Method for determining diethylamine based on derivatization reaction-HPLC (High Performance Liquid Chromatography) and application of method

Abstract

The invention belongs to the field of chemical analysis, and particularly discloses a method for determining diethylamine based on derivatization reaction-HPLC (high performance liquid chromatography) and application thereof, and the method comprises the following steps: adding excessive 1-naphthyl isocyanate into a diethylamine solution, carrying out nucleophilic addition reaction to generate an ultraviolet absorption derivative 1-naphthyl diethylurea, and carrying out column chromatography on the 1-naphthyl diethylurea to obtain 1-naphthyl diethylurea; the method comprises the following steps: determining the peak area of 1-naphthyl diethylurea by adopting a high performance liquid chromatography, drawing a calibration curve by taking the mass concentration of diethylamine as a horizontal coordinate and the peak area of 1-naphthyl diethylurea as a vertical coordinate, and realizing quantitative determination of diethylamine based on the calibration curve, the molar ratio of the 1-naphthyl isocyanate to the diethylamine is 5: 1. Specific recognition of derivatization reaction is realized by utilizing the chemical structure difference between diethylamine (secondary amine) and triethylamine (tertiary amine), triethylamine cannot react with 1-naphthyl isocyanate, matrix interference is thoroughly eliminated in cooperation with physical separation of HPLC gradient elution, and the content of diethylamine can be specifically recognized and measured.

Description

Technical Field

[0001] This invention belongs to the field of chemical analysis technology, specifically relating to a method for determining diethylamine based on derivatization reaction-HPLC and its application. Background Technology

[0002] Amine compounds are widely used in chemical production and pharmaceutical synthesis. Industrially, triethylamine is mostly prepared by ammonolysis of ethanol and ammonia under catalysis, high temperature, and high pressure. This synthetic route is a stepwise amination reaction: ethanol first reacts with ammonia to produce ethylamine, which then adds to ethanol to produce diethylamine, which further reacts to produce triethylamine. Due to reaction kinetics and equilibrium limitations, the ammonolysis reaction cannot achieve complete directional conversion, and the intermediate product diethylamine is difficult to react completely, thus remaining as a characteristic byproduct in the finished triethylamine product. Triethylamine is an important raw material in pharmaceutical synthesis and fine chemicals, and its residual diethylamine content directly affects product quality and safety. According to the relevant provisions of the current national standard GB / T23964-2023 "Industrial Triethylamine", the residual diethylamine content in industrial triethylamine products must be ≤0.1% (equivalent to a concentration of 1000 μg / mL). To strictly control product purity and meet compliance quality control requirements, accurate and efficient detection of diethylamine residue in triethylamine is necessary.

[0003] In the field of amine detection, existing technologies mainly rely on gas chromatography and ion chromatography to determine diethylamine residues. However, both methods have significant limitations: when detecting amines using gas chromatography, amines tend to form strong adsorption on the chromatographic column, leading to peak tailing, which seriously affects detection sensitivity and quantitative accuracy. Furthermore, the sample pretreatment steps are cumbersome, and the detection effect is poor for low-volatility amines. Ion chromatography, on the other hand, has difficulty achieving complete separation of the chromatographic peaks of structurally similar diethylamine and triethylamine, limiting the detection concentration range, and requiring a high level of professional skills from laboratory personnel.

[0004] The chemical derivatization combined with HPLC detection strategy provides a new approach for the determination of amines without UV absorption. While there are reports of using 1-naphthyl isocyanate as a derivatization reagent to detect some amine compounds, no mature HPLC method has yet been publicly disclosed for the detection of diethylamine residues in a triethylamine matrix. The core challenge lies in the fact that both diethylamine and triethylamine are aliphatic amines. Diethylamine molecules lack a conjugated system and chromophore, exhibiting no significant absorption in the UV spectral region, making direct HPLC detection impossible. Furthermore, their structures are highly similar, and conventional detection methods struggle to effectively eliminate interference from the triethylamine matrix, resulting in insufficient specificity and accuracy.

[0005] In summary, the development of a highly specific, sensitive, and easy-to-operate method for the determination of diethylamine that can effectively eliminate interference from the triethylamine matrix has become an urgent need in the fields of chemical product quality control and drug synthesis raw material detection. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for the determination of diethylamine based on derivatization reaction-HPLC and its application. This method boasts high specificity, high detection sensitivity, and accurate and reliable results, enabling efficient and precise determination of diethylamine residues in triethylamine. This invention uses 1-naphthyl isocyanate as the derivatization reagent, utilizing the specific nucleophilic addition reaction between diethylamine and this reagent to generate 1-naphthyldiethylurea with UV absorption. Then, high-performance liquid chromatography-UV detection combined with external standard method is used to achieve precise determination of diethylamine. Simultaneously, leveraging the characteristic that triethylamine (tertiary amine) lacks active hydrogen and cannot react with the derivatization reagent, chromatographic separation effectively eliminates interference from the triethylamine matrix, significantly improving detection specificity.

[0007] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: One of the technical solutions A method for determining diethylamine based on derivatization reaction-HPLC is characterized by the following steps: adding excess 1-naphthyl isocyanate to a diethylamine solution to induce a nucleophilic addition reaction to generate the ultraviolet-absorbing derivative 1-naphthyldiethylurea; determining the peak area of ​​the 1-naphthyldiethylurea using high-performance liquid chromatography; plotting a calibration curve with the mass concentration of diethylamine on the x-axis and the peak area of ​​1-naphthyldiethylurea on the y-axis; and performing quantitative determination of diethylamine based on the calibration curve; wherein the molar ratio of 1-naphthyl isocyanate to diethylamine is 5:1.

[0008] Furthermore, in the nucleophilic addition reaction, the molar ratio of diethylamine to 1-naphthyldiethylurea is 1:1. Based on this molar ratio, the present invention can accurately calculate the initial content of diethylamine by the amount of product generated.

[0009] Furthermore, the chromatographic conditions for the high-performance liquid chromatography (HPLC) are as follows: C18 column; column temperature 22-28℃; sample chamber temperature 5℃; flow rate 0.99-1.01 mL·min. -1 The detector was an ultraviolet detector with a detection wavelength of 236 nm; the injection volume was 5 μL; 1‰ phosphoric acid water was used as mobile phase A and acetonitrile as mobile phase B, and gradient elution was used.

[0010] Furthermore, the gradient elution procedure is as follows: 0 min: 70% mobile phase A, 30% mobile phase B; 10 min: 40% mobile phase A, 60% mobile phase B; 12 min: 10% mobile phase A, 90% mobile phase B; 15 min: 10% mobile phase A, 90% mobile phase B; 16 min: 70% mobile phase A, 30% mobile phase B; 23 min: 70% mobile phase A, 30% mobile phase B.

[0011] The above chromatographic conditions enable efficient separation of 1-naphthyldiethylurea, with good peak shape, no tailing, no interference from extraneous peaks, and accurate detection results.

[0012] Technical Solution Two: An application of the above-mentioned method based on derivatization reaction-HPLC for the determination of diethylamine residues in triethylamine.

[0013] Technical Solution 3: A method for determining the residual amount of diethylamine in triethylamine includes the following steps: adding an excess of 1-naphthyl isocyanate derivatization reagent to a triethylamine sample containing residual diethylamine, reacting at 20-30℃ for 10-15 min to allow diethylamine and 1-naphthyl isocyanate to fully generate 1-naphthyldiethylurea, and then performing high performance liquid chromatography detection according to the method of the above-mentioned technical solution one, and calculating the residual amount of diethylamine in the triethylamine sample based on the calibration curve.

[0014] In this invention, triethylamine is a pharmaceutical raw material or industrial-grade triethylamine. When this method is used for determination, the detection limit of diethylamine is 0.7256 μg / mL and the quantitation limit is 2.6187 μg / mL, which meets the limit control requirement of ≤0.1% for diethylamine residue in triethylamine in the national standard GB / T 23964-2023.

[0015] The experimental principle of this invention for determining the residual diethylamine in triethylamine based on derivatization reaction-HPLC is as follows: Diethylamine, as a typical aliphatic secondary amine, contains only C, C, CH, and CN single bonds in its molecular structure. Due to the lack of a conjugated system and chromophores, it has no obvious absorption characteristics in the ultraviolet spectral region, making it impossible to directly detect by HPLC (high performance liquid chromatography). To address this problem, chemical derivatization strategy becomes an effective solution. Triethylamine belongs to the tertiary amine class, and its nitrogen atom lacks an active hydrogen atom, thus it cannot chemically react with 1-naphthyl isocyanate; however, the nitrogen atom in diethylamine is bonded to a hydrogen atom, enabling it to undergo a nucleophilic addition reaction with 1-naphthyl isocyanate, ultimately generating 1-naphthyldiethylurea with a characteristic structure.

[0016] This invention utilizes an excess of 1-naphthyl isocyanate to undergo a nucleophilic addition reaction with diethylamine. By leveraging the well-defined stoichiometric relationship between the reactants and products, precise quantitative analysis of the initial amount of diethylamine can be achieved based on the amount of product formed. Figure 1 The reaction of naphthyl isocyanate with amine compounds is shown.

[0017] In the above nucleophilic addition reaction, 1-naphthyl isocyanate is in excess, and the stoichiometric ratio of diethylamine to the product is 1:1. When the reaction is complete, n(product) = n(reactant), that is, n(1-naphthyldiethylurea) = n(diethylamine).

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: 1. Highly specific: Utilizing the difference in chemical structure between diethylamine (secondary amine) and triethylamine (tertiary amine), it achieves specific identification of derivatization reactions. Triethylamine cannot react with 1-naphthyl isocyanate. Combined with the physical separation of HPLC gradient elution, matrix interference is completely eliminated, allowing for specific identification and determination of diethylamine content. 2. High sensitivity and accuracy: The method for determining diethylamine based on derivatization reaction-HPLC provided by this invention has a detection limit as low as 0.7256 μg / mL and a quantification limit of 2.6187 μg / mL for diethylamine. It exhibits good linearity (R² = 0.9995) within the concentration range of 13.72–205.8 μg / mL. The recovery rate is 98.01%–100.77%, and the precision RSD ≤ 0.96%. The detection results are accurate and reliable, meeting the requirements of national standards and the detection requirements for pharmaceutical synthesis raw materials. According to the relevant provisions of the current national standard GB / T 23964-2023 "Industrial Triethylamine", the residual limit of diethylamine in industrial triethylamine products must be ≤ 0.1% (equivalent to a concentration of 1000 μg / mL). The quantitation limit of the method of this invention is far lower than the threshold limit of the above-mentioned national standard, and the sensitivity fully covers the detection requirements of actual samples. It can accurately realize the quantitative analysis and quality control of diethylamine residue in industrial triethylamine raw materials, meet the practical requirements of industrial batch detection, and provide a convenient and reliable technical means for the quality control of chemical products and the detection of raw materials for drug synthesis. It has strong practicality and market application value. 3. Simple operation and strong practicality: No complicated sample pretreatment steps are required. The derivatization reaction conditions are mild (reaction at 20-30℃ for 10-15 min). The HPLC detection conditions are easy to achieve. The instrument has a high penetration rate and is suitable for daily quality control in enterprises and rapid detection in scientific research laboratories. It can be widely used in the detection of diethylamine in chemical, pharmaceutical and other fields. 4. Filling a technological gap: For the first time, the derivatization of 1-naphthyl isocyanate coupled with HPLC was applied to the determination of diethylamine residue in triethylamine, which solved the technical problem that diethylamine has no ultraviolet absorption and is difficult to detect due to its similar structure to triethylamine, thus providing a new and effective means for the quality control of triethylamine products. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a reaction between naphthyl isocyanate and amine compounds; Figure 2 Chromatograms of blank solution, diethylamine, and the reaction solution of 1-naphthyl isocyanate; Figure 3 Chromatogram of 1-naphthyldiethylurea standard solution; Figure 4 Chromatograms of a blank solution, a reaction solution of diethylamine and 1-naphthyl isocyanate, and a reaction solution of triethylamine and 1-naphthyl isocyanate; Figure 5 The chromatogram for the linearity test of 1-naphthyldiethylurea; Figure 6 This is the standard curve for diethylamine; Figure 7 Chromatogram for repeatability test 1; Figure 8 Chromatogram for repeatability test 2; Figure 9 Chromatogram for accuracy test 1; Figure 10 Chromatogram for accuracy test 2; Figure 11 For the accuracy test, chromatogram 3 is used; Figure 12 Chromatogram for stability test; Figure 13 For flow rate robust chromatography Figure 1 ; Figure 14 For flow rate robust chromatography Figure 2 ; Figure 15 For flow rate robust chromatography Figure 3 ; Figure 16 For column temperature robust chromatography Figure 1 ; Figure 17 For column temperature robust chromatography Figure 2 ; Figure 18 For column temperature robust chromatography Figure 3 . Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Combining national standard quality control requirements with existing testing challenges, the current detection of diethylamine residue in triethylamine faces multiple bottlenecks: on the one hand, according to GB / T 23964-2023 "Industrial Triethylamine", diethylamine residue must be strictly controlled to ≤0.1% (1000 μg / mL), imposing stringent requirements on detection sensitivity and accuracy; on the other hand, existing technologies suffer from poor specificity, low sensitivity, and cumbersome operation. Furthermore, diethylamine lacks ultraviolet absorption, making direct HPLC detection impossible, thus failing to meet compliance quality control requirements. Therefore, this invention provides a method for determining diethylamine based on derivatization reaction-HPLC.

[0027] This invention provides a method for the determination of diethylamine based on derivatization reaction-HPLC, aiming to solve the technical problem of poor selectivity and insufficient accuracy of traditional detection methods for trace diethylamine impurities in triethylamine due to their similar structure and lack of UV absorption. This invention uses 1-naphthyl isocyanate as a derivatization reagent, utilizing the specific nucleophilic addition reaction of diethylamine with this reagent to generate 1-naphthyldiethylurea with UV absorption. Then, high-performance liquid chromatography-UV detection combined with external standard method is used to achieve accurate determination of diethylamine. Simultaneously, relying on the characteristic that triethylamine (tertiary amine) has no active hydrogen and cannot react with the derivatization reagent, combined with chromatographic separation, effectively eliminates interference from the triethylamine matrix, significantly improving detection specificity. Methodological validation shows that this method has a detection limit as low as 0.7256 μg / mL and a quantification limit of 2.6187 μg / mL for diethylamine; it exhibits good linearity in the concentration range of 13.72~205.8 μg / mL, with a coefficient of determination R0. 2 =0.9995; the recovery rate of the sample was 98.01%~100.77%, and the precision, stability and durability all met the requirements of analysis and detection, with excellent overall detection performance.

[0028] Example 1 The chromatographic conditions used in this embodiment are as follows: Column: Symmetry C18: 4.6 mm × 250 mm, 5 μm; Column temperature: 25℃; Sample chamber temperature: 5℃; 1 mL of chromatographic grade phosphoric acid was dissolved in 1000 mL of deionized water and stirred to obtain 1‰ phosphoric acid solution as mobile phase A; chromatographic grade acetonitrile was used as mobile phase B; Gradient elution program is shown in Table 1; Flow rate: 1.0 mL·min -1 Detector: UV 236nm; Injection volume: 5μL; Needle washing solvent: chromatographic grade acetonitrile; Run time: 23min; Data acquisition: Empower 3 software; High performance liquid chromatograph: Waters e2695. Table 1 Solution preparation: Preparation of 1-naphthyl isocyanate solution: Accurately weigh 0.7782 g of 1-naphthyl isocyanate using an analytical balance, place it in a 10 mL beaker, add 50 mL of chromatographic grade acetonitrile, and mix thoroughly. Preparation of diethylamine solution: Accurately weigh 0.0686 g of diethylamine standard into a 50 mL volumetric flask using an analytical balance, and dilute to volume with deionized water.

[0029] Determining the retention period: Preparation of blank solution: Use a pipette to transfer 1 mL of 1-naphthyl isocyanate solution and 0.5 mL of deionized water into a 10 mL volumetric flask; Preparation of the reaction solution of diethylamine and 1-naphthyl isocyanate: Pipette 5 mL of diethylamine solution into a 50 mL volumetric flask, dilute to volume with deionized water, and mix thoroughly to obtain a diethylamine solution; Pipette 1 mL of the above 1-naphthyl isocyanate solution and 0.5 mL of the above diethylamine solution into a 10 mL volumetric flask to obtain the reaction solution of diethylamine and 1-naphthyl isocyanate. After thoroughly mixing the blank solution and the reaction solution of diethylamine and 1-naphthyl isocyanate, let them stand at 60°C for 1 hour with occasional shaking. Then, dilute to volume with deionized water. After dilution, pipette 1 mL of each sample solution into a 0.22 μm syringe filter, filter the solution through the syringe, and then fill the syringe into a 2 mL vial. Inject the vial into the high-performance liquid chromatograph to determine the retention time of 1-naphthyldiethylurea.

[0030] Figure 2 The chromatograms are of the blank solution, diethylamine, and the reaction solution of 1-naphthyl isocyanate. Figure 2 As can be seen, when the retention time tR of 1-naphthyl diethylurea, the product of the reaction between diethylamine and 1-naphthyl isocyanate, is 11.187 min, this peak is not present in the blank solution. Furthermore, the peak with a retention time tR of approximately 11.187 min, as determined by liquid chromatography-mass spectrometry (LC-MS), is the peak of 1-naphthyl diethylurea, a 1-naphthylurea product generated by the reaction of 1-naphthyl isocyanate and diethylamine, detected by an ultraviolet detector.

[0031] System adaptability test Preparation of blank solution: Use a pipette to transfer 2 mL of 1-naphthyl isocyanate solution and 0.1 mL of deionized water from Example 1 into a 10 mL volumetric flask to obtain a blank solution.

[0032] Preparation of 1-naphthyldiethylurea standard solution: Pipette 2 mL of 1-naphthyl isocyanate solution and 0.1 mL of diethylamine solution into a 10 mL volumetric flask to obtain the 1-naphthyldiethylurea standard solution.

[0033] After thoroughly mixing the two solutions, let them stand at 60°C for 1 hour, occasionally shaking, and then dilute to volume with deionized water. Pipette 1 mL of the diluted blank solution into an injection tube, filter it through a 0.2 μm syringe filter, and then fill the tube into a 2 mL vial. Inject the vial into the high-performance liquid chromatograph (HPLC) syringe, checking for baseline drift, noise, or other signs of system instability. Repeat until stable.

[0034] After adjusting the volume of the 1-naphthyldiethylurea standard solution, 1 mL of injection solution was drawn up, filtered through a 0.22 μm needle filter, and then filled into a 2 mL injection vial. The vial was then placed in the injector of the high performance liquid chromatograph for injection. The theoretical plate number and resolution were then examined.

[0035] When performing qualitative identification or quantitative analysis using high-performance liquid chromatography (HPLC), the resolution between the analyte peak and adjacent peaks should be greater than 1.5. Figure 3 The chromatogram of the 1-naphthyldiethylurea standard solution shows that the resolution between 1-naphthyldiethylurea and the adjacent peak with retention time tR=4.09318 min is 25.88; the resolution between 1-naphthyldiethylurea and the adjacent peak with retention time tR=15.78289 min is 16.69, both meeting the requirements. The system shows that the theoretical plate number of 1-naphthyldiethylurea is 25774.774.

[0036] The formula for calculating the separation degree is as follows: ; The formula for calculating the theoretical plate number of a chromatographic column is: 2 ; Specificity test Preparation of blank solution: Use a pipette to transfer 2 mL of 1-naphthyl isocyanate solution and 0.5 mL of deionized water from Example 1 into a 10 mL volumetric flask to obtain a blank solution.

[0037] Preparation of the reaction solution of diethylamine and 1-naphthyl isocyanate: Pipette 5 mL of diethylamine solution into a 50 mL volumetric flask, dilute to volume with deionized water, mix well to obtain a diethylamine solution; Pipette 1 mL of 1-naphthyl isocyanate solution and 0.5 mL of the above diethylamine solution into a 1 mL volumetric flask to obtain the reaction solution of diethylamine and 1-naphthyl isocyanate.

[0038] The reaction solution of triethylamine and 1-naphthyl isocyanate: Accurately weigh 0.5 g of triethylamine standard into a 100 mL beaker using an analytical balance, add 50 mL of chromatographic grade acetonitrile, and mix thoroughly to obtain a triethylamine solution; use a pipette to transfer 1 mL of 1-naphthyl isocyanate solution and 0.5 mL of the above triethylamine solution into a 10 mL volumetric flask to obtain the reaction solution of triethylamine and 1-naphthyl isocyanate.

[0039] After thoroughly mixing the above solution, let it stand at 60℃ for 1 hour, shaking occasionally, and then dilute to volume with deionized water. Pipette 1 mL of the diluted solution into an injection tube, filter it through a 0.22 μm syringe filter, and then fill the tube into a 2 mL injection vial for injection into the high-performance liquid chromatograph.

[0040] Figure 4 Chromatograms of a blank solution, a reaction solution of diethylamine and 1-naphthyl isocyanate, and a reaction solution of triethylamine and 1-naphthyl isocyanate are shown below. Figure 4 It can be seen that the blank solution, the reaction solution of triethylamine and 1-naphthyl isocyanate do not interfere with the detection of 1-naphthyldiethylurea, the reaction product of diethylamine and 1-naphthyl isocyanate, proving that triethylamine and 1-naphthyl isocyanate do not react.

[0041] Linear range and correlation coefficient test Use a pipette to transfer seven 2 mL aliquots of 1-naphthyl isocyanate solution into 10 mL volumetric flasks; then, sequentially measure 0.1 mL, 0.12 mL, 0.5 mL, 0.8 mL, 1 mL, 1.2 mL, and 1.5 mL of diethylamine solution into the same seven 10 mL volumetric flasks. Shake the solutions thoroughly and let them stand at 60 °C for 1 h, occasionally shaking, and then dilute to volume with deionized water.

[0042] After adjusting the volume, take 1 mL of the solution into 7 vials, filter them through a 0.22 μm needle filter, fill them into 2 mL injection vials, and inject them into the injector of the high performance liquid chromatograph.

[0043] Plot the concentration of diethylamine x (unit: μg / mL) on the x-axis and the peak area y of 1-naphthyldiethylurea (unit: μV) on the y-axis. Plot a calibration curve with seconds as the ordinate. In the content determination method, the correlation coefficient R... 2 The general requirement is ≥0.998.

[0044] Table 2 shows the linearity results of the diethylamine test, where the peak area is the peak area of ​​1-naphthyldiethylurea and the concentration is the concentration of diethylamine. Figure 5 The chromatogram for the linearity test of 1-naphthyldiethylurea; Figure 6 The standard curve for diethylamine is shown in Table 2. Figure 5 and Figure 6 As can be seen from the data, within the concentration range of 13.72 μg / mL to 205.8 μg / mL, the correlation coefficient R of the diethylamine standard curve is [value missing]. 2 =0.9995, the concentration of diethylamine and the peak area of ​​1-naphthyldiethylurea showed a good linear relationship.

[0045] Table 2 Limit of detection and limit of quantitation tests Accurately measure 1 mL of the reaction solution of diethylamine and 1-naphthyl isocyanate, and dilute it serially with deionized water. After thoroughly mixing the solution, let it stand at 60°C for 1 h with occasional shaking, and then make up to volume with deionized water. Pipette 1 mL of the diluted sample solution into an injection tube, filter it through a 0.22 μm syringe filter, and then fill the tube into a 2 mL injection vial. Inject the vial into the high-performance liquid chromatograph (HPLC) and record the peak area. The limits of detection and quantitation are the corresponding concentrations of diethylamine at signal-to-noise ratios of 3:1 and 10:1, respectively.

[0046] The detection limit for diethylamine was 0.7256 μg / mL (S / N=3), and the quantitation limit was 2.6187 μg / mL (S / N=10).

[0047] Precision test Repeatability: The same analyst used a pipette to transfer six 2 mL aliquots of 1-naphthyl isocyanate solution and six 0.5 mL aliquots of diethylamine solution into six 10 mL volumetric flasks. After thoroughly mixing the solutions, they were allowed to stand at 60 °C for 1 h with occasional shaking, and then diluted to volume with deionized water. Each of the six diluted solutions was then filtered through a 0.22 μm syringe filter using a 1 mL syringe, and then filled into 2 mL injection vials. These vials were then injected into the high-performance liquid chromatograph (HPLC) syringe to determine the concentration of the test solution and calculate the RSD.

[0048] Intermediate precision (repeatability test 2): On different days, different analysts used pipettes to transfer six 2 mL aliquots of 1-naphthyl isocyanate solution and six 0.5 mL aliquots of diethylamine solution into six 10 mL volumetric flasks. After thoroughly mixing the solutions, they were allowed to stand at 60°C for 1 hour with occasional shaking, and then diluted to volume with deionized water. Each of the six diluted solutions was then pipetted into a 1 mL syringe, filtered through a 0.22 μm syringe filter, and then filled into 2 mL injection vials. These vials were then injected into the high-performance liquid chromatograph (HPLC) syringe to determine the concentration of the test solution. The results were combined with the repeatability results, and the RSD was calculated.

[0049] Table 3 shows the results of repeatability experiment 1, and Table 4 shows the results of intermediate precision experiment; Figure 7 Chromatogram for repeatability test 1; Figure 8 The chromatogram is for repeatability test 2.

[0050] In the content determination, the chromatographic method requires repeatability of 12 test solutions with an intermediate precision RSD ≤ 2%; and repeatability of 6 test solutions with an intermediate precision RSD ≤ 1%. For diethylamine, the repeatability RSD was 0.9425% (n=6); the intermediate precision RSD was 0.746% (n=6), and the intermediate precision RSD was 0.811% (n=12). The test results show that diethylamine has good repeatability and intermediate precision, meeting the requirements.

[0051] Table 3 Table 4 Accuracy test Use a pipette to transfer nine 2 mL aliquots of 1-naphthyl isocyanate solution and three aliquots each of 0.8 mL, 1 mL, and 1.2 mL diethylamine solution into nine 10 mL volumetric flasks.

[0052] After thoroughly mixing the above solution, let it stand at 60℃ for 1 hour with occasional shaking, and then dilute to volume with deionized water. Take 1 mL of the diluted solution, filter it through a 0.22 μm syringe filter, and then fill it into 2 mL injection vials. Inject the vials into the high-performance liquid chromatograph (HPLC) syringe and calculate the recovery rate and RSD for each concentration.

[0053] In the content determination, the chromatographic method requires a recovery rate between 98% and 101%, with a recovery RSD ≤ 2%. As shown in Table 5, the recoveries of diethylamine at theoretical concentrations of 109.76 μg / mL, 137.20 μg / mL, and 164.64 μg / mL were 100.766% (RSD = 0.9330%, n = 3), 98.2793% (RSD = 0.9175%, n = 3), and 98.0064% (RSD = 1.486%, n = 3), respectively. Figure 9 , Figure 10 , Figure 11 The accuracy meets the requirements.

[0054] Table 5 Stability test Pipette 2 mL of 1-naphthyl isocyanate solution and 1.5 mL of diethylamine solution into a 10 mL volumetric flask. Shake the solution thoroughly and allow it to stand at 60 °C for 1 h, occasionally shaking. Dilute to volume with deionized water. Pipette 1 mL of the diluted solution containing 1-naphthyl isocyanate and diethylamine into a syringe, filter through a 0.22 μm syringe filter, and then fill the syringe into a 2 mL vial. Inject the vial at 0 h, 12 h, 24 h, 36 h, 48 h, and 60 h, and calculate the recovery rate and RSD.

[0055] Table 6 shows the results of the stability test. Figure 12 The chromatogram is for the stability test. In the content determination, the chromatographic method requires the ratio of the main peak area at each time point to 0 h to be 98%–102%. The results show that the recoveries of diethylamine at 0 h, 12 h, 24 h, 36 h, 48 h, and 60 h were 100.426%, 98.988%, 99.591%, 99.683%, and 99.344%, respectively (RSD = 0.5334%, n = 6). The recoveries of diethylamine are all between 98% and 102%, with an RSD ≤ 2%. The stability of diethylamine meets the requirements.

[0056] Table 6 Durability test Effect of flow rate variation: The flow rate was set to 0.99 mL·min. -1 1.00 mL·min -1 1.01 mL·min -1 Use a pipette to transfer 2 mL of 1-naphthyl isocyanate solution and 0.9 mL of diethylamine solution into a 10 mL volumetric flask. Repeat the injection three times at various flow rates, record the retention time and peak area of ​​1-naphthyl diethylurea, and determine the concentration of diethylamine using the external standard method.

[0057] Effect of column temperature variation: The column temperature was set to 22℃, 25℃, and 28℃, respectively. 2 mL of 1-naphthyl isocyanate solution and 0.9 mL of diethylamine solution were pipetted into 10 mL volumetric flasks. At each column temperature, the injections were repeated three times. The retention time and peak area of ​​1-naphthyl diethylurea were recorded, and the mass concentration of diethylamine was determined using the external standard method.

[0058] Figure 13-15 These are flow rate robustness chromatography. Figure 1-3 ; Figure 16-18 Column temperature durability chromatography Figure 1-3 Table 7 shows the results of the durability test. As can be seen from the attached figures and Table 7, the RSD of the diethylamine concentration remained constant at a flow rate of 0.582% (n=9), indicating that this chromatographic method is effective at flow rates between 0.99 and 1.01 mL / min.-1 The method exhibits good robustness when varying within a certain range. The RSD of diethylamine concentration with changes in column temperature is 1.842% (n=9), and the effect of column temperature variations between 22 and 28°C on the diethylamine content determination results is within an acceptable range.

[0059] Table 7 The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for determining diethylamine based on derivatization reaction-HPLC, characterized in that, Includes the following steps: An excess of 1-naphthyl isocyanate was added to a diethylamine solution, resulting in a nucleophilic addition reaction to generate the UV-absorbing derivative 1-naphthyldiethylurea. The peak area of ​​the 1-naphthyldiethylurea was determined by high-performance liquid chromatography (HPLC). A calibration curve was plotted with the mass concentration of diethylamine on the x-axis and the peak area of ​​1-naphthyldiethylurea on the y-axis. The quantitative determination of diethylamine was achieved based on the calibration curve. The molar ratio of 1-naphthyl isocyanate to diethylamine was 5:

1.

2. The method for determining diethylamine based on derivatization reaction-HPLC according to claim 1, characterized in that, In the nucleophilic addition reaction, the molar ratio of diethylamine to 1-naphthyldiethylurea is 1:

1.

3. The method for determining diethylamine based on derivatization reaction-HPLC according to claim 1, characterized in that, The chromatographic conditions for the high-performance liquid chromatography (HPLC) method are as follows: C18 column; column temperature 22-28℃; sample chamber temperature 5℃; flow rate 0.99-1.01 mL / min. -1 The detector was an ultraviolet detector with a detection wavelength of 236 nm; the injection volume was 5 μL; 1‰ phosphoric acid water was used as mobile phase A and acetonitrile as mobile phase B, and gradient elution was used.

4. The method for determining diethylamine based on derivatization reaction-HPLC according to claim 3, characterized in that, The gradient elution procedure is as follows: 0 min: 70% mobile phase A, 30% mobile phase B; 10 min: 40% mobile phase A, 60% mobile phase B; 12 min: 10% mobile phase A, 90% mobile phase B; 15 min: 10% mobile phase A, 90% mobile phase B; 16 min: 70% mobile phase A, 30% mobile phase B; 23 min: 70% mobile phase A, 30% mobile phase B.

5. The method for determining diethylamine based on derivatization reaction-HPLC according to claim 3, characterized in that, The C18 column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm.

6. The application of the method for determining diethylamine based on derivatization reaction-HPLC according to any one of claims 1-5 in the determination of diethylamine residue in triethylamine.

7. A method for determining the residual amount of diethylamine in triethylamine, characterized in that, Includes the following steps: An excess of 1-naphthyl isocyanate is added to a triethylamine sample containing residual diethylamine, and the mixture is reacted at 20-30°C for 10-15 min. Then, high-performance liquid chromatography is performed according to the method described in any one of claims 1-5, and the residual amount of diethylamine in the triethylamine sample is calculated based on the calibration curve.

Images