous proton transfer reaction occurs with volatile biogenic amines. Since the proton affinity of most volatile compounds is less than that of ammonia, common volatile substances such as ketones, aldehydes, esters, alcohols, and aromatic hydrocarbons cannot be ionized, and most of the interfering peaks can be excluded. The present invention realizes the rapid detection of trimethylamine and volatile biogenic amines with high sensitivity, high selectivity, and strong specificity, while also reducing the interference of other chemical substances, making the spectrum cleaner.

[0022] The present invention will be further described with reference to the accompanying drawings: BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The schematic diagram of the structure of the ion migration tube used in the present invention includes: 1. micro-injection pump; 2. tail gas outlet; 3. migration gas inlet; 4. electrospray ionization source; 5. injection pipeline; 6. ion gate; 7. detector; 8. migration zone; 9. ionization zone; 10. insulating ceramic ring; 11. annular electrode sheet; 12. gas generating device.

[0024] Figure 2 The response spectrum of trimethylamine when the electrospray solution is ammonium acetate, ammonium bicarbonate, and ammonia.

[0025] Figure 3 Response spectra of trimethylamine with and without ammonium acetate.

[0026] Figure 4 Standard curve of trimethylamine response.

[0027] Figure 5 Distribution of volatile biogenic amines on ion mobility spectra.

[0028] Figure 6 The method used in the present invention detects the signal intensity changes of various volatile biogenic amines in grass carp stored at 4°C for 12 days. DETAILED DESCRIPTION

[0029] Example 1

[0030] The device used in the present invention is as follows Figure 1As shown. The total length of the ion migration tube in the present invention is 25 cm, of which the ionization zone 9 is 8.8 cm long, the migration zone 8 is 16.2 cm long, and the inner diameter of the migration tube is 5 cm. The ionization source of the ion migration tube is an electrospray ionization source 4; the ion gate 6 is a Bradbury-Nielsen type ion gate; the entire ionization zone and migration zone are formed by the annular electrode sheet 11 and the insulating ceramic ring 10 overlapping each other. The annular electrode sheets are connected end to end through resistors to form a voltage divider resistor and are connected to a high voltage power supply and the ground. The electrospray high voltage is 4.5 kV, the ion migration tube migration voltage is 10.5 kV, and the migration tube temperature is 160°C.

[0031] An exhaust gas outlet 2 is provided on the outer wall of the migration tube of the ionization region 9 close to the electrospray ionization source 4 ; and a migration gas inlet 3 is provided on the outer wall of the ion migration tube of the migration region 8 close to the detector 7 .

[0032] The flow rate of the electrospray solution in the microinjection pump 1 is 0.5 μl / min, and the concentration is 100 mg / L. The electrospray solution forms reactive ions through the electrospray ionization source 4.

[0033] The trimethylamine and volatile biogenic amine sample gas to be tested is volatilized from the gas generating device 12 at a gas flow rate of 30 ml / min and enters the ionization zone 9 through the sample inlet line 5 .

[0034] The migration gas is dry purified air with a flow rate of 400 ml / min. It enters the migration tube from the migration gas inlet 3, flows through the migration zone 8 and ionization zone 9 of the ion migration tube, and then flows out of the ion migration tube through the tail gas outlet 2.

[0035] In the positive ion mode, the electrospray solution forms charged droplets ((NH3)H + ), the sample gas (trimethylamine, etc.) enters the ionization region 9 and reacts with the charged droplets ((NH3)H + ) and ions interact to form protonated biogenic amine ions, which then enter the migration zone through the periodically opened ion gate 6 and collide with the migrating gas molecules under the action of the electric field in the migration zone 8. The ions reach the detector 7 under the action of the electric field in the migration zone, forming a spectrum of ion current intensity corresponding to ion migration time, thereby realizing the identification and quantification of trimethylamine and volatile biogenic amines.

[0036] Figure 2 The effect of different electrospray solutions ammonium acetate, ammonium bicarbonate, and ammonia solution on the signal intensity of trimethylamine is presented. It can be seen from the figure that when the electrospray solution is ammonium acetate, the signal intensity of trimethylamine is the highest, and from the perspective of material stability, ammonium acetate is relatively stable, ammonium bicarbonate is easy to decompose, and ammonia is easy to volatilize, so it is more appropriate to use ammonium acetate as the electrospray solution.

[0037] Figure 3It shows the distribution of ion migration spectra peaks of methanol water, adding trimethylamine and methanol water, adding only ammonium acetate, and adding ammonium acetate and trimethylamine. When only methanol water is added and when trimethylamine + methanol water is added, the peak shape and peak position in these two cases are consistent, indicating that the trimethylamine peak may be covered by the reaction ion peak, resulting in its undetected. When only ammonium acetate is added, the reaction ion peak has a better peak shape, and the peaks of other chemical interferences are also reduced. On the basis of using ammonium acetate as the electrospray solution, the injection detection of trimethylamine is performed. As shown in the figure, the peak of trimethylamine is completely separated from the reaction ion peak, and it can be identified and quantified.

[0038] Figure 4 The linear response range of trimethylamine detection using the method of the present invention is shown. The linear range of trimethylamine is 0.05-5ppmv, which can meet the basic requirements for detecting the content of trimethylamine in aquatic products.

[0039] Figure 5 The peak distribution of each volatile biogenic amine detected by the method of the present invention is reflected. As shown in the figure, each biogenic amine and the reaction ion peak can be completely separated, and the peak shape is good, indicating that this method can detect volatile biogenic amines such as β-phenylethylamine, spermidine, and putrescine in addition to trimethylamine.

[0040] Figure 6 The method of the present invention was used to detect the signal intensity changes of volatile biogenic amines in grass carp samples stored at 4°C for 12 days. In the grass carp samples, two volatile biogenic amines, trimethylamine and putrescine, were detected using the method of the present invention, and both of these biogenic amines showed a trend of gradual accumulation with the increase of storage time. This shows that the method of the present invention can be used to identify and quantify volatile biogenic amines in actual samples.

Claims

1. A method for identifying and quantifying trimethylamine and volatile biogenic amines based on high selectivity and high sensitivity, characterized in that: The device used in the method is composed of two parts: a specific gaseous ammonium ion generation and proton transfer reaction device and an ion migration spectrometer; the ion migration spectrometer is divided into four parts: an ionization zone, an ion gate, a migration zone, and a detector; the ion gate is located between the ionization zone and the migration zone, and the ions can enter the migration zone from the ionization zone by periodically controlling the switch of the ion gate; the area between the ion gate and the ionization source is the ionization zone, and the area between the ion gate and the detector is the migration zone; a migration gas inlet is provided on the outer wall of the migration tube close to the detector, and a tail gas outlet is provided on the outer wall of the ionization zone of the migration tube close to the ionization source; the electrospray ionization source and the sample gas channel together constitute the specific gaseous ammonium ion generation and proton transfer reaction device; the electrospray device is composed of an electrospray ionization source and a micro-injection pump; the sample gas channel includes an injection pipeline and a gas generating device; The sample gas inlet is directly above the electrospray ionization source; The trimethylamine and volatile biogenic amine gases to be tested enter the ionization region through the sample gas channel; The migration gas is one of air and nitrogen; the migration gas flow rate is 200-600 ml / min; the electrospray solution is one or more combinations of ammonium acetate, ammonium bicarbonate, and ammonia solution; and the detection is performed by combining a gas injection method with an electrospray ionization method.

2. The method for highly selective and sensitive identification and quantification of trimethylamine and volatile biogenic amines according to claim 1, characterized in that: The ion gate is a Bradbury-Nielsen type ion gate or a TP type ion gate.

3. The method for highly selective and sensitive identification and quantification of trimethylamine and volatile biogenic amines according to claim 1, characterized in that: The sample gas flow rate was 20-60 ml / min.

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