A liquid phase method for simultaneous detection of dibutyl lauroyl glutamine raw material, intermediate and product
By using a C18 column and specific mobile phase conditions, the problem of separating raw materials, intermediates and products in the reaction solution of butyllauroyl glutamine was solved by HPLC, achieving efficient and accurate detection and reaction monitoring.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHANGSHA PUJI BIOTECH
- Filing Date
- 2026-04-27
- Publication Date
- 2026-06-26
AI Technical Summary
Existing technologies lack effective methods for detecting raw materials, intermediates, and products in dibutyllauroyl glutamine reaction solutions, especially in esterification and urethane exchange reactions where efficient separation and monitoring are difficult to achieve.
The HPLC method was used with a C18 column and isocratic elution with disodium hydrogen phosphate solution and a high proportion of acetonitrile as the mobile phase. The ionic strength and pH value were adjusted to enhance the difference in hydrophobic interaction between the compound and the stationary phase, so as to achieve efficient separation of raw materials, intermediates and products.
It achieves efficient separation of dibutyllauroyl glutamine raw materials, intermediates and products, with high sensitivity and accuracy, and can monitor the reaction process in real time to ensure reaction quality.
Smart Images
Figure CN122283004A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of chemical analysis and relates to a method for simultaneous detection of raw materials, intermediates and products in the reaction solution for the synthesis of dibutyllauroyl glutamine, which belongs to the field of chemical analysis technology. Background Technology
[0002] With technological innovation and upgraded consumer demand in the beauty industry, lip makeup products are showing a significant trend towards high-end and diversified development. Gel agents are the core functional ingredients in lip makeup formulations, capable of being combined with waxes, oils, and pigments to construct the product matrix. Small molecule gel agents, in particular, can form gel networks with organic solvents, possessing excellent oil-phase thickening properties and are widely used in lipsticks, lip balms, and various cream-based cosmetics. Amide compounds are a typical class of small molecule gelling agents, which can self-assemble through hydrogen bonding interactions of the amide groups in their molecules, thereby forming a stable gel system. N-lauroyl-L-glutamic acid derivatives are representative of this type of gelling agent, while also possessing the functional properties of amino acid surfactants. Butyllauroyl glutamine, as the core functional monomer of this derivative, is currently the most widely used gelling agent product in lip makeup formulations.
[0003] However, there are currently no reports on detection methods for butyllauroyl glutamine. Summary of the Invention
[0004] To address the shortcomings of existing technologies, the present invention aims to provide a liquid phase analysis method for detecting the raw material (lauroyl glutamic acid), intermediate (lauroyl glutamic acid trifluoroethyl ester), and product (dibutyllauroyl glutamamide) in a reaction solution for the synthesis of dibutyllauroyl glutamamide. Specifically, it addresses the reaction system in which lauroyl glutamic acid undergoes an esterification reaction with trifluoroethanol to obtain the intermediate (lauroyl glutamic acid trifluoroethyl ester), which is then subjected to an aminotransfer reaction with n-butylamine to obtain the product dibutyllauroyl glutamamide, allowing for real-time monitoring of the reaction progress.
[0005] To achieve the above-mentioned technical objectives, the present invention provides a liquid chromatography method for simultaneous detection of dibutyllauroyl glutamine raw material, intermediate and product. The method involves performing HPLC analysis on a reaction solution containing dibutyllauroyl glutamine raw material, intermediate and product. The dibutyllauroyl glutamine raw material is lauroyl glutamic acid; the intermediate is lauroyl glutamic acid trifluoroethyl ester; and the product is dibutyllauroyl glutamine. The HPLC chromatographic conditions are as follows: a C18 column, a mobile phase of disodium hydrogen phosphate solution and acetonitrile in a volume ratio of (30~10):(70~90), and isocratic elution of the mobile phase.
[0006] Because lauroyl glutamate, lauroyl glutamate trifluoroethyl ester, and dibutyllauroyl glutamine have highly similar structures, all containing long-chain lauroyl groups and glutamate skeletons, with only slight differences in terminal functional groups (carboxyl, trifluoroethyl, and dibutylamide groups), their polarities are similar and their retention behaviors overlap, making baseline separation difficult to achieve with conventional reversed-phase chromatography. This invention employs a C18 column and uses disodium hydrogen phosphate solution and a high proportion of acetonitrile as the mobile phase for isocratic elution. The phosphate buffer solution is used to adjust the ionic strength and pH value, significantly enhancing the difference in hydrophobic interactions between the three compounds and the stationary phase. Simultaneously, the high proportion of acetonitrile optimizes the elution intensity, thereby differentiating the retention times and achieving efficient separation of the raw materials, intermediates, and products. Furthermore, this mobile phase maintains a stable baseline and good peak shape.
[0007] Further preferably, the mobile phase is a disodium hydrogen phosphate solution and acetonitrile in a volume ratio of 20:80.
[0008] As a preferred embodiment, the concentration of disodium hydrogen phosphate solution in the mobile phase is 20-25 mmol / L. Experiments have shown that when the concentration is below 20 mmol / L, the buffering capacity is weak, failing to effectively shield the interactions of residual silanol groups on the stationary phase surface, resulting in tailing of the dibutyllauroyl glutamine peak and poor retention time reproducibility. Conversely, when the concentration is above 25 mmol / L, it not only increases column pressure and the risk of salting out but also reduces the selectivity for separating raw materials and intermediates. Within the preferred concentration range of this invention, buffering capacity, resolution, peak shape, and column pressure can be balanced. More preferably, the concentration is 20-22 mmol / L, and even more preferably, it is 20 mmol / L.
[0009] As a preferred embodiment, the pH of the disodium hydrogen phosphate solution is adjusted to 2.6-3.0 with phosphoric acid. In this invention, when the pH is below 2.6, the low pH environment provided by excess phosphoric acid will affect the reproducibility of butyllauroyl glutamine retention and may also shorten column life. Conversely, when the pH is above 3.0, it will cause peak tailing of butyllauroyl glutamine. A further preferred pH is 2.8.
[0010] As a preferred embodiment, the flow rate of the mobile phase is 0.8~1.2 mL / min. Within the flow rate range of this invention, good separation between the dibutyllauroyl glutamine feedstock, intermediate, and product can be ensured, and baseline stability can also be maintained.
[0011] As a preferred embodiment, the reaction solution containing dibutyllauroyl glutamine raw material, intermediate and product is sampled, filtered directly, and then diluted to a certain concentration with mobile phase before being put into the machine.
[0012] As a preferred embodiment, the column temperature is 35~45℃, and the injection volume is 20μL. Within the column temperature range of this invention, both rapid mass transfer and sharp peak shape are ensured, while the hydrophobic distribution balance of each component on the C18 column is stably maintained, thereby further enhancing the retention time differences among the three components and achieving baseline separation. More preferably, the column temperature is 35~38℃, and even more preferably 35℃.
[0013] As a preferred embodiment, the detector is an ultraviolet detector with a wavelength of 200-220 nm. More preferably, it is 210 nm.
[0014] As a preferred embodiment, the concentration range of lauroyl glutamic acid is 1.192 μg / mL to 5960 μg / mL, and the linear equation is: y1 = 2289396.18x1 + 7000.78, where x1 is the concentration of lauroyl glutamic acid and y1 is the peak area of lauroyl glutamic acid; the concentration range of lauroyl glutamine trifluoroethyl ester is 2.56 μg / mL to 12810 μg / mL, and the linear equation is y2 = 1025347. 0.44x2-56005.09, where x2 is the concentration of lauroyl glutamine trifluoroethyl ester, and y2 is the peak area of lauroyl glutamine trifluoroethyl ester; the concentration range of the dibutyllauroyl glutamic acid is 1.058 μg / mL to 5290 μg / mL, and the linear equation is: y3=2579326.8337x3+7139.3053; where x3 is the concentration of dibutyllauroyl glutamic acid, and y3 is the peak area of dibutyllauroyl glutamic acid. Within the concentration range of this invention, all three substances exhibit good linearity, R 2 A value greater than 0.99 allows for the quantitative detection of three substances.
[0015] Compared with the prior art, the present invention has the following beneficial effects:
[0016] (1) Compared with the normal phase system, the reversed phase chromatography system used in this invention has the advantages of stable chromatographic medium performance, strong separation ability and simple operation process.
[0017] (2) The present invention uses an isocratic mobile phase for elution, and can be tested by both single-pump and dual-pump liquid phases. Liquid phase analysis and detection are not limited by instruments.
[0018] (3) This invention achieves the effective separation of raw material lauroyl glutamic acid, trifluoroethyl ester intermediate and product in the synthesis reaction of dibutyllauroyl glutamine. While detecting the main component dibutyllauroyl glutamine, it can also locate the raw material lauroyl glutamic acid. The ratio of raw material to product can be obtained by detecting the reaction solution, which has good practicality.
[0019] (4) This invention uses a C18 column and isocratic elution with disodium hydrogen phosphate solution and a high proportion of acetonitrile as the mobile phase. The ionic strength and pH value are adjusted by using phosphate buffer, which significantly enhances the difference in hydrophobic interaction between the three compounds and the stationary phase. At the same time, the high proportion of acetonitrile optimizes the elution intensity, thereby differentiating the retention time and achieving efficient separation of raw materials, intermediates and products. Moreover, under this mobile phase, the baseline can be kept stable and the peak shape can be well maintained. It has high sensitivity and accuracy and can well control the reaction quality. Attached Figure Description
[0020] Figure 1 This is the detection chromatogram of lauroyl glutamic acid localization under the chromatographic conditions of Example 1 of the present invention.
[0021] Figure 2 The detection chromatogram of intermediate localization under chromatographic conditions in Example 1 of the present invention.
[0022] Figure 3 The detection chromatogram of dibutyllauroyl glutamine under chromatographic conditions in Example 1 of this invention.
[0023] Figure 4 This is the detection chromatogram of the system adaptability solution under the chromatographic conditions of Example 3 of the present invention.
[0024] Figure 5 This is the detection chromatogram of the control solution under the chromatographic conditions of Example 1 of the present invention.
[0025] Figure 6 This is the detection chromatogram of the blank solvent under the chromatographic conditions of Example 1 of the present invention.
[0026] Figure 7 This is the detection chromatogram of the control solution under the chromatographic conditions of Example 2 of the present invention.
[0027] Figure 8 This is the detection chromatogram of the control solution under the chromatographic conditions of Comparative Example 1 of this invention.
[0028] Figure 9 This is the detection chromatogram of the control solution under the chromatographic conditions of Comparative Example 2 of this invention.
[0029] Figure 10 This is the detection chromatogram of the control solution under the chromatographic conditions of Comparative Example 3 of this invention.
[0030] Figure 11 This is a linear regression equation graph for dibutyllauroyl glutamine.
[0031] Figure 12 This is a linear regression equation graph for lauroyl glutamate.
[0032] Figure 13This is the linear regression equation graph for lauroyl glutamate trifluoroethyl ester. Detailed Implementation
[0033] The following specific embodiments are intended to further illustrate the content of the present invention, rather than to limit the scope of protection of the claims.
[0034] Unless otherwise stated, the terms used in this invention generally have the meanings commonly understood by those skilled in the art.
[0035] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0036] Unless otherwise specified, all reagents, materials, instruments, etc. used in the following examples are commercially available.
[0037] Example 1
[0038] Preparation of the mobile phase: Weigh 3.12 g of disodium hydrogen phosphate, dissolve it in 1 L of pure water, and then adjust the pH to 2.8 with phosphoric acid to obtain a 20 mmol / L disodium hydrogen phosphate solution. Mix the disodium hydrogen phosphate solution and acetonitrile at a volume ratio of 20:80 to obtain the mobile phase.
[0039] Positioning solutions: Take lauroyl glutamate standard, lauroyl glutamate trifluoroethyl ester standard and dibutyl lauroyl glutamine standard respectively and add them to the mobile phase to dilute to 590 μg / mL, 2560 μg / mL and 520 μg / mL, respectively, and set aside for use.
[0040] Central control solution: Take 1 mL of the central control reaction solution of the reaction system in which lauroyl glutamic acid and trifluoroethanol are esterified to obtain intermediate (lauroyl glutamic acid trifluoroethyl ester), and then undergo amine transesterification reaction with n-butylamine to obtain product dibutyllauroyl glutamine. Dilute with mobile phase in a 10 mL volumetric flask, filter through a 0.45 μm filter membrane, and set aside for use.
[0041] High performance liquid chromatography determination:
[0042] After the instrument stabilizes, under the given instrument conditions, inject the prepared positioning solution and central control solution into the chromatographic column in sequence.
[0043] Chromatographic conditions:
[0044] Liquid chromatography column: Ultimate AQ-C18 (250 mm × 4.6 mm, 5 μm) reversed-phase column
[0045] Mobile phase and its ratio: 20 mmol / L disodium hydrogen phosphate solution : acetonitrile = 20 : 80
[0046] Chromatography instrument: SSI 1500 high-performance liquid chromatograph
[0047] Detector and wavelength: UV-210
[0048] Flow rate: 1.1 mL / min
[0049] Column temperature: 30℃
[0050] Injection volume: 20 μl
[0051] Diluent: Mobile phase
[0052] The obtained chromatogram is as follows Figure 1 , 2 As shown in Figures 3, 5, and 6, under the conditions of Example 1, lauroyl glutamic acid, the intermediate, and dibutyllauroyl glutamine eluted sequentially, with good separation and peak shape. Dibutyllauroyl glutamine showed moderate retention, and the blank solvent did not interfere. Figure 6 As shown.
[0053] Example 2
[0054] The only difference between this embodiment and Embodiment 1 is that the mobile phase ratio is changed to 15:85; the column temperature is changed to 35°C; and the mobile phase pH is changed to 3.0. The remaining steps and parameters are the same as in Embodiment 1.
[0055] The obtained chromatogram is as follows Figure 7 As shown in this embodiment, adaptive adjustments within the scope of the present invention can achieve good separation of lauroyl glutamic acid, intermediates, and dibutyllauroyl glutamine.
[0056] Example 3
[0057] The only difference between this embodiment and Example 1 is that the concentration of the disodium hydrogen phosphate solution in the mobile phase is changed to 5, 10, 20, 25 and 30 mmol / L, respectively. The remaining steps and parameters are the same as in Example 1.
[0058] The conclusions are shown in Table 1.
[0059]
[0060] In Table 1, RT1 represents the retention time of the raw material (lauroyl glutamic acid), RT2 represents the retention time of the intermediate (trifluoroethyl lauroyl glutamic acid), RT3 represents the retention time of the product (butyllauroyl glutamine), R1 represents the separation degree between the raw material and the intermediate, R2 represents the separation degree between the intermediate and the product, and S represents the symmetry factor of the product (butyllauroyl glutamine).
[0061] As shown in Table 1, the concentration of disodium hydrogen phosphate solution in this embodiment has a significant impact on the resolution between peaks and the symmetry factor of the product peak. With increasing disodium hydrogen phosphate concentration, the peak resolution between the raw material and intermediate first increases and then decreases. Excessively high concentrations of disodium hydrogen phosphate product lead to increased column pressure, resulting in more similar retention times for the raw material and intermediate, thus reducing resolution. Conversely, excessively low concentrations of disodium hydrogen phosphate have weak buffering capacity, causing product peak tailing and inaccurate quantitative analysis results. Overall, a disodium hydrogen phosphate concentration of 20–25 mmol / L is suitable, with 20 mmol / L being more preferably preferred.
[0062] Example 4
[0063] The only difference between this embodiment and Embodiment 1 is that the pH value in the mobile phase is changed; the remaining steps and parameters are the same as in Embodiment 1.
[0064] The conclusions are shown in Table 2.
[0065]
[0066] In Table 2, RT1 represents the retention time of the raw material (lauroyl glutamic acid), RT2 represents the retention time of the intermediate (trifluoroethyl lauroyl glutamic acid), RT3 represents the retention time of the product (butyllauroyl glutamine), R1 represents the separation degree between the raw material and the intermediate, R2 represents the separation degree between the intermediate and the product, and S represents the symmetry factor of the product (butyllauroyl glutamine).
[0067] As shown in Table 2, the pH value and solution concentration have a significant impact on the resolution between peaks and the symmetry factor of the product peak. With the increase of pH, the resolution between the raw material and intermediate peaks first increases and then decreases, while the symmetry factor of the product peak shows a trend of first increasing and then decreasing. The range of 2.6 to 3.0 is more suitable. Further optimization shows that a mobile phase pH of 2.8 is the most suitable.
[0068] Comparative Example 1
[0069] Preparation of mobile phase: Take 1 mL of phosphoric acid and add 1 L of water to obtain a 0.1 vol% phosphoric acid aqueous solution. Mix the solution with acetonitrile at a volume ratio of 20:80 to obtain the mobile phase.
[0070] The central control solution was prepared in the same manner as in Example 1.
[0071] High performance liquid chromatography determination:
[0072] After the instrument stabilizes, the prepared central control solution is injected sequentially into the chromatographic column under the given instrument conditions.
[0073] Chromatographic conditions:
[0074] Liquid chromatography column: Ultimate AQ-C18 (250mm × 4.6mm, 5μm) reversed-phase column
[0075] Mobile phase and its ratio: 0.1% phosphoric acid aqueous solution : acetonitrile = 20 : 80
[0076] Chromatography instrument: SSI 1500 high-performance liquid chromatograph
[0077] Detector and wavelength: UV-210
[0078] Flow rate: 1.1 mL / min
[0079] Column temperature: 30℃
[0080] Injection volume: 20 μl
[0081] Diluent: Mobile phase.
[0082] The obtained chromatogram is as follows Figure 8 As shown in Comparative Example 1, the peak shapes of the principal components and intermediates are poor, and the peak symmetry is poor. At the same time, a significant baseline rise occurs in the 7.5~12.5 min range. The baseline noise and drift exceed the methodological requirements and cannot meet the test requirements for accurate sample quantification. Therefore, this method cannot be used for sample analysis.
[0083] Comparative Example 2
[0084] Preparation of mobile phase: Take 1 mL of ammonia water and add 1 L of water to obtain a 0.1 vol% ammonia solution. Mix the ammonia solution and acetonitrile in a volume ratio of 20:80 to obtain the mobile phase.
[0085] The central control solution was prepared in the same manner as in Example 1.
[0086] High performance liquid chromatography determination:
[0087] Chromatographic conditions:
[0088] Liquid chromatography column: Ultimate AQ-C18 (250mm × 4.6mm, 5μm) reversed-phase column
[0089] Mobile phase and its ratio: 0.1% ammonia solution : acetonitrile = 2080
[0090] Chromatography instrument: SSI 1500 high-performance liquid chromatograph
[0091] Detector and wavelength: UV-210nm
[0092] Flow rate: 1.1 mL / min
[0093] Column temperature: 30℃
[0094] Injection volume: 20 μL
[0095] Diluent: Mobile phase.
[0096] Centralized reaction solution: Same as in Example 1.
[0097] The obtained chromatogram is as follows Figure 9 As shown in Comparative Example 2, the two peaks did not return to the baseline for separation, making accurate integration impossible and failing to distinguish between the main component, intermediates, and raw materials.
[0098] Comparative Example 3
[0099] The central control solution was prepared in the same manner as in Example 1.
[0100] High performance liquid chromatography determination:
[0101] Chromatographic conditions:
[0102] Liquid chromatography column: Ultimate AQ-C18 (250 mm × 4.6 mm, 5 μm) reversed-phase column
[0103] Mobile phases: Phase A is 20 mmol / L disodium hydrogen phosphate solution (pH adjusted to 2.8 with phosphoric acid), and Phase B is acetonitrile.
[0104] The flow ratios are shown in Table 3:
[0105]
[0106] Chromatography instrument: Agilent 1260 Infinity II HPLC
[0107] Detector and wavelength: UV-210
[0108] Flow rate: 1.1 mL / min
[0109] Column temperature: 30℃
[0110] Injection volume: 20 μL
[0111] Diluent: Mobile phase.
[0112] The obtained chromatogram is as follows Figure 10 As shown, in the gradient mobile phase of Comparative Example 3, the retention times of lauroyl glutamate and the trifluoroethyl ester intermediate were similar and not separated, resulting in peak distortion, significant fronting phenomenon, and poor peak symmetry. This does not meet the system adaptability requirements, and the method is not suitable as a method for detecting the raw material (lauroyl glutamate), intermediate (lauroyl glutamate trifluoroethyl ester), and product (dibutyllauroyl glutamine).
[0113] Example 5
[0114] The chromatographic conditions for the preliminary analysis of dibutyllauroyl glutamine were established. The results showed that dibutyllauroyl glutamine could be effectively separated from the raw materials and intermediates. Further investigation of the detection methodology will be conducted below.
[0115] Chromatographic conditions:
[0116] Chromatography instrument: SSI 1500 high-performance liquid chromatograph
[0117] Column: Ultimate AQ-C18 (250mm × 4.6mm, 5μm) reversed-phase column
[0118] Mobile phase: 20 mmol / L disodium hydrogen phosphate solution (3.12 g of disodium hydrogen phosphate was dissolved in 1 L of water, and the pH was adjusted to 2.8 with phosphoric acid) : acetonitrile = 20 : 80
[0119] Column temperature: 30℃
[0120] Detector and wavelength: UV-210
[0121] Flow rate: 1.1 mL / min
[0122] Injection volume: 20 μl
[0123] (1) System applicability and specificity
[0124] System suitability solution: Take appropriate amounts of lauroyl glutamic acid reference standard, intermediate reference standard and dibutyllauroyl glutamine reference standard, dissolve and dilute them with mobile phase to prepare a mixed solution containing 2.58 mg lauroyl glutamic acid, 1.54 mg trifluoroethyl ester intermediate and 1.16 mg dibutyllauroyl glutamine per 1 mL, as the system suitability solution.
[0125] Blank solvent: same as mobile phase.
[0126] Dibutyllauroyl glutamine reference solution: Accurately weigh an appropriate amount of dibutyllauroyl glutamine reference standard, dissolve and dilute it with the mobile phase to prepare a solution containing approximately 520 μg per 1 mL, which is used as the reference solution.
[0127] Lauroyl glutamic acid positioning solution: Take an appropriate amount of lauroyl glutamic acid reference standard, dissolve and dilute it with the mobile phase to prepare a solution containing 590 μg of lauroyl glutamic acid per 1 mL, which is used as the lauroyl glutamic acid positioning solution.
[0128] Lauroyl glutamate trifluoroethyl ester positioning solution: Take an appropriate amount of lauroyl glutamate trifluoroethyl ester reference standard, dissolve and dilute it with the mobile phase to prepare a solution containing 2560 μg of lauroyl glutamate trifluoroethyl ester per 1 mL, as the positioning solution.
[0129] Dibutyllauroyl glutamine positioning solution: Take an appropriate amount of dibutyllauroyl glutamine reference standard, dissolve and dilute it with the mobile phase to prepare a solution containing about 520 μg per 1 mL, as the dibutyllauroyl glutamine positioning solution.
[0130] Control solution for the synthesis reaction of dibutyllauroyl glutamine: Take 1 mL of control solution for the synthesis reaction of dibutyllauroyl glutamine (containing dibutyllauroyl glutamine raw material, intermediate and product), place it in a 10 mL volumetric flask, dilute to the mark with the mobile phase, and sonicate to mix.
[0131] Inject 20 μl of the above solution into the liquid chromatograph and record the chromatogram. The chromatogram is shown below. Figure 1 , 2 As shown in 3, 4, 5, and 6, the system applicability results are shown in Table 4.
[0132]
[0133] The results showed that the product dibutyllauroyl glutamine was retained moderately in the system suitability solution, and the separation degree from the raw materials and intermediates was greater than 1.5, indicating good separation. Moreover, the blank solvent and blank excipients did not interfere with the determination.
[0134] (2) Linearity and range
[0135] Under these chromatographic conditions, the concentration of the butyllauroyl glutamine reference solution (5290 μg / mL) was used as the maximum linear concentration, with concentration points set at 0.02%, 1%, 5%, 10%, 50%, and 100%. Linear solutions of butyllauroyl glutamine reference standard were prepared and diluted to concentrations of 1.058 μg / mL, 52.9 μg / mL, 264.5 μg / mL, 529 μg / mL, 2645 μg / mL, and 5290 μg / mL. 20 μL of each linear solution was accurately measured and injected into the liquid chromatograph, and chromatograms were recorded. A graph was plotted showing the concentration and peak area of butyllauroyl glutamine.
[0136] The maximum linear concentration of lauroyl glutamic acid (5960 μg / mL) was used as the reference solution concentration. Linear solutions of lauroyl glutamic acid were prepared at concentrations of 0.02%, 1%, 5%, 10%, 50%, and 100%. Lauroyl glutamic acid reference linear solutions with concentrations of 1.192 μg / mL, 59.6 μg / mL, 298.0 μg / mL, 596 μg / mL, 2980 μg / mL, and 5960 μg / mL were accurately injected into the liquid chromatograph, and chromatograms were recorded. The concentration and peak area of lauroyl glutamic acid were plotted.
[0137] The maximum linear concentration was set at the reference solution concentration of lauroyl glutamine trifluoroethyl ester (12810 μg / mL), with concentration points set at 0.02%, 1%, 5%, 10%, 50%, and 100%. Linear solutions of lauroyl glutamine trifluoroethyl ester reference standard were prepared and diluted to concentrations of 2.56 μg / mL, 128.1 μg / mL, 256.2 μg / mL, 1281 μg / mL, 2562 μg / mL, and 12810 μg / mL. 20 μL of each linear solution was accurately measured and injected into the liquid chromatograph. Chromatograms were recorded, and the concentration and peak area of lauroyl glutamine trifluoroethyl ester were plotted.
[0138] like Figure 11 As shown, the results indicate that butyllauroyl glutamine exhibits a good linear relationship with the peak area response in the concentration range of 1.058 μg / mL to 5290 μg / mL, with the linear equation being y3 = 2579326.8337x3 + 7139.3053 and the correlation coefficient r = 0.9995.
[0139] like Figure 12 As shown, lauroyl glutamate exhibited a good linear relationship with the peak area response in the concentration range of 1.192 μg / mL to 5960 μg / mL, with the linear equation being y1 = 2289396.18x1 + 7000.78 and the correlation coefficient r = 0.9995.
[0140] like Figure 13 As shown, lauroyl glutamine trifluoroethyl ester exhibited a good linear relationship with the peak area response in the concentration range of 2.56 μg / mL to 12810 μg / mL, with the linear equation being y2 = 1025347.44 x2 - 56005.09 and the correlation coefficient r = 0.9992.
[0141] (3) Limit of detection and limit of quantitation
[0142] Take the dibutyllauroyl glutamine reference solution under linearity verification, and quantitatively dilute it stepwise to prepare a solution with the limit of quantitation (signal-to-noise ratio S / N of approximately 10) and a solution with the limit of detection (signal-to-noise ratio S / N of approximately 3), and inject them in parallel twice.
[0143] The results showed that the limit of quantitation (LOQ) of butyllauroyl glutamine was 2.645 μg / mL (equivalent to 0.05% of the concentration of the reference solution), and the limit of detection (LOD) was 1.058 μg / mL (equivalent to 0.02% of the concentration of the reference solution). The method has high detection sensitivity.
[0144] (4) Precision
[0145] a. Sample injection precision test
[0146] A 5.29 mg / mL dibutyllauroyl glutamine standard solution was injected six times consecutively under the linearity test. The RSD of the peak area was calculated to examine the injection precision of the system.
[0147] The results showed that the RSD of the peak area of dibutyllauroyl glutamine was 0.98% with 6 injections, indicating that the injection precision of the system was good.
[0148] b. Repeatability test
[0149] Intermediate reaction solution: Take 1 mL of the intermediate reaction solution for the synthesis of dibutyllauroyl glutamine, place it in a 10 mL volumetric flask, dilute to the mark with the mobile phase, and sonicate to mix. Prepare 6 parallel solutions as the test solution.
[0150] The results showed that the determination results of dibutyllauroyl glutamine in the six intermediate control reaction solutions were basically consistent, and the RSD of the calculated peak area was 0.35%, indicating that the method has good repeatability.
Claims
1. A liquid phase method for simultaneous detection of dibutyllauroyl glutamine raw material, intermediate and product, characterized in that: The reaction solution containing dibutyllauroyl glutamine raw material, intermediate and product was determined by HPLC; The dibutyllauroyl glutamine raw material is lauroyl glutamic acid; the intermediate is lauroyl glutamic acid trifluoroethyl ester; the product is dibutyllauroyl glutamine. The HPLC chromatographic conditions are as follows: the chromatographic column is C18, the mobile phase is a mixture of disodium hydrogen phosphate solution and acetonitrile in a volume ratio of (30~10):(70~90), and isocratic elution is used for the mobile phase.
2. The liquid phase method for simultaneous detection of dibutyllauroyl glutamine raw material, intermediate and product according to claim 1, characterized in that: The concentration of disodium hydrogen phosphate solution in the mobile phase is 20-25 mmol / L.
3. A liquid phase method for simultaneous detection of dibutyllauroyl glutamine raw material, intermediate and product according to claim 1 or 2, characterized in that: The pH of the disodium hydrogen phosphate solution was adjusted to 2.6-3.0 with phosphoric acid.
4. The liquid phase method for simultaneous detection of dibutyllauroyl glutamine raw material, intermediate and product according to claim 3, characterized in that: The flow rate of the mobile phase is 0.8~1.2 mL / min.
5. The liquid phase method for simultaneous detection of dibutyllauroyl glutamine raw material, intermediate and product according to claim 4, characterized in that: The column temperature is 35~45℃.
6. The liquid phase method for simultaneous detection of dibutyllauroyl glutamine raw material, intermediate and product according to claim 1, characterized in that: The detector is an ultraviolet detector with a wavelength of 200~220nm.
7. A liquid phase method for simultaneous detection of dibutyllauroyl glutamine raw material, intermediate and product according to claim 1 or 6, characterized in that: The concentration range of the lauroyl glutamic acid is 1.192 μg / mL to 5960 μg / mL, and the linear equation is: y1 = 2289396.18x1 + 7000.78, where x1 is the concentration of lauroyl glutamic acid and y1 is the peak area of lauroyl glutamic acid. The concentration range of the lauroyl glutamine trifluoroethyl ester is 2.56 μg / mL to 12810 μg / mL, and the linear equation is y2 = 1025347.44x2 - 56005.09, where x2 is the concentration of lauroyl glutamine trifluoroethyl ester and y2 is the peak area of lauroyl glutamine trifluoroethyl ester. The concentration range of the dibutyllauroyl glutamic acid is 1.058 μg / mL to 5290 μg / mL, and the linear equation is: y3=2579326.8337x3+7139.3053; x3 is the concentration of dibutyllauroyl glutamic acid, and y3 is the peak area of dibutyllauroyl glutamic acid.