Method for simultaneously detecting lysine, aminocaprolactam and derivatives thereof and application

Abstract

The invention relates to the field of detection, and discloses a method and application for simultaneously detecting lysine, aminocaprolactam and derivatives thereof, and the method comprises the following steps: carrying out qualitative detection and / or quantitative detection on a sample solution containing lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam by adopting high performance liquid chromatography; the high performance liquid chromatography is hydrophilic interaction chromatography. The method is simple, convenient and rapid to operate, accurate in quantitative result, high in precision and good in reproducibility.

Description

Technical Field

[0001] The present invention relates to the detection field, and in particular to a method and application for simultaneously detecting lysine, aminocaprolactam and derivatives thereof. Background Art

[0002] Amino acids are important bio-based materials, among which lysine is the largest amino acid fermentation product after glutamic acid. It is currently mainly used as food, medicine and feed additives, and the added value of the product is relatively low. With the continuous development of fermentation technology, the lysine industry has seen a new booming development, but it has also led to serious overcapacity. Therefore, the development of new technologies for producing functionalized caprolactam derivatives using lysine as raw material, and the manufacture of high-value-added bio-based polymers or polymer precursors through biological, chemical and physical means, are of great significance to sustainable development and industrial upgrading.

[0003] Lysine obtained by fermentation can be dehydrated and cyclized to obtain lysine-based α-amino-ε-caprolactam (aminocaprolactam for short); in order to avoid the adverse effects of active hydrogen on the amino group on amide polymerization, a new functional bio-based polymerizable monomer, dimethylaminocaprolactam, is created by replacing the active hydrogen of the amino group; it is then copolymerized with caprolactam prepared by fossil methods to create bio-based / antibacterial nylon-6 fiber and plastic new materials, promoting the industrial application of bulk bio-based products and the formation of a biomanufacturing industry chain. In the development of a large-scale preparation technology route from bio-based lysine to functionalized caprolactam derivative monomers, due to the complex reaction process, the system covers many different types of biochemical components such as biomass raw materials, small molecular compounds, and oligomers. Therefore, the accurate quantitative analysis of the main compounds lysine, aminocaprolactam, monomethylaminocaprolactam, and dimethylaminocaprolactam in the biomass reaction solution is an important basis for analyzing and evaluating the reaction path and optimizing the reaction process parameters. There is no analytical method for the simultaneous detection of lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam reported so far. Therefore, it is urgent to develop a rapid determination method with simple operation, accurate determination and high precision.

[0004] Lysine, aminocaprolactam, monomethylaminocaprolactam, and dimethylaminocaprolactam are all highly polar compounds. Among them, since amino acids contain two functional groups, amino and carboxyl, in their molecular structure, they are very polar and difficult to retain in reversed-phase mode; and most of them have no ultraviolet absorption, making it difficult to obtain a highly sensitive response on an ultraviolet detector. They usually need to be derivatized before or after the column, which is cumbersome to operate and has high requirements for the derivatization environmental conditions and amino acid structure. For biomass systems, the introduction of derivatization agents will further complicate the system and increase the difficulty of analysis. In addition, the molecular structures and properties of amide compounds, including aminocaprolactam, monomethylaminocaprolactam, and dimethylaminocaprolactam, are not much different, and it is difficult to achieve a satisfactory separation effect using conventional columns. Therefore, it is difficult to achieve rapid and convenient separation and detection of similar polar compounds in a biomass reaction system where amino acids and amide compounds coexist. Patent CN112824890B discloses an analytical method for simultaneously detecting caprolactam, lysine and α-dimethylaminocaprolactam in the process of preparing caprolactam using lysine as a raw material. A C18 column capped with a special polar functional group is used to separate polar compounds. However, the ultraviolet detector has a low response to amino acids and is not suitable for biomass reaction liquid systems with lower concentrations. This method also cannot simultaneously separate and detect monomethylaminocaprolactam and dimethylaminocaprolactam.

[0005] Therefore, there is a need for a method that can simultaneously detect lysine, aminocaprolactam and their derivatives, and can also achieve separation of the substances in the sample solution to be detected. Summary of the invention

[0006] The purpose of the present invention is to overcome the problem in the prior art that functionalized caprolactam derivative monomers are difficult to detect and separate quickly, and to provide a method and application for simultaneously detecting lysine, aminocaprolactam and their derivatives. The method is simple and rapid to operate, and the quantitative results are accurate, highly precise and reproducible.

[0007] In order to achieve the above object, the first aspect of the present invention provides a method for simultaneously detecting lysine, aminocaprolactam and their derivatives, wherein the method comprises: using high performance liquid chromatography to perform qualitative and / or quantitative detection on a sample solution containing lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam; The high performance liquid chromatography is hydrophilic interaction chromatography.

[0008] Preferably, the filler of the stationary phase of the chromatographic column of the high performance liquid chromatography is a hydrophilic filler, preferably at least one selected from the group consisting of unbonded silica gel, polar bonded silica gel and polar bonded polymer.

[0009] Preferably, the polar bonded silica gel and / or polar bonded polymer is obtained by bonding silica gel and / or polymer with at least one of amino group, cyano group, diol group, amide group, ether group, zwitterion and hydrophilic macromolecule.

[0010] Preferably, the filler is selected from at least one of amino-bonded silica gel, amide-bonded silica gel, glycol-bonded silica gel, zwitterion-bonded silica gel, amino-bonded polymer, amide-bonded polymer, glycol-bonded polymer, zwitterion-bonded polymer and unbonded silica gel.

[0011] Preferably, the mobile phase of the HPLC is gradient elution and / or isocratic elution.

[0012] Preferably, under gradient elution, the volume proportion of the initial mobile phase B is 75-95%, preferably 80-90%, and the volume proportion of the terminal mobile phase B is 15-35%, preferably 20-30%.

[0013] The second aspect of the present invention provides an application of the method described in the first aspect to monitoring the substance types and content changes in the reaction solution in the synthesis process of preparing functionalized caprolactam derivative monomers from lysine.

[0014] The above technical solution has the following beneficial effects: (1) The detection method provided by the present invention is based on hydrophilic interaction liquid chromatography, which has a strong retention effect on lysine in the sample solution, and the other three components have suitable retention times on the chromatographic column, which can achieve efficient separation of different substances; (2) In the present invention, preferably, a universal detector is used to achieve the separation, detection and efficient response of underivatized lysine in the sample solution. The analytical method is simple and rapid to operate, accurate in testing, high in precision and good in reproducibility, and provides a simple and easy testing method for the preparation of aminocaprolactam derivatives from biomass. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the standard curve of lysine; Figure 2 is the standard curve of aminocaprolactam; Figure 3 is the standard curve of monomethylaminocaprolactam; Figure 4 is the standard curve of dimethylaminocaprolactam; Figure 5 This is the HPLC test result diagram for qualitative and quantitative detection of the sample solution. DETAILED DESCRIPTION

[0016] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0017] The first aspect of the present invention provides a method for simultaneously detecting lysine, aminocaprolactam and their derivatives, wherein the method comprises: using high performance liquid chromatography to perform qualitative and / or quantitative detection on a sample solution containing lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam; The high performance liquid chromatography is hydrophilic interaction chromatography.

[0018] The first aspect of the present invention provides a method for simultaneously detecting lysine, aminocaprolactam and their derivatives, wherein the method comprises: using high performance liquid chromatography to perform qualitative and / or quantitative detection on a sample solution containing lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam; The chromatographic column of the high performance liquid chromatography is a hydrophilic interaction chromatography column.

[0019] In the present invention, the detection method provided can simultaneously perform qualitative and / or quantitative analysis on lysine, aminocaprolactam and their derivatives. The lysine, aminocaprolactam and their derivatives include at least one of lysine (Lys), aminocaprolactam (ACL), monomethylaminocaprolactam (MMAC) and dimethylaminocaprolactam (DMAC).

[0020] In the present invention, a hydrophilic interaction chromatography column is selected as a chromatographic column of high performance liquid chromatography, which has a strong retention effect on lysine in a sample solution, and the other three components have a suitable retention time on the chromatographic column, and can realize efficient separation of different substances in the sample solution, especially can separate monomethylaminocaprolactam and dimethylaminocaprolactam.

[0021] According to the present invention, preferably, the method comprises the following steps: (1) Preparation of standard samples: prepare a series of standard samples of lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam with different concentrations; (2) Qualitative and / or quantitative detection: Analyze standard samples and sample solutions using high performance liquid chromatography, and perform qualitative analysis based on the retention time of the standard samples; derive the regression equation of the standard curve and use the external standard method to perform quantitative analysis on each substance in the sample solution.

[0022] According to the present invention, preferably, the filler of the stationary phase of the chromatographic column of the high performance liquid chromatography is a hydrophilic filler, preferably at least one selected from the group consisting of unbonded silica gel, polar bonded silica gel and polar bonded polymer.

[0023] According to the present invention, preferably, the polar bonded silica gel and / or polar bonded polymer is obtained from at least one bonded silica gel and / or polymer selected from the group consisting of amino, cyano, diol, amide, ether, zwitterion and hydrophilic macromolecule.

[0024] In the present invention, the zwitterion has the conventional meaning in the art, and refers to a molecule having both a positively charged region and a negatively charged region. The hydrophilic macromolecule has the conventional meaning in the art, and refers to a macromolecule containing multiple polar groups, preferably cyclodextrin and / or cyclofructan.

[0025] In the present invention, at least one of amino, cyano, diol, amide, ether, zwitterion and hydrophilic macromolecule is used to bond silica gel and / or polymer, which can provide good hydrophilicity for the stationary phase.

[0026] In the present invention, there is no particular limitation on the type of silica gel, as long as it can be used as a stationary phase filler for a hydrophilic interaction chromatography column, and those skilled in the art can select it according to actual detection conditions.

[0027] In the present invention, the type of the polymer is not particularly limited, and is a conventional polymer used for chromatographic column stationary phase filler in the art, preferably polystyrene-divinylbenzene and / or polymethacrylate. The above polymer is polarly bonded to effectively improve the hydrophilicity of the above polymer, and is suitable for hydrophilic interaction chromatography.

[0028] According to the present invention, preferably, the filler is selected from at least one of amino-bonded silica gel, amide-bonded silica gel, diol-bonded silica gel, zwitterion-bonded silica gel, amino-bonded polymer, amide-bonded polymer, diol-bonded polymer, zwitterion-bonded polymer and unbonded silica gel. Using the above chromatographic column as the stationary phase helps to improve the retention and separation selectivity of highly polar substances.

[0029] In the present invention, the source of the filler is not particularly limited, and it can be commercially available or prepared by an existing preparation method, as long as the stationary phase obtained by using the filler can be used for hydrophilic interaction chromatography, meets the detection method of the present invention, and can achieve the separation of polar substances.

[0030] According to the present invention, preferably, the mobile phase of the HPLC comprises mobile phase A and mobile phase B.

[0031] According to the present invention, preferably, the mobile phase A is an acidic aqueous solution and / or a buffered salt solution.

[0032] According to the present invention, preferably, the acidic substance in the acidic aqueous solution is selected from at least one of formic acid, acetic acid, sulfuric acid, phosphoric acid and perchloric acid, and the volume fraction of the acidic substance is ≤0.2%, preferably 0.05-0.1%.

[0033] According to the present invention, preferably, the buffer salt in the buffer saline solution is selected from at least one of ammonium formate buffer salt, ammonium acetate buffer salt, ammonium phosphate buffer salt, potassium dihydrogen phosphate buffer salt, sodium dihydrogen phosphate buffer salt, ammonium dihydrogen phosphate buffer salt and ammonium bicarbonate buffer salt, and the concentration of the buffer salt in the buffer saline solution is 5-30mmol / L, for example, 5mmol / L, 10mmol / L, 15mmol / L, 20mmol / L, 25mmol / L, 30mmol / L, and any range between any two values, preferably 10-20mmol / L.

[0034] According to the present invention, preferably, the pH value of the mobile phase A is 1-6, preferably 2-3.

[0035] In the present invention, the mobile phase B is not particularly limited and is a conventional mobile phase in the art. According to the present invention, preferably, the mobile phase B is selected from at least one of methanol, ethanol, acetonitrile and isopropanol.

[0036] According to the present invention, preferably, the mobile phase comprises a buffered saline solution and acetonitrile. The mobile phase of the above combination is used for elution, which can reduce the ion exchange effect and avoid the situation where the result curve has a peak tailing, bifurcation and deformation. When the curve has a peak tailing, bifurcation and deformation, when the peak height is used to calculate the minimum detection limit, the calculation result will be affected, and it is also difficult to determine the end point of the chromatographic peak, which will also affect the calculation result of the peak area, thereby affecting the accuracy of the results of qualitative analysis and quantitative analysis.

[0037] According to the present invention, preferably, the mobile phase of the HPLC is in the form of gradient elution and / or isocratic elution.

[0038] According to the present invention, preferably, under isocratic elution, the volume proportion of mobile phase A is 5-40%, for example 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, and any range composed of any two values, preferably 20-30%, and the volume proportion of mobile phase B is 60-95%, for example 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and any range composed of any two values, preferably 70-80%.

[0039] In the present invention, the isocratic elution has a conventional interpretation in the art, and refers to an elution method in which the composition ratio and flow rate of the mobile phase remain constant during the analysis cycle of the sample solution. There is no particular limitation on the time of the isocratic elution, as long as the elution and separation of each substance in the sample solution can be achieved. Those skilled in the art can make adaptive adjustments according to the elution situation. Preferably, the isocratic elution time is 5-55 minutes.

[0040] In the present invention, under the above isocratic elution mode, the separation effect of lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam in the sample solution is better.

[0041] In the present invention, the gradient elution has the conventional meaning in the art, which means that the volume ratio of the mobile phase is continuously changed to a certain extent in the same analysis cycle. In the gradient elution process, the change of the volume ratio of the mobile phase over time is not particularly limited, and those skilled in the art can adjust the gradient elution time and the change of the volume ratio of the mobile phase over time according to the elution situation.

[0042] According to the present invention, preferably, under gradient elution, the volume ratio of the initial mobile phase B is 75-95%, such as 75%, 80%, 85%, 90%, 95%, and any range between any two values, preferably 80-90%, and the volume ratio of the termination mobile phase B is 15-35%, 15%, 20%, 25%, 30%, 35%, and any range between any two values, preferably 20-30%. In the present invention, the use of the above-mentioned method for gradient elution can shorten the analysis cycle, improve the separation ability, and improve the situation of peak tailing.

[0043] According to a preferred embodiment of the present invention, the gradient program of the gradient elution is set as follows: the volume ratio of the initial mobile phase, the volume ratio of the termination mobile phase, and the gradient time from the initial to the termination are set in the high performance liquid chromatograph, and the volume ratio of the mobile phase changes gradually from the initial setting value to the termination setting value.

[0044] According to the present invention, there is no particular limitation on the detection injection volume of the HPLC, and those skilled in the art can make adaptive adjustments according to the detection situation. Preferably, the detection injection volume of the HPLC is 1-30 μL, for example, 1 μL, 2 μL, 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, and any range between any two values, preferably 5-10 μL.

[0045] According to the present invention, there is no particular limitation on the flow rate of the mobile phase of the HPLC, and those skilled in the art can make adaptive adjustments according to the detection situation. Preferably, the flow rate of the mobile phase of the HPLC is 0.1-2 mL / min, for example, 0.1 mL / min, 0.2 mL / min, 0.4 mL / min, 0.6 mL / min, 0.8 mL / min, 1 mL / min, 1.2 mL / min, 1.4 mL / min, 1.6 mL / min, 1.8 mL / min, 2 mL / min, and any range between any two values, preferably 0.4-1 mL / min.

[0046] According to the present invention, there is no particular limitation on the column temperature of the HPLC column, and those skilled in the art can make adaptive adjustments according to the detection conditions. Preferably, the column temperature of the HPLC column is 20-45° C., such as 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., and any range between any two values, preferably 25-35° C. Detection within the above temperature range has little effect on the separation degree and peak shape of the test substance in the sample solution, and can achieve a good separation effect.

[0047] According to the present invention, preferably, the detector of the high performance liquid chromatography is selected from at least one of a differential refractive index detector, an evaporative light scattering detector, an ultraviolet detector and a mass spectrometry detector. According to the present invention, preferably, the temperature of the differential refractive index detector is 20-50° C., such as 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., and any range between any two values, preferably 30-35° C. In the present invention, the elution mode of the differential refractive index detector is isocratic elution.

[0048] In the present invention, the elution mode of the evaporative light scattering detector, the ultraviolet detector and the mass spectrometer detector is not particularly limited, and can be either isocratic elution or gradient elution, as long as the effect of separating the sample solution can be achieved.

[0049] According to the present invention, preferably, the atomization temperature of the evaporative light scattering detector is 30-70°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and any range between any two values, preferably 30-50°C.

[0050] According to the present invention, preferably, the evaporation temperature of the evaporative light scattering detector is 30-70°C, for example, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, and any range between any two values, preferably 30-50°C.

[0051] According to the present invention, preferably, the carrier gas pressure of the evaporative light scattering detector is 100-700 kPa, for example, 100 kPa, 200 kPa, 300 kPa, 400 kPa, 500 kPa, 600 kPa, 700 kPa, and any range between any two values, preferably 400-600 kPa.

[0052] According to the present invention, preferably, the carrier gas flow rate of the evaporative light scattering detector is 1-2.5 L / min, 1 L / min, 1.2 L / min, 1.5 L / min, 2 L / min, 2.5 L / min, and any range between any two values, preferably 1.2-2 L / min.

[0053] In the present invention, under the above conditions, an evaporative light scattering detector is used to detect the sample solution, and the test sensitivity and precision of the content of each substance in the sample solution are higher.

[0054] According to the present invention, preferably, the detection wavelength range of the ultraviolet detector is 190-400 nm, for example 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 260 nm, 280 nm, 300 nm, 350 nm, 400 nm, and any range between any two values, preferably 210-240 nm.

[0055] In the present invention, preferably, the mass spectrometer uses an electrospray ion source, both positive and negative ion modes are possible, multiple reaction monitoring mode or full scan mode is used for sample qualitative analysis, and selective ion detection is used for sample quantitative analysis.

[0056] According to the present invention, preferably, the detector of the high performance liquid chromatography is a differential refractive index detector and / or an evaporative light scattering detector. In the present invention, preferably a differential refractive index detector and / or an evaporative light scattering detector is used, and the test method is fast and accurate, with high precision, and has a good response to substances without or with weak ultraviolet absorption.

[0057] In the present invention, preferably, the detector of the high performance liquid chromatography is a differential refractive index detector and / or an evaporative light scattering detector in combination with a mass spectrometer detector.

[0058] According to the present invention, preferably, in step (2), the qualitative analysis comprises: analyzing the standard sample and the sample solution by high performance liquid chromatography, performing qualitative analysis according to the retention time of the standard sample, drawing concentration-peak area standard curves of lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam, and obtaining a regression equation for the standard curve.

[0059] In the present invention, preferably, the process of qualitative analysis includes: accurately measuring the standard solution and the sample solution, respectively injecting and analyzing them, and recording the liquid chromatogram; comparing with the chromatogram of the standard solution, observing whether the corresponding chromatographic peak appears at the corresponding retention time in the chromatogram of the sample solution; if a mass spectrometer is used, judging the mass number of the test product according to the mass-to-charge ratio of the molecular ion peak in the mass spectrum.

[0060] According to the present invention, preferably, the quantitative analysis comprises: performing quantitative analysis on each substance in the sample solution by an external standard method to calculate the contents of lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam in the sample solution.

[0061] In the present invention, the sample solution is separated during the elution process, and the separation efficiency of the HPLC is evaluated according to the separation index. A separation of ≥1.5 can be considered to have achieved baseline separation and achieved separation effect. The separation has a conventional interpretation in the art, and in the present invention refers to the degree of separation between the substance to be tested and the substance to be separated. It can be calculated according to the respective corresponding formulas by the retention time and the baseline peak width or the retention time and the half-peak width parameters, which will not be repeated here.

[0062] According to the present invention, there is no particular limitation on the concentration of each substance in the sample solution, as long as it can meet the concentration range of detection. Preferably, the concentration of lysine in the sample solution is 5-600 mg / g, preferably 10-100 mg / g, the concentration of aminocaprolactam is 5-600 mg / g, preferably 10-100 mg / g, the concentration of monomethylaminocaprolactam is 1-200 mg / g, preferably 2-100 mg / g, and the concentration of dimethylaminocaprolactam is 1-200 mg / g, preferably 2-100 mg / g.

[0063] In the present invention, preferably, when the concentration of each substance in the sample solution exceeds the concentration limit of the detection range, the sample solution is appropriately diluted so that the concentration of each substance in the diluted sample solution meets the detection range limit; when the concentration of each substance in the sample solution is lower than the concentration limit of the detection range, the sample solution is appropriately concentrated so that the concentration of each substance in the diluted sample solution meets the detection range limit. In the present invention, the dilution and concentration methods are not particularly limited, and those skilled in the art can make adaptive selections as needed.

[0064] According to a preferred embodiment of the present invention, the sample solution is diluted with a diluent, wherein the diluent is acetonitrile or a mixture of acetonitrile and water with acetonitrile accounting for ≥50%, preferably a mixture of acetonitrile and water, V 乙腈 / V 水 = 1:1.

[0065] According to the present invention, there is no particular limitation on the source of the sample solution, which can be an existing reaction solution or a prepared solution. Preferably, the sample solution can be obtained by preparing functionalized caprolactam derivative monomers from lysine.

[0066] The second aspect of the present invention provides an application of the method described in the first aspect to monitoring changes in the content of key compounds in the synthesis process of functionalized caprolactam derivative monomers prepared from lysine.

[0067] In the present invention, preferably, a universal detector is used to achieve separation detection and efficient response of functionalized caprolactam derivative monomers prepared from lysine. The analytical method provided by the present invention is simple and fast to operate, has accurate testing, high precision and good reproducibility, and provides a simple and easy testing method for qualitative / quantitative analysis of aminocaprolactam derivatives prepared from biomass.

[0068] According to a particularly preferred embodiment of the present invention, a method for simultaneously detecting lysine, aminocaprolactam and derivatives thereof comprises: using high performance liquid chromatography to perform qualitative and / or quantitative detection on a sample solution containing lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam; The high performance liquid chromatography is hydrophilic interaction chromatography; The filler of the chromatographic column stationary phase of the high performance liquid chromatography is selected from at least one of amino-bonded silica gel, amide-bonded silica gel, diol-bonded silica gel, zwitterion-bonded silica gel, amino-bonded polymer, amide-bonded polymer, diol-bonded polymer, zwitterion-bonded polymer and unbonded silica gel; The mobile phase comprises a buffered salt solution and acetonitrile; Under gradient elution, the volume proportion of the initial mobile phase B is 80-90%, and the volume proportion of the ending mobile phase B is 20-30%; The detector of the high performance liquid chromatography is an evaporative light scattering detector; The atomization temperature of the evaporative light scattering detector is 30-50°C; the evaporation temperature is 30-50°C; the carrier gas pressure is 400-600 kPa; the carrier gas flow rate is 1.2-2 L / min; The column temperature of the HPLC is 25-35°C.

[0069] The present invention will be described in detail below through examples and comparative examples. In the following examples and comparative examples, the raw materials of lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam can be obtained from the market, and the purity is analytically pure; The high performance liquid chromatograph was an Agilent 1260 Infinity II high performance liquid chromatograph; Unless otherwise specified, all reagents and materials used were commercially available, and the room temperature was 25°C.

[0070] Example 1 (1) Preparation of sample solution: Accurately weigh 0.1 g each of lysine (Lys), aminocaprolactam (ACL), monomethylaminocaprolactam (MMAC), and dimethylaminocaprolactam (DMAC) into a 100 mL volumetric flask, dissolve and dilute to the mark with a mixed solvent of acetonitrile and water in a volume ratio of 1:1, and shake well to prepare the sample solution. (2) HPLC detection conditions Instrument: Agilent 1260 Infinity II HPLC; Detector: Evaporative light scattering detector (carrier gas flow rate 2L / min, carrier gas pressure 500kPa, evaporation temperature 50℃, atomization temperature 50℃); Chromatographic column: The filler of hydrophilic interaction chromatography is silica gel, the length of the chromatographic column is 100 mm, the inner diameter is 4.6 mm, and the filler particle diameter is 2.7 μm; Elution mode: gradient elution, mobile phase A: 10mmol / L ammonium formate buffer, pH = 3, solvent: deionized water, mobile phase B: acetonitrile; Gradient elution program: Set the volume ratio of mobile phase B to 80% at 0 min and 65% at 5 min. The volume ratio of mobile phase B changes from 80% to 65% during the elution process from 0 to 5 min. During the elution process of 5-10 min, the volume proportion of mobile phase B was gradually changed from 65% to 45%; During the elution process of 10-15 min, the volume proportion of mobile phase B was gradually changed from 45% to 30%; Mobile phase flow rate: 0.8 mL / min; column temperature: 30°C; injection volume: 5 μL.

[0071] Example 2 The method of Example 1 was followed, except that: in the HPLC detection conditions, the elution mode was isocratic elution, the volume ratio of mobile phase A and B was 85:15; the elution time was 15 min; the detector was a differential refractometer, and the temperature was 35°C.

[0072] Example 3 The method of Example 2 is followed, except that in the HPLC detection conditions, the filler of the chromatographic column is amino-bonded silica gel, the length of the chromatographic column is 150 mm, the inner diameter is 4.6 mm, and the filler particle diameter is 3.5 μm.

[0073] Example 4 The method of Example 1 was followed, except that in the HPLC detection conditions, mobile phase A was 20 mmol / L potassium dihydrogen phosphate buffer, pH = 2.5, the detector was an ultraviolet detector, and the detection wavelength was 210 nm.

[0074] Example 5 The method of Example 1 is followed, except that: in the HPLC detection conditions, the gradient elution program is set as follows: the volume ratio of mobile phase B in 0-10 min is reduced from 70% to 60%; the volume ratio of mobile phase B in 10-15 min is reduced from 60% to 20%.

[0075] Comparative Example 1 The method of Example 1 is followed, except that in the HPLC detection conditions, the chromatographic column adopts a Waters ACQUITY UPLC (SKU: 186005608) chromatographic column, the chromatographic column has a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle diameter of 1.7 μm.

[0076] Preparation of standard samples (1) A series of standard solutions of Lys, ACL, MMAC and DMAC were accurately prepared at concentrations of 0.03 mg / g, 0.2 mg / g, 0.5 mg / g, 0.8 mg / g, 1 mg / g and 1.3 mg / g, respectively, and analyzed by high performance liquid chromatography. The chromatographic conditions were the same as those in Example 1.

[0077] (2) Linear regression analysis was performed with mass concentration as the horizontal axis and peak area as the vertical axis to obtain a linear equation. A standard curve was drawn within the corresponding linear range, and the signal-to-noise ratio of 3 and 10 were used as the detection limit (LOD) and quantification limit (LOQ) of the method, respectively. The results are shown in Table 1. Figure 1 is the standard curve of lysine; Figure 2 is the standard curve of aminocaprolactam; Figure 3 is the standard curve of monomethylaminocaprolactam; Figure 4 This is the standard curve of dimethylaminocaprolactam.

[0078] Table 1 Linear range, linear equation, correlation coefficient, detection limit and quantification limit of four substances Test Example 1 Qualitative and quantitative determination of samples (1) Qualitative analysis: Accurately measure the standard solution and the sample solution for analysis and record the liquid chromatogram. Compare the chromatogram of the sample solution with that of the standard solution to observe whether the corresponding chromatographic peak appears at the corresponding retention time. If a mass spectrometer is used, determine the mass of the sample based on the mass-to-charge ratio of the molecular ion peak in the mass spectrum.

[0079] (2) Quantitative analysis: Test of the content of Lys, ACL, MMAC and DMAC in the caprolactam reaction solution prepared with lysine as raw material. Preparation of sample solution: Weigh about 0.25 g of the reaction solution sample, dilute it to 10 g with acetonitrile / water (v / v = 1:1), shake it well and inject it into liquid chromatography for sample analysis. Record the chromatographic peak area and calculate the concentration of Lys, ACL, MMAC and DMAC in the sample solution according to the standard curve equation. Further calculate the content of Lys, ACL, MMAC and DMAC in the reaction solution.

[0080] According to the method of the present invention, five determinations were carried out, and the chromatographic conditions were the same as in Example 1. The results are shown in Table 2.

[0081] Table 2 Content of each component in the reaction solution and relative standard deviation RSD It can be seen from the results in Table 2 that the relative standard deviation of the test method adopted by the present invention is less than 2%, and the repeatability of the test results is good.

[0082] Test Case 2 Method Verification The reaction solution samples in Test Example 1 in which the contents of each substance have been accurately determined (including lysine mass percentage 2.85%, aminocaprolactam mass percentage 5.00%, monomethylaminocaprolactam (MMAC), monomethylaminocaprolactam mass percentage 0.56%, dimethylaminocaprolactam mass percentage 0.31%) were selected, and the corresponding substances were added to the reaction solution at three addition levels of low, medium and high (the corresponding mass percentages are shown in Table 3) (for example, the addition level of lysine Lys is 1.5%, which means that 1.5% of lysine is added to the reaction solution), and the spike recovery experiment was performed. When the injection concentration exceeded the maximum value of the linear range, it was diluted to the linear range, and each addition level was repeated 6 times, and the recovery rate was calculated after deducting the background content. The chromatographic conditions were the same as those in Example 1, and the results were shown in Table 3.

[0083] Average recovery rate (%) = (measured value of sample after spike-measured value of sample before spike) / spike amount 100% Table 3 Average recovery and relative standard deviation of four substances As shown in Table 3, the average spiked recoveries of the four components at three levels range from 98.2 to 104.2%, and the relative standard deviations are 1.1 to 3.5%, which meet the analysis requirements. Therefore, the analytical method for the dehydration cyclization process of lysine developed in the technical route of the present invention is accurate and reliable, and can be used for the rapid quantitative analysis of lysine, aminocaprolactam and its derivatives.

[0084] Test Example 3 Separation Test In the process of qualitative / quantitative analysis using the methods of the embodiments and comparative examples, separation is performed by elution. The specific separation method has been described in the aforementioned embodiments and comparative examples and will not be repeated here. The separation results of each component are shown in Table 4.

[0085] Table 4 Separation data between components under different conditions It can be seen from Table 4 that the methods used in Examples 1-4 can separate the four components in the sample solution with good separation effect, while the method in Comparative Example 1 leaves almost no retention on the substances and cannot achieve complete separation.

[0086] Figure 5 The HPLC test result diagram of the qualitative and quantitative detection of the sample solution using the method of Example 1 is shown in FIG. Figure 5 It can be seen that the curve does not show peak tailing and bifurcation, the sample components can achieve good baseline separation, the peak shape is good; the detection is fast and highly sensitive.

Claims

1. A method for simultaneously detecting lysine, aminocaprolactam and their derivatives, characterized in that: The method comprises: using high performance liquid chromatography to perform qualitative and / or quantitative detection on a sample solution containing lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam; The high performance liquid chromatography is hydrophilic interaction chromatography.

2. The method according to claim 1, wherein: The method comprises the following steps: (1) Preparation of standard samples: prepare a series of standard samples of lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam with different concentrations; (2) Qualitative and / or quantitative detection: Analyze standard samples and sample solutions using high performance liquid chromatography, and perform qualitative analysis based on the retention time of the standard samples; derive the regression equation of the standard curve and use the external standard method to perform quantitative analysis on each substance in the sample solution.

3. The method according to claim 1 or 2, wherein: The filler of the stationary phase of the chromatographic column of the high performance liquid chromatography is a hydrophilic filler, preferably at least one selected from unbonded silica gel, polar bonded silica gel and polar bonded polymer; Preferably, the polar bonded silica gel and / or polar bonded polymer is obtained from at least one bonded silica gel and / or polymer selected from the group consisting of amino, cyano, diol, amide, ether, zwitterion and hydrophilic macromolecule; Preferably, the filler is selected from at least one of amino-bonded silica gel, amide-bonded silica gel, glycol-bonded silica gel, zwitterion-bonded silica gel, amino-bonded polymer, amide-bonded polymer, glycol-bonded polymer, zwitterion-bonded polymer and unbonded silica gel.

4. The method according to claim 1 or 2, wherein: The mobile phase of the high performance liquid chromatography comprises mobile phase A and mobile phase B; Preferably, the mobile phase A is an acidic aqueous solution and / or a buffered saline solution; Preferably, the acidic substance in the acidic aqueous solution is selected from at least one of formic acid, acetic acid, sulfuric acid, phosphoric acid and perchloric acid, and the volume fraction of the acidic substance is ≤0.2%, preferably 0.05-0.1%; Preferably, the buffer salt in the buffer salt solution is selected from at least one of ammonium formate buffer salt, ammonium acetate buffer salt, ammonium phosphate buffer salt, potassium dihydrogen phosphate buffer salt, sodium dihydrogen phosphate buffer salt, ammonium dihydrogen phosphate buffer salt and ammonium bicarbonate buffer salt, and the concentration of the buffer salt in the buffer salt solution is 5-30 mmol / L, preferably 10-20 mmol / L; Preferably, the pH value of the mobile phase A is 1-6, preferably 2-3; Preferably, the mobile phase B is selected from at least one of methanol, ethanol, acetonitrile and isopropanol; Preferably, the mobile phase comprises a buffered saline solution and acetonitrile.

5. The method according to claim 3 or 4, wherein: The mobile phase of the high performance liquid chromatography is a gradient elution and / or an isocratic elution mode; Preferably, under isocratic elution, the volume ratio of mobile phase A is 5-40%, preferably 20-30%, and the volume ratio of mobile phase B is 60-95%, preferably 70-80%; Preferably, under gradient elution, the volume proportion of the initial mobile phase B is 75-95%, preferably 80-90%, and the volume proportion of the terminal mobile phase B is 15-35%, preferably 20-30%.

6. The method according to any one of claims 1 to 5, wherein: The detection injection volume of the high performance liquid chromatography is 1-30 μL, preferably 5-10 μL; The flow rate of the mobile phase of the HPLC is 0.1-2 mL / min, preferably 0.4-1 mL / min; Preferably, the column temperature of the HPLC column is 20-45°C, preferably 25-35°C.

7. The method according to any one of claims 1 to 6, wherein: The detector of the high performance liquid chromatography is selected from at least one of a differential refractive index detector, an evaporative light scattering detector, an ultraviolet detector and a mass spectrometer detector; Preferably, the temperature of the differential refractive index detector is 20-50°C, preferably 30-35°C; Preferably, the atomization temperature of the evaporative light scattering detector is 30-70°C, preferably 30-50°C; the evaporation temperature is 30-70°C, preferably 30-50°C; the carrier gas pressure is 100-700 kPa, preferably 400-600 kPa; the carrier gas flow rate is 1-2.5 L / min, preferably 1.2-2 L / min; Preferably, the detection wavelength range of the ultraviolet detector is 190-400 nm, preferably 210-240 nm; Preferably, the detector of the HPLC is a differential refractive index detector and / or an evaporative light scattering detector.

8. The method according to any one of claims 2 to 7, wherein: In step (2), the qualitative analysis includes: analyzing the standard sample and the sample solution by high performance liquid chromatography, qualitatively analyzing the standard sample according to the retention time, drawing the concentration-peak area standard curve of lysine, aminocaprolactam, monomethylaminocaprolactam, and dimethylaminocaprolactam, and obtaining the standard curve regression equation; Preferably, the quantitative analysis comprises: performing quantitative analysis on each substance in the sample solution by an external standard method, and calculating the contents of lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam in the sample solution.

9. The method according to any one of claims 1 to 8, wherein: The lysine, aminocaprolactam and derivatives thereof include at least one of lysine, aminocaprolactam, monomethylaminocaprolactam and dimethylaminocaprolactam; Preferably, the concentration of lysine is 5-600 mg / g, preferably 10-100 mg / g, the concentration of aminocaprolactam is 5-600 mg / g, preferably 10-100 mg / g, the concentration of monomethylaminocaprolactam is 1-200 mg / g, preferably 2-100 mg / g, and the concentration of dimethylaminocaprolactam is 1-200 mg / g, preferably 2-100 mg / g; Preferably, the sample solution can be obtained by preparing functionalized caprolactam derivative monomers from lysine.

10. Use of the method according to any one of claims 1 to 9 in monitoring the substance types and content changes in the reaction solution in the synthesis process of functionalized caprolactam derivative monomers prepared from lysine.

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