Method for detecting related substances of dihydroxypropyl arginine hydrochloride

Abstract

The invention provides a method for detecting related substances of dihydroxypropyl arginine hydrochloride, which is characterized in that a high performance liquid chromatography method is adopted, a chromatographic column is a reversed-phase, cation exchange and hydrophilic interaction mixed type chromatographic column, and a mobile phase is used for gradient elution. The detection method provided by the invention can effectively separate and detect main components, starting materials and process impurities in dihydroxypropyl arginine hydrochloride, has the advantages of high sensitivity, good degree of separation, simple operation, fast appearance time, short detection time, good method reproducibility and stability, and strong specificity, meets the requirements of product quality control, and is suitable for industrial production. The method has the advantages of high sensitivity and high precision, is favorable for ensuring the safety and effectiveness of the product, reduces the production cost, and has remarkable technical advantages and application value.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical analysis, and particularly to a method for detecting related substances of dihydroxypropyl arginine hydrochloride. Background Art

[0002] Dihydroxypropyl arginine, also known as dihydroxypropyl arginine hydrochloride, has a structure as shown in Formula I and is a common skin care product ingredient. It mainly has the effects of moisturizing, repairing damaged skin cells, reducing cellular oxidative damage, and soothing the skin. Dihydroxypropyl arginine has the following application scenarios in skin care products: facial cream, eye cream, facial mask, and essence. By correctly selecting and using skin care products containing dihydroxypropyl arginine, the moisture retention of the skin can be improved, damaged cells can be repaired, skin aging can be delayed, and the skin can become healthier and younger.

[0003]

[0004] The patent EP1584320A1 of Nippon Seika Chemical Co., Ltd. discloses the synthesis method of dihydroxypropyl arginine hydrochloride, and also discloses the in-process control and detection methods. There is no detection method for the related substances of the existing dihydroxypropyl arginine. To ensure the quality and safety of the product, it is urgent to establish a detection method for its control. Therefore, the development of a method for detecting related substances of dihydroxypropyl arginine by high performance liquid chromatography has always been a new problem to be solved urgently.

[0005] To accurately detect the related substances of dihydroxypropyl arginine hydrochloride, the detection method of high performance liquid chromatography is generally used, but there are the following problems: firstly, using a C18 chromatographic column with a reverse phase method, neither the main peak nor its starting materials are retained; secondly, when the finished product encounters pure organic reagents, solids will precipitate and block the normal phase chromatographic column; while using an amino column, dihydroxypropyl arginine and its starting material arginine cannot be separated. Also, because the ultraviolet absorption of dihydroxypropyl arginine is weak, an unconventional liquid phase detector needs to be used, and the mobile phase requirements of the unconventional liquid phase detector are harsh, and the separation degree cannot be improved by adjusting the mobile phase additives. It can be seen that because there are too many limiting conditions for developing the HPLC method, it is difficult to achieve simultaneous retention and separation of multiple components for detection in an ordinary reverse phase chromatographic system. Summary of the Invention

[0006] Based on this, the purpose of the present invention is to overcome the prior art and provide a method for detecting related substances of dihydroxypropyl arginine hydrochloride, which can effectively separate and detect the main component, starting materials and process impurities in dihydroxypropyl arginine hydrochloride. It has the advantages of high sensitivity, good separation degree, simple operation, fast peak emergence time, short detection time, good method reproducibility, stability and low cost.

[0007] The present invention provides a method for detecting related substances of dihydroxypropyl arginine hydrochloride. The high performance liquid chromatography method is adopted, and the chromatographic column is a reversed-phase, cation-exchange and hydrophilic interaction mixed chromatographic column, and gradient elution is carried out using a mobile phase.

[0008] Preferably, the chromatographic column is Comixsil RP-100.

[0009] Preferably, the inner diameter of the chromatographic column is 2.7 mm to 4.6 mm, the length is 100 mm to 150 mm, and the filler particle size is 3 μm to 5 μm.

[0010] Preferably, the inner diameter of the chromatographic column is 4.6 mm, the length is 150 mm, and the filler particle size is 3 μm.

[0011] Preferably, the column temperature of the chromatographic column is 30 °C to 40 °C, preferably 30 °C.

[0012] Preferably, the flow rate of the chromatographic column is 0.8 mL / min to 1.2 mL / min, preferably 0.8 mL / min to 1.0 mL / min, and more preferably 1.0 mL / min.

[0013] Preferably, the injection volume of the chromatographic column is 10 μL to 30 μL, preferably 20 μL.

[0014] Preferably, the mobile phase is mobile phase A: ammonium formate solution; mobile phase B: acetonitrile; mobile phase C: water.

[0015] Preferably, the gradient elution program is as follows:

[0016]

[0017] Preferably, the concentration of the ammonium formate solution is 0.1 mol / L to 0.2 mol / L, preferably 0.2 mol / L.

[0018] Preferably, the pH of the ammonium formate solution is 3.0 to 4.0, preferably pH 3.0.

[0019] Preferably, the pH regulator of the ammonium formate solution is formic acid.

[0020] Preferably, the detector used in the high performance liquid chromatography method is an evaporative light scattering detector.

[0021] Preferably, the conditions of the evaporative light scattering detector are: the carrier gas is nitrogen, the carrier gas flow rate is 1.6 L / min to 3.0 L / min, the nebulizer temperature is 40 °C to 60 °C, and the evaporator temperature is 50 °C to 60 °C.

[0022] Preferably, the conditions of the evaporative light scattering detector are as follows: the carrier gas flow rate is 1.6 L / min, the nebulizer temperature is 50 °C, and the evaporator temperature is 55 °C.

[0023] Preferably, the related substances include process impurities and the starting material arginine.

[0024] Compared with the prior art, the present invention provides a method for detecting related substances of dihydroxypropyl arginine hydrochloride, which can effectively separate and detect the main component, starting material and process impurities in dihydroxypropyl arginine hydrochloride, has high sensitivity and good resolution, is simple to operate, has a fast peak emergence time, a short detection time, good reproducibility and stability of the method, strong specificity, meets the requirements of product quality control, has good sensitivity and precision, helps to ensure the safety and effectiveness of the product, and at the same time reduces the production cost, having significant technical advantages and application value. Description of the Drawings

[0025] Figure 1 It is the HPLC chromatogram of the test solution in Example 2 of the present invention, wherein the marked chromatographic peaks are chloride ion, process impurity, main component, and arginine; the process impurity peak is the by-product peak generated during the synthesis process; the main component peak is the dihydroxypropyl arginine peak in dihydroxypropyl arginine hydrochloride (meanwhile, the hydrochloride salt will generate a chloride ion peak, and the present invention does not detect the chloride ion); the arginine peak is the arginine peak of the starting material in the synthesis route.

[0026] Figure 2 It is the HPLC chromatogram of the arginine series reference solution in Example 3 of the present invention. Detailed Embodiments

[0027] For the convenience of understanding the present invention, the present invention will be described more comprehensively below with reference to the relevant drawings. Preferred embodiments of the present invention are shown in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided so that the understanding of the disclosure of the present invention is more thorough and comprehensive.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs. The terms used in the description of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0029] The present invention provides a method for detecting related substances of dihydroxypropyl arginine hydrochloride, which adopts high performance liquid chromatography. The chromatographic column is a reversed-phase, cation exchange and hydrophilic interaction mixed chromatographic column, and gradient elution is carried out using the mobile phase.

[0030] In some embodiments, the chromatographic column is Comixsil RP-100.

[0031] In some embodiments, the inner diameter of the chromatographic column is 2.7 mm to 4.6 mm, the length is 100 mm to 150 mm, and the particle size of the packing is 3 μm to 5 μm.

[0032] In some embodiments, the inner diameter of the chromatographic column is 4.6 mm, the length is 150 mm, and the particle size of the packing is 3 μm.

[0033] In some embodiments, the column temperature of the chromatographic column is 30°C to 40°C.

[0034] In some embodiments, the column temperature of the chromatographic column is 30°C.

[0035] In some embodiments, the flow rate of the chromatographic column is 0.8 mL / min to 1.2 mL / min

[0036] In some embodiments, the flow rate of the chromatographic column is 0.8 mL / min to 1.0 mL / min.

[0037] In some embodiments, the flow rate of the chromatographic column is 1.0 mL / min.

[0038] In some embodiments, the injection volume of the chromatographic column is 10 μL to 30 μL.

[0039] In some embodiments, the injection volume of the chromatographic column is 20 μL.

[0040] In some embodiments, the mobile phase is mobile phase A: ammonium formate solution; mobile phase B: acetonitrile; mobile phase C: water.

[0041] In some embodiments, the gradient elution program is as follows:

[0042]

[0043] In some embodiments, the concentration of the ammonium formate solution is 0.1 mol / L to 0.2 mol / L.

[0044] In some embodiments, the concentration of the ammonium formate solution is 0.2 mol / L.

[0045] In some embodiments, the pH of the ammonium formate solution is 3.0 to 4.0.

[0046] In some embodiments, the pH of the ammonium formate solution is 3.0.

[0047] In some embodiments, the pH regulator of the ammonium formate solution is formic acid.

[0048] In some embodiments, the detector used in the high performance liquid chromatography is an evaporative light scattering detector.

[0049] In some embodiments, the conditions of the evaporative light scattering detector are as follows: the carrier gas is nitrogen, the carrier gas flow rate is 1.6 L / min to 3.0 L / min, the nebulizer temperature is 40 °C to 60 °C, and the evaporator temperature is 50 °C to 60 °C.

[0050] In some embodiments, the conditions of the evaporative light scattering detector are as follows: the carrier gas flow rate is 1.6 L / min, the nebulizer temperature is 50 °C, and the evaporator temperature is 55 °C.

[0051] In some embodiments, the related substances include process impurities and the starting material arginine.

[0052] In some embodiments, the detection method includes the following steps:

[0053] (1) Preparation of a series of reference substance solutions: Accurately weigh the arginine reference substance, dissolve and dilute it with water to obtain a series of reference substance solutions. The specific solution concentration parameters are shown in Table 1;

[0054] Table 1 Concentration parameters of the series of reference substance solutions

[0055] Solution name Concentration (mg / mL) Series reference solution 1 0.1 Series reference solution 2 1.5 Series reference solution 3 2.0

[0056] (2) Preparation of the test sample solution: Accurately weigh the test sample, place it in a 20 mL volumetric flask, and dilute it to the mark with water to obtain a test sample solution with a concentration of 25 mg / mL;

[0057] (3) Use a high performance liquid chromatograph to detect the series of reference substance solutions 1 - 3 to obtain a linear equation;

[0058] (4) Use a high performance liquid chromatograph to detect the test sample solution, and obtain the content of arginine in the test sample solution according to the linear equation and the measured peak area. Except for the chloride ion peak, other impurities are calculated by the area normalization method.

[0059] In some embodiments, the detection method further includes a maintenance step for the chromatographic column. The maintenance step is: after the detection is completed, do not flush the chromatographic column, and directly store the chromatographic column in the mobile phase.

[0060] For the experimental methods without specific conditions noted in the following examples, they are usually in accordance with conventional experimental conditions.

[0061] The instruments, reagents and samples used in the present invention are all conventional products that can be obtained commercially, including but not limited to the instruments, reagents and samples used in the examples of this application, as specifically shown in Table 2 - Table 3.

[0062] Table 2 Instruments and Reagents Used in This Detection Method

[0063] name Model / Specification Manufacturer High performance liquid chromatography 1260 Agilent Evaporative light detector 1260Infinity II Agilent One hundred thousandth electronic balance BCE951-10CN Sartorius Ammonium formate HPLC grade TEDIA Formic acid HPLC grade TEDIA Acetonitrile HPLC grade Adamas water Ultrapure water self made

[0064] Table 3 Samples Used in This Detection Method

[0065] name content(%) Manufacturer hydrochloric acid 37 Adamas Arginine Reference 98 Aladdin Dihydroxypropylarginine hydrochloride 27～33 Suzhou Boyan Pharmaceutical Technology Co., Ltd.

[0066] Example 1: A Detection Method for Related Substances of Dihydroxypropyl Arginine Hydrochloride

[0067] 1. Chromatographic Conditions

[0068] Instrument: High Performance Liquid Chromatograph;

[0069] Chromatographic Column: Comixsil RP - 100 4.6mm×150mm, particle size 3μm;

[0070] Mobile Phase A: 0.2mol / L ammonium formate solution (adjusted to pH 3.0 with formic acid);

[0071] Mobile Phase B: Acetonitrile;

[0072] Mobile Phase C: Water;

[0073] Gradient Elution Program:

[0074]

[0075] Flow Rate: 1.0mL / min;

[0076] Column Temperature: 30°C;

[0077] Detector: Evaporative Light Scattering Detector (reference conditions: nebulizer temperature 50°C, evaporator temperature 55°C, carrier gas is nitrogen, carrier gas flow rate is 1.6L / min);

[0078] Injection Volume: 20μL;

[0079] Solvent: Water.

[0080] 2. Solution Preparation

[0081] (1) Preparation of Hydrochloric Acid Positioning Solution: Accurately measure 1mL of hydrochloric acid and place it in a 10mL volumetric flask, dilute to the mark with water, and shake well to obtain.

[0082] (2) Preparation of arginine positioning solution: Accurately weigh 10 mg of arginine reference substance and place it in a 10 mL volumetric flask. Weigh accurately, dissolve it with water and dilute to the scale, shake well to obtain the solution.

[0083] (3) Preparation of a series of reference substance solutions: Accurately weigh arginine reference substance, dissolve it with water and dilute to obtain a series of reference substance solutions. The specific concentration parameters are shown in Table 4.

[0084] Table 4 Concentration parameters of arginine series reference substance solutions

[0085] Solution name Concentration (μg / mL) Reference solution 1 102.3 Reference solution 2 1535 Reference solution 3 2046

[0086] (4) Preparation of test solution: Accurately weigh about 500 mg of the test substance (dihydroxypropyl arginine hydrochloride) and place it in a 20 mL volumetric flask, dilute to the scale with water, shake well to obtain the solution.

[0087] Example 2: Specificity test

[0088] Accurately measure 20 μL of the positioning solution and the test solution prepared in Example 1, inject them into the liquid chromatograph respectively, and perform detection according to the chromatographic conditions in Example 1. Record the chromatograms respectively to obtain Figure 1 , and the results of the specificity test are shown in Table 5.

[0089] As Figure 1 shown, the process impurity peak, the main component dihydroxypropyl arginine peak, and the arginine peak are completely separated. Among them, the process impurity peak is the by-product peak generated during the synthesis process; the main component peak is the dihydroxypropyl arginine peak in dihydroxypropyl arginine hydrochloride (at the same time, the hydrochloride will generate a chloride ion peak); the arginine peak is the arginine peak of the starting material in the synthesis route.

[0090] Table 5 Results of the specificity test

[0091]

[0092]

[0093] As shown in Table 5, using the chromatographic conditions of the present invention can effectively separate the main component of dihydroxypropyl arginine hydrochloride from the process impurities, and can also ensure that the starting material arginine does not interfere with the detection, meeting the requirements of specificity.

[0094] Example 3: Sensitivity and precision test

[0095] Accurately measure 20 μL of the series of reference substance solutions prepared in Example 1, inject them into the liquid chromatograph respectively, and perform detection according to the chromatographic conditions in Example 1. Record the chromatograms respectively to obtain Figure 2 , and the results of the sensitivity and precision test are shown in Table 6.

[0096] Table 6 Results of Sensitivity and Precision Tests

[0097]

[0098] As shown in Table 6, the present invention can detect arginine residues at a relative concentration of 0.4% of the test sample, and the sensitivity meets the detection requirements for arginine residues in the process, and the precision is good.

[0099] Example 4: Detection of Test Sample Solution

[0100] Precisely measure 20 μL of reference solution 1, reference solution 2, reference solution 3 and test sample solution prepared as in Example 1, and inject them into the liquid chromatograph respectively. Detect according to the chromatographic conditions of Example 1, and record the chromatogram.

[0101] Taking the logarithm of the concentration as the abscissa and the logarithm of the peak area as the ordinate, draw a standard curve. Through linear regression analysis, the results are shown in Table 7.

[0102] Table 7 Standard Curve of Arginine Series Reference Substances

[0103]

[0104] Note: Calculate the arginine content of the starting material.

[0105] Calculation formula:

[0106] Where: A i : Peak area of arginine in the test sample solution;

[0107] b: Intercept of the linear equation of the arginine series reference solution;

[0108] V: Dilution volume of the test sample solution, mL;

[0109] a: Slope of the linear equation of the arginine series reference solution;

[0110] m: Sampling amount of the test sample, μg.

[0111] After calculation and analysis of the test results of the test sample solution, the arginine content of the starting material is 5.35% (external standard method), and the content of process impurities is 4.99% (area normalization method).

[0112] It should be understood that the above embodiments are all exemplary and do not cover all possible implementation manners included in the claims. Without departing from the scope of the present disclosure, various deformations and changes can be made on the basis of the above embodiments. Similarly, any combination of the technical features of the above embodiments can be made to form additional embodiments of the present invention that may not be explicitly described. Therefore, the above embodiments only represent several implementation manners of the present invention and do not limit the protection scope of the present invention patent.

Claims

1. A method for detecting related substances of dihydroxypropyl arginine hydrochloride, characterized in that, The high performance liquid chromatography method is adopted. The chromatographic column is a reverse phase, cation exchange and hydrophilic interaction mixed chromatographic column, and gradient elution is carried out using a mobile phase.

2. The detection method according to claim 1, wherein The chromatographic column is Comixsil RP-100.

3. The detection method according to claim 1, wherein The inner diameter of the chromatographic column is 2.7 mm to 4.6 mm, the length is 100 mm to 150 mm, and the filler particle size is 3 μm to 5 μm; Preferably, the inner diameter of the chromatographic column is 4.6 mm, the length is 150 mm, and the filler particle size is 3 μm.

4. The detection method according to claim 1, characterized in that, The column temperature of the chromatographic column is 30 °C to 40 °C, preferably 30 °C.

5. The detection method according to claim 1, wherein The flow rate of the chromatographic column is 0.8 mL / min to 1.2 mL / min, preferably 0.8 mL / min to 1.0 mL / min, more preferably 1.0 mL / min; Preferably, the injection volume of the chromatographic column is 10 μL to 30 μL, preferably 20 μL.

6. The detection method according to claim 1, wherein The mobile phase is mobile phase A: ammonium formate solution; mobile phase B: acetonitrile; mobile phase C: water; Preferably, the gradient elution program is as follows:

7. The detection method according to claim 6, wherein The concentration of the ammonium formate solution is 0.1 mol / L to 0.2 mol / L, preferably 0.2 mol / L; Preferably, the pH of the ammonium formate solution is 3.0 to 4.0, preferably pH 3.0; Preferably, the pH regulator of the ammonium formate solution is formic acid.

8. The detection method according to claim 1, characterized in that The detector used in the high performance liquid chromatography method is an evaporative light scattering detector.

9. The detection method according to claim 8, wherein The conditions of the evaporative light scattering detector are: the carrier gas is nitrogen, the carrier gas flow rate is 1.6 L / min to 3.0 L / min, the nebulizer temperature is 40 °C to 60 °C, and the evaporator temperature is 50 °C to 60 °C; Preferably, the conditions of the evaporative light scattering detector are: the carrier gas flow rate is 1.6 L / min, the nebulizer temperature is 50 °C, and the evaporator temperature is 55 °C.

10. The detection method according to claim 1, characterized in that, The related substances include process impurities and the starting material arginine.

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