Method for accurately determining contents of trimethylamine, diethylamine and triethylamine in animal-derived traditional Chinese medicinal materials

Abstract

The invention discloses a method for accurately determining the content of trimethylamine, diethylamine and triethylamine in animal-derived traditional Chinese medicinal materials, which comprises the following steps: mixing alkali liquor and n-tetracosane in a headspace bottle, heating to completely dissolve the n-tetracosane, and standing until the n-tetracosane layer is solidified to seal the alkali liquor below the n-tetracosane layer; animal-derived traditional Chinese medicinal materials are crushed, sieved and dispersed in a trichloroacetic acid aqueous solution, centrifugation is performed after homogenization, supernate is collected, volume metering is performed, and a sample extracting solution is obtained; adding the sample extracting solution above the n-tetracosane layer in the headspace bottle, sealing by a gland, placing on an automatic sampler, sucking gas at the upper part of the headspace bottle, analyzing by a gas chromatograph-triple quadrupole mass spectrometer, and obtaining the contents of trimethylamine, diethylamine and triethylamine in the animal-derived traditional Chinese medicinal materials by an external standard method. The method is accurate and reliable in quantitative result, high in sensitivity and high in analysis efficiency, derivation is assisted by the phase change material n-tetracosane, the phenomenon that the quantitative accuracy is poor due to the volatile and escape characteristics of trimethylamine, diethylamine and triethylamine is effectively improved, the false positive phenomenon can be effectively eliminated through headspace sampling and a multi-reaction monitoring mode, and the method is suitable for large-scale popularization and application. The peak appearance of trimethylamine, diethylamine and triethylamine is completed within 6.5 minutes.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis and detection, and particularly relates to a method for detecting the contents of trimethylamine, diethylamine and triethylamine in animal-derived traditional Chinese medicinal materials. Background Art

[0002] Animal-derived Chinese medicines are generally derived from the whole body, organs or tissues, physiological or pathological products, excrement or secretions, processed products, etc. of animals, and contain a large number of volatile compounds such as amines, ketones, aldehydes, alcohols, low-level fatty acids, sulfides, etc., which work together to make animal-derived medicinal materials produce an unacceptable fishy odor. Volatile amine compounds, such as trimethylamine, diethylamine and triethylamine, as components of fishy odor, play an important synergistic role in the fishy odor of animal medicines. Detecting the content of volatile amine compounds is of vital importance to controlling the quality of animal-derived Chinese medicines, supervising the safety of Chinese medicines, and ensuring the health of consumers.

[0003] At present, the determination methods of trimethylamine in food are relatively mature, mainly including headspace gas chromatography, headspace gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, etc., and the national standard GB 5009.179-2016 "National Food Safety Standard Determination of Trimethylamine in Food" has been established. The testing principle of this national standard is that the sample extract is first added to the headspace bottle, and after the cap is sealed, the alkali solution is injected into the headspace bottle through a syringe to mix with the sample solution. After the trimethylamine hydrochloride in the sample extract is mixed with the alkali solution, a chemical reaction occurs and it is converted into trimethylamine. The boiling point of trimethylamine is 2.87℃, and it has a high volatility characteristic. It will evaporate to the upper part of the headspace bottle. The gas in the upper part of the headspace bottle is absorbed and injected into the gas chromatography-mass spectrometry for detection. However, the following problems exist in the actual experimental operation: ① If the lid is sealed after the injection of alkali solution, trimethylamine will escape from the headspace bottle mouth before the lid is sealed; ② If the alkali solution is injected into the headspace bottle through a syringe after the lid is sealed, trimethylamine will escape from the pinhole left in the headspace bottle cap when the alkali solution is injected; both of the above situations will cause poor linearity of the standard curve, poor repeatability of the test results, and affect the quantitative accuracy. In addition, no reports have been found in the prior art on the simultaneous detection of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for accurately determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials in view of the deficiencies in the above-mentioned prior art, which can effectively improve the phenomenon of poor quantitative accuracy caused by the volatility and easy escape characteristics of trimethylamine, diethylamine and triethylamine.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for accurately determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials comprises the following steps:

[0007] (1) mixing a sodium hydroxide aqueous solution and n-tetracosane in a headspace injection bottle, heating to completely dissolve the n-tetracosane, and then standing until a n-tetracosane layer solidifies on the upper part of the sodium hydroxide aqueous solution, so that the sodium hydroxide aqueous solution is sealed under the n-tetracosane layer, presenting a state in which the sodium hydroxide aqueous solution is concentrated in the lower part of the headspace bottle and the n-tetracosane layer is sealed on the surface layer of the sodium hydroxide aqueous solution;

[0008] (2) using trichloroacetic acid solution to prepare a series of mixed standard solutions of trimethylamine hydrochloride, diethylamine hydrochloride and triethylamine hydrochloride;

[0009] (3) Grinding the animal-derived Chinese medicinal materials as sample powder, taking a small amount of the sample powder and dispersing it in a trichloroacetic acid aqueous solution, homogenizing and centrifuging, collecting the supernatant obtained by centrifugation and making up the volume with a trichloroacetic acid aqueous solution to obtain the sample extract;

[0010] (4) Accurately measure the mixed standard solution of step (2) or the sample extract of step (3), add it to the n-tetracosane layer in the headspace injection bottle obtained in step (1), and seal it with a cap;

[0011] (5) placing each headspace injection bottle obtained in step (4) on a headspace automatic sampler, heating it to 70-85° C., absorbing the gas above the headspace injection bottle, and analyzing it by gas chromatography-triple quadrupole mass spectrometry to obtain each MRM spectrum, and then establishing a standard curve of trimethylamine, diethylamine and triethylamine, and obtaining the content of trimethylamine, diethylamine and triethylamine in animal-derived traditional Chinese medicine by external standard method.

[0012] More specifically, the specific process of step (5) is as follows: placing the headspace injection bottles of each mixed standard solution in step (4) on the headspace automatic injector, heating to 70-85° C., absorbing the gas above the headspace injection bottle, and analyzing it by gas chromatography-triple quadrupole mass spectrometry to obtain an MRM spectrum, and then using the concentration of trimethylamine, diethylamine or triethylamine as the horizontal coordinate and the corresponding quantitative ion pair peak area as the vertical coordinate to obtain the standard curves of trimethylamine, diethylamine and triethylamine respectively;

[0013] Then, the headspace injection bottle of the sample extract in step (4) is placed on a headspace automatic sampler, heated to 70-85° C., the gas above the headspace injection bottle is absorbed, and the MRM spectrum is obtained by gas chromatography-triple quadrupole mass spectrometry analysis. The corresponding quantitative ion pair peak area is substituted into each standard curve to obtain the concentration of trimethylamine, diethylamine and triethylamine in the sample extract, and then the content of trimethylamine, diethylamine and triethylamine in the animal-derived traditional Chinese medicine is converted.

[0014] In the present invention, the animal-derived Chinese medicinal materials include one or more of leeches, American cockroaches, nine-scented insects, chicken gizzard lining, earthworms, silkworm pupa, cicada shells, antelope horns, buffalo horns, tortoise shells, turtle shells, etc.

[0015] According to the above scheme, the sodium hydroxide aqueous solution can be replaced by other water-soluble inorganic alkali aqueous solutions, such as potassium hydroxide aqueous solution, which can be used in the present invention generally within a mass concentration range of 10%-20%.

[0016] Further preferably, the ratio between the volume of the sodium hydroxide aqueous solution and the mass of n-tetracosane is (0.2-1) mL: (0.5-1) g.

[0017] Further preferably, in step (1), heating is performed in an oven and the mixture is completely dissolved by placing the mixture in an oven at 75-85° C. for 15-60 min; and standing is generally performed at room temperature for 20-60 min.

[0018] Further preferably, in step (2) and step (3), the mass concentration of the trichloroacetic acid aqueous solution is 0.5%-5%.

[0019] Further preferably, the concentrations of trimethylamine, diethylamine and triethylamine in the mixed standard solution are all between 0.5 mg / L and 10 mg / L, and five to six concentrations are generally prepared.

[0020] According to the above scheme, in step (3), the sample powder is added to the trichloroacetic acid aqueous solution at a concentration of 20-100 mg / mL for extraction.

[0021] According to the above scheme, in step (3), the dispersion, homogenization and centrifugation steps can be repeated multiple times, specifically: after each centrifugation, the trichloroacetic acid aqueous solution is added to the part where the supernatant is removed for dispersion, and then homogenization and centrifugation are performed; the supernatants obtained from each centrifugation are combined and fixed to volume with the trichloroacetic acid aqueous solution to obtain the sample extract.

[0022] According to the above scheme, in step (4), the sample extract is located above the n-tetracosane layer, and the sodium hydroxide aqueous solution is located below the n-tetracosane layer. Therefore, the sample extract and the sodium hydroxide aqueous solution serving as a derivatization reagent are separated by the n-tetracosane layer and sealed in a headspace injection bottle.

[0023] According to the above scheme, a capillary column is used for gas chromatography (GC), and the column oven is programmed to heat up. The starting temperature of the column oven is 35-45°C, and the temperature is increased to 180-220°C at a rate of 15-25°C / min, and maintained for 1-4min (preferably 2min, which can effectively meet the separation requirements and is relatively efficient and saves time). The injection port temperature is 180-220°C, the carrier gas is controlled in constant flow mode, the column flow rate is 1-2mL / min (preferably 1.5mL / min), and the injection method is split injection, with the split ratio set to (5-20):1.

[0024] According to the above scheme, the triple quadrupole mass spectrometer uses electron impact ionization (EI) and multiple reaction monitoring mode (MRM) detection, the ion source temperature is set to 180°C-230°C, the chromatographic mass spectrometry interface temperature is set to 220°C-250°C, and the detector voltage is set to the tuning voltage +0.4kV-0.7KV. Further, the selected specific parent ions are subjected to collision-induced dissociation, and the detection ion pairs of trimethylamine, diethylamine and triethylamine are all 2 pairs. Specifically, the detection ion pair of trimethylamine is 58.00>42.00, collision voltage 15-25V and 58.00>30.00, collision voltage 4-10V; the detection ion pair of diethylamine is 73.00>58.10, collision voltage 4-8V and 58.00>42.10, collision voltage 24-36V; the detection ion pair of triethylamine is 86.00>58.10, collision voltage 4-8V and 101.00>86.10, collision voltage 4-8V. Two ion pairs are monitored for each compound, the peak area of ​​the first ion pair is used for quantitative analysis, and the second ion pair is used for auxiliary qualitative analysis. For example, the peak area of ​​the ion pair 58.00>42.00 of trimethylamine is used for quantitative analysis, and the ion pair 58.00>30.00 is used for auxiliary qualitative analysis.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention adopts a single compound n-tetracosane (boiling point of 49-52°C, density of 0.7791 g / mL) as a phase change material, which has an appropriate melting point (between room temperature and 75°C), a density lower than water (1 g / mL), is solid at room temperature, and is liquid at about 75°C. The good solid-liquid phase change property is utilized to separate the sample extract and the derivatization reagent alkali solution (sodium hydroxide aqueous solution) in a headspace bottle, and control it not to undergo a derivatization reaction. After the headspace injection bottle is sealed by a pressure cap, it is placed in a headspace automatic sampler and heated to about 80°C, the phase change material n-tetracosane is converted into a liquid, the sample extract and the derivatization reagent begin to contact and mix, and a derivatization reaction occurs. At this time, the derivative products trimethylamine, diethylamine and triethylamine are all sealed in the headspace bottle and cannot escape, thereby ensuring the accuracy of the quantitative results of trimethylamine, diethylamine and triethylamine from the perspective of sample preparation.

[0027] (2) The present invention adopts a headspace injection method, heats the headspace bottle to about 70-85° C., converts n-tetracosane into liquid, mixes the sample extract and the alkali solution (sodium hydroxide aqueous solution), converts trimethylamine hydrochloride, diethylamine hydrochloride and triethylamine hydrochloride into volatile trimethylamine, diethylamine and triethylamine, and absorbs the gas in the upper part of the headspace bottle and injects it into GC for analysis; and the boiling point of the phase change material n-tetracosane is 391.1° C., which is difficult to volatilize to the upper space of the headspace bottle and enter the GC, thereby reducing the interference of the phase change material on the analysis result and the pollution to the GC, thereby ensuring the stability of the instrument and the reliability of the analysis result.

[0028] ⑶ In the present invention, the triple quadrupole mass spectrometer uses electron impact ionization (EI) and multiple reaction monitoring (MRM) modes to perform collision-induced dissociation of the selected specific parent ions, remove interference from other daughter ions, and only collect mass spectrometry signals of the selected specific daughter ions. It has high sensitivity and can effectively eliminate false positive phenomena. Trimethylamine, diethylamine and triethylamine all complete peak emission within 6.5 minutes, and the analysis efficiency and accuracy are high. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the MRM spectrum of the mixed standard solution in the example (the concentration is 0.5 mg / L).

[0030] Figure 2 It is the standard curve of trimethylamine, diethylamine and triethylamine in the example.

[0031] Figure 3 It is the MRM spectrum of the earthworm sample in the embodiment.

[0032] Figure 4 It is the MRM spectrum of the nine-scented insect sample in the embodiment.

[0033] Figure 5 The MRM spectra of the mixed standard solutions of different concentrations in the comparative example are compared (wherein, the blue line: the concentrations of diethylamine and triethylamine are both 5 mg / L, and the black line: the concentrations of diethylamine and triethylamine are both 10 mg / L). DETAILED DESCRIPTION

[0034] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the present invention is not limited to the following embodiments.

[0035] In the following examples, trimethylamine hydrochloride, diethylamine hydrochloride, and triethylamine hydrochloride standards were purchased from Shanghai Anpu Experimental Technology Co., Ltd., trichloroacetic acid and sodium hydroxide were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; pure water was homemade in the laboratory, and the pure water machine model was ADVANTAGE A10 (Milli-Q, USA); analytical balance model AP135W (Shimadzu, Japan), centrifuge model AllegraTM 64R (BECKMAN COULTER, USA); homogenizer model ULTRA-TURRAX T25 (IKA, Germany); oven MOV-112F (SANYO, Japan); high-speed Chinese medicine pulverizer model 150B swing type, purchased from Zhejiang Ruian Yongli Pharmaceutical Machinery Co., Ltd.

[0036] In the following examples, the concentration of the sodium hydroxide aqueous solution is 10%, and the preparation method is: accurately weigh 10 g of sodium hydroxide, dissolve it in water, and make up to volume in a 100 mL volumetric flask.

[0037] In the following examples, the concentration of the trichloroacetic acid solution is 1%, and the preparation method is: accurately weigh 10 g of trichloroacetic acid, dissolve it in water, and make up to volume in a 1000 mL volumetric flask.

[0038] In the following examples, the prepared standard sample headspace vials, earthworm sample headspace vials and nematode sample headspace vials were placed on the headspace automatic sampler AOC-6000, and the AOC-6000 headspace injection method was used for analysis.

[0039] In the following embodiments, the capillary column model used for gas chromatography is SH-Volatile Amine, 30m×0.32mm×5μm, the starting temperature of the column oven is 35°C, the temperature is increased to 200°C at 20°C / min, and maintained for 2min, the injection port temperature is 200°C, the carrier gas control adopts constant flow mode, the column flow rate is 1.5mL / min, the injection method adopts split injection, and the split ratio is set to 10:1.

[0040] In the following embodiments, the triple quadrupole mass spectrometer uses an electron impact ionization source (EI) and adopts a multiple reaction monitoring mode (MRM) for detection, the ion source temperature is set to 220°C, the chromatographic mass spectrometry interface temperature is set to 230°C, the detector voltage is set to a tuning voltage of +0.6kV, and the selected specific parent ion is subjected to collision-induced dissociation to remove the interference of other daughter ions, and only the selected specific daughter ions are subjected to mass spectrometry signal collection, which has high sensitivity and can effectively exclude false positive phenomena. Specifically, the trimethylamine detection ion pair is 58.00>42.00, collision voltage 20V and 58.00>30.00, collision voltage 7V; the diethylamine detection ion pair is 73.00>58.10, collision voltage 6V and 58.00>42.10, collision voltage 30V; the triethylamine detection ion pair is 86.00>58.10, collision voltage 6V and 101.00>86.10, collision voltage 6V.

[0041] In the following examples, the retention time of trimethylamine is 3.045 min, the retention time of diethylamine is 5.088 min, and the retention time of triethylamine is 6.483 min.

[0042] In the following embodiments, after the animal-derived Chinese medicinal materials are crushed, they are generally passed through a 5-15 mesh sieve, such as a No. 1 sieve; if they are not in powder form after crushing, the sieving can be omitted, for example, earthworms are in a flocculent state after crushing; the homogenization time is 1-2 min, the centrifugal speed is 6000 rpm-10000 rpm, and the centrifugal time is 5 min-10 min.

[0043] Example

[0044] A method for accurately determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials comprises the following steps:

[0045] (1) Accurately weigh 0.5 mL of 10% sodium hydroxide aqueous solution and 0.5 g of n-tetracosane (melting point 49-52° C.) into a 20 mL headspace injection bottle (hereinafter referred to as headspace bottle), place the headspace bottle in a 75° C. oven for 30 min, and after the n-tetracosane is completely dissolved, take out the headspace bottle and place it at room temperature for 30 min. The n-tetracosane layer gradually solidifies on the upper part of the sodium hydroxide aqueous solution, and seal the sodium hydroxide aqueous solution at the lower part of the headspace bottle;

[0046] This step was carried out 7 times in parallel, and 7 headspace bottles were obtained accordingly, which were used in the subsequent steps (4), (5) and (6) respectively;

[0047] (2) A series of mixed standard solutions of trimethylamine hydrochloride, diethylamine hydrochloride and triethylamine hydrochloride were prepared using a 1% trichloroacetic acid solution, a total of 5; in the 5 mixed standard solutions, the concentrations of trimethylamine hydrochloride, diethylamine hydrochloride and triethylamine hydrochloride were 0.5, 1, 2, 5 and 10 mg / L, respectively.

[0048] (3) Crush the animal-derived Chinese medicinal materials (earth dragon and nine-scented worm samples, respectively) as much as possible until there is no obvious block of powder or fiber, as the sample powder; accurately weigh 1g of the sample powder into a 50mL stoppered centrifuge tube, add 20mL of 1% trichloroacetic acid solution, homogenize for 1min using a homogenizer, and centrifuge at 10000r / min for 5min using a centrifuge; transfer the supernatant obtained by centrifugation to a new 50mL volumetric flask, and repeat the above extraction process with 15mL and 10mL of 1% trichloroacetic acid solution, respectively, combine the supernatants and make up to 50mL, let it stand for 30min, and the sample extracts (earth dragon sample extracts and nine-scented worm sample extracts, respectively) are obtained;

[0049] (4) Accurately measure 1 mL of each of the five mixed standard solutions and place them on the top of the n-tetracosane layer of the five headspace bottles obtained in step (1), respectively, and seal them with a cap. The mixed standard solutions of various concentrations and the sodium hydroxide aqueous solution are separated by the n-tetracosane layer in the sealed headspace bottles. At this time, the preparation of the standard sample headspace bottles is completed;

[0050] (5) Accurately measure 1 mL of the earthworm sample extract and place it on the top of the n-tetracosane layer of the headspace bottle obtained in step (1), press the cap to seal, and the earthworm sample extract and the sodium hydroxide aqueous solution are separated by the n-tetracosane layer in the sealed headspace bottle. The preparation of the earthworm sample headspace bottle is completed;

[0051] (6) Accurately measure 1 mL of the above-mentioned nine-scented insect sample extract and place it on the top of the n-tetracosane layer of the headspace bottle obtained in step (1), press the cover to seal, and the nine-scented insect sample extract and the sodium hydroxide aqueous solution are separated by the n-tetracosane layer in the sealed headspace bottle. The preparation of the nine-scented insect sample headspace bottle is completed;

[0052] (7) The five standard sample headspace bottles obtained in step (4) are placed on the headspace autosampler in sequence, heated to 80° C., and after equilibration for 30 min, 1 mL of the gas above the headspace bottle is aspirated, and each MRM spectrum is obtained by gas chromatography-triple quadrupole mass spectrometry analysis. Then, the concentration of trimethylamine, diethylamine or triethylamine is used as the horizontal coordinate (trimethylamine hydrochloride, diethylamine hydrochloride and triethylamine hydrochloride are completely converted into trimethylamine, diethylamine and triethylamine, respectively, so the concentration of trimethylamine, diethylamine or triethylamine here is calculated based on the concentration of the mixed standard solution, which is 0.5, 1, 2, 5, and 10 mg / L, respectively), and the corresponding quantitative ion pair peak area is used as the vertical coordinate to obtain a standard curve, such as Figure 2 shown.

[0053] by Figure 1 For example, Figure 1 This is the MRM spectrum of the mixed standard solution when the concentrations of trimethylamine, diethylamine and triethylamine are all 0.5 mg / L. At this concentration, the quantitative ion pair peak areas of trimethylamine, diethylamine and triethylamine are 2490906, 367213 and 658228, respectively.

[0054] (8) Place the headspace bottle of the earthworm sample obtained in step (5) on a headspace automatic sampler, heat it to 80° C., draw 1 mL of the gas above the headspace bottle, and analyze it using a gas chromatography-triple quadrupole mass spectrometer to obtain the following: Figure 3 The MRM spectra of the earthworm sample shown are the mass chromatograms of trimethylamine, diethylamine and triethylamine, and the corresponding peak areas are 4758304, 418238 and 637362 respectively. Figure 2 After following the standard curves, the contents of trimethylamine, diethylamine and triethylamine in the headspace bottle of the earthworm sample were calculated to be 0.976 mg / L, 0.565 mg / L and 0.580 mg / L, respectively. Combined with the pretreatment steps, the contents in the earthworm powder sample were converted to 48.8 mg / kg, 28.2 mg / kg and 29.0 mg / kg, respectively.

[0055] (9) Place the headspace bottle of the nine-scented insect sample obtained in step (6) on a headspace automatic sampler, heat it to 80° C., absorb 1 mL of the gas above the headspace bottle, and analyze it using a gas chromatography-triple quadrupole mass spectrometer to obtain Figure 4 The spectra of the nine-scented insect sample shown are the mass chromatograms of trimethylamine, diethylamine and triethylamine, and the corresponding peak areas are 283546, 35971 and 48425 respectively. Figure 2 After following the standard curves, the contents of trimethylamine, diethylamine and triethylamine in the headspace bottle of the nine-scented insect sample were calculated to be 0.0700 mg / L, 0.400 mg / L and 0.255 mg / L, respectively. Combined with the pretreatment steps, the contents in the earthworm powder sample were converted to 3.70 mg / kg, 20.0 mg / kg and 12.5 mg / kg, respectively.

[0056] In order to investigate the accuracy and precision of the method for determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials provided by the present invention, a spike recovery experiment was conducted. The specific steps are as follows: select a sample of earthworm powder, add a mixed standard solution of trimethylamine hydrochloride, diethylamine hydrochloride and triethylamine hydrochloride, and the corresponding spike concentrations of trimethylamine, diethylamine and triethylamine are all 100 mg / kg, then perform sample pretreatment according to the above steps (3)-(5), and perform determination according to step (8), repeat the determination 6 times, and the average determination results are shown in Table 1.

[0057]

[0058] Comparative Example

[0059] The specific steps of the comparative example are carried out according to steps (1), (2), (4) and (7) of the embodiment, except that: instead of using n-tetracosane, a phase change material paraffin is used.

[0060] like Figure 5 As shown, the comparative example uses phase change material paraffin, and the peak areas of diethylamine and triethylamine spectra obtained therefrom are unstable during repeated injections. When 10 mg / L mixed standard solution is injected, the peak areas of diethylamine are 15844857, and the peak areas of triethylamine are 1875335; and when 5 mg / L mixed standard solution is injected, the peak areas of diethylamine are 3750521, and the peak areas of triethylamine are 1743561. The peak area ratio of diethylamine at the above two concentrations is 4.22 times, and the peak area ratio of triethylamine at the above two concentrations is 10.75, which is quite different from the concentration ratio of the two compounds of 2 times, and there is no linear relationship between the peak area and the concentration.

[0061] Therefore, although the comparative example only replaces n-tetracosane with the phase change material paraffin, it is unable to achieve accurate quantification of diethylamine and triethylamine, and furthermore, it is even more impossible to achieve accurate simultaneous determination of the contents of trimethylamine, diethylamine and triethylamine.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and changes without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials, characterized in that: The following steps are involved: (1) mixing an alkali solution and n-tetracosane in a headspace injection bottle, heating to completely dissolve the n-tetracosane, and then standing until a layer of n-tetracosane solidifies above the alkali solution, so that the alkali solution is sealed below the n-tetracosane layer; (2) using trichloroacetic acid solution to prepare a series of mixed standard solutions of trimethylamine hydrochloride, diethylamine hydrochloride and triethylamine hydrochloride; (3) After the animal-derived Chinese medicinal materials are crushed, they are dispersed in a trichloroacetic acid aqueous solution, homogenized and then centrifuged, and the supernatant obtained by centrifugation is collected and fixed to volume with a trichloroacetic acid aqueous solution to obtain a sample extract; (4) Accurately measure the mixed standard solution of step (2) or the sample extract of step (3), add it to the headspace injection bottle obtained in step (1) above the n-tetracosane layer, and seal it with a cap; (5) placing each headspace injection bottle obtained in step (4) on a headspace automatic injector, heating it to 70-85° C., absorbing the gas above the headspace injection bottle, and analyzing it by gas chromatography-triple quadrupole mass spectrometry to obtain an MRM spectrum, and using an external standard method to obtain the contents of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials.

2. The method for determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials according to claim 1, characterized in that: The specific process of step (5) is as follows: placing the headspace injection bottles of each mixed standard solution in step (4) on the headspace automatic injector respectively, heating to 70-85° C., absorbing the gas above the headspace injection bottle, and analyzing it by gas chromatography-triple quadrupole mass spectrometry to obtain an MRM spectrum, and then using the concentration of trimethylamine, diethylamine or triethylamine as the horizontal coordinate and the corresponding quantitative ion pair peak area as the vertical coordinate to obtain the standard curves of trimethylamine, diethylamine and triethylamine respectively; The headspace injection bottle of the sample extract in step (4) is placed on a headspace automatic injector, heated to 70-85° C., the gas above the headspace injection bottle is aspirated, and the MRM spectrum is obtained by gas chromatography-triple quadrupole mass spectrometry analysis. Then, the corresponding quantitative ion pair peak area is substituted into each standard curve to obtain the concentration of trimethylamine, diethylamine and triethylamine in the sample extract, and then further converted to obtain the content of trimethylamine, diethylamine and triethylamine in animal-derived traditional Chinese medicine.

3. The method for determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials according to claim 1, characterized in that: Animal-derived Chinese medicinal materials include one or more of the following: leeches, American cockroaches, nine-scented insects, chicken gizzard lining, earthworms, silkworm pupae, cicada shells, antelope horns, buffalo horns, tortoise shells, and turtle shells.

4. The method for determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials according to claim 1, characterized in that: The alkali solution is a sodium hydroxide aqueous solution or a potassium hydroxide aqueous solution with a mass concentration of 10%-20%. The ratio between the volume of the alkali solution and the mass of n-tetracosane is (0.2-1) mL: (0.5-1) g.

5. The method for determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials according to claim 1, characterized in that: In step (1), heating is performed in an oven and the mixture is placed in an oven at 75-85° C. for 15-60 minutes to achieve complete dissolution; and the mixture is allowed to stand at room temperature for 20-60 minutes.

6. The method for determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials according to claim 1, characterized in that: In step (2) and step (3), the mass concentration of the trichloroacetic acid aqueous solution is 0.5%-5%.

7. The method for determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials according to claim 1, characterized in that: The concentrations of trimethylamine, diethylamine and triethylamine in the series of mixed standard solutions are all set between 0.5 mg / L and 10 mg / L.

8. The method for determining the contents of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials according to claim 1, characterized in that: In step (3), after the animal-derived Chinese medicinal materials are crushed, the concentration of the materials added to the trichloroacetic acid aqueous solution is 20-100 mg / mL.

9. The method for determining the content of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials according to claim 1, characterized in that: The gas chromatography used a capillary column and the column oven was programmed to heat up. The starting temperature of the column oven was 35-45°C, and the temperature was increased to 180-220°C at a rate of 15-25°C / min and maintained for 1-4 min. The injection port temperature was 180-220°C. The carrier gas was controlled in constant flow mode. The column flow rate was 1-2 mL / min. The injection method was split injection, and the split ratio was set to (5-20):

1.

10. The method for determining the contents of trimethylamine, diethylamine and triethylamine in animal-derived Chinese medicinal materials according to claim 1, characterized in that: The triple quadrupole mass spectrometer used electron impact ionization and multiple reaction monitoring mode for detection; the detection ion pairs for trimethylamine, diethylamine and triethylamine were all 2 pairs, the detection ion pairs for trimethylamine were 58.00>42.00, collision voltage 15-25V and 58.00>30.00, collision voltage 4-10V; the detection ion pairs for diethylamine were 73.00>58.10, collision voltage 4-8V and 58.00>42.10, collision voltage 24-36V; the detection ion pairs for triethylamine were 86.00>58.10, collision voltage 4-8V and 101.00>86.10, collision voltage 4-8V.

Images