Method for purifying Z-phenylalanyl tryptophan
Through a multi-step extraction and washing purification method combined with high-performance liquid chromatography determination, the problem of by-product residues during Z-phenylalanyl tryptophan synthesis is solved, the product is high purity and safety, simplified operation and improved detection accuracy and stability.
Patent Information
- Application Number
- CN202510221804.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-27
- Publication Date
- 2025-06-17
AI Technical Summary
During the synthesis of Z-phenylalanyl tryptophan, it is difficult to effectively control the residual amount of by-products, which affects the purity of the product.
Using a purification method including multi-step extraction and washing steps, high performance liquid chromatography is performed through gradient elution procedures to ensure the removal of by-products and purification of the product.
The efficient purification of Z-phenylalanyl tryptophan is achieved, ensuring high purity and safety of the product, simplifying operation and improving the accuracy and stability of the detection.
Smart Images
Figure CN120161135A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for detecting the purity of polypeptide amino acids, and particularly to a method for purifying Z-phenylalanyltryptophan. Background Art
[0002] Tryptophan, as a polypeptide amino acid, is an essential amino acid for the human body, which has functions such as regulating mood and promoting sleep; it also has a unique taste and flavor, which can enhance the freshness and flavor of food. By adding tryptophan, the deficiency of amino acids in food can be made up, and the nutritional value of food can be improved; as a food additive, the safety of Z-phenylalanyltryptophan cannot be ignored. Therefore, when used as a food additive, its purity and unknown impurities should be strictly controlled to ensure it is within a safe range; the technical problem of difficult removal of related by-products generated during the production process needs to be solved. And Z-phenylalanyltryptophan is prone to side reactions and some organic impurities in the synthesis process, and currently, the residual amount of by-products cannot be controlled. Summary of the Invention
[0003] The present invention provides a method for purifying Z-phenylalanyltryptophan, which mainly solves the technical problems that the by-products generated in the production affect the purity of the main component of the product, and thus accurately controls the residual amount of by-products.
[0004] The present invention adopts the following technical solutions: A method for purifying Z-phenylalanyltryptophan, comprising the following steps: Step 1, prepare the reagents for preparation: acetonitrile - for preparation, deionized water, chromatographic grade - formic acid.
[0005] Step 2: Treatment of the sample: Weigh a certain amount of the sample and place it in a beaker, add water and stir to dissolve at room temperature. Take an appropriate amount of petroleum ether for extraction. Adjust the pH of the aqueous phase to 5 with a citric acid aqueous solution, then extract with ethyl acetate twice. Wash the extract with water twice and wash once with saturated brine. Rotavapor to dry the organic phase to obtain a dry product; the dry product is then stirred with a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1 for 20 minutes, and finally filtered to obtain a wet product. After drying in a blast oven at 45 °C for 60 minutes, the final finished product is obtained. Take an appropriate amount of the finished product, dissolve it with acetonitrile by ultrasonic wave at room temperature, add deionized water, and finally filter the sample with a polypropylene organic filter membrane for detection.
[0006] Step 3, perform high-performance liquid chromatography determination: The chromatographic conditions are that the sample chamber temperature is room temperature, an automatic injector, and the chromatographic column is: The chromatographic column uses Waters Sun Fire C18, the column temperature: 35 °C, the flow rate: 1.0 mL / min, the mobile phase A is an aqueous solution of 0.05% formic acid in molar concentration; the mobile phase B is a 100% acetonitrile solution in mass percentage concentration, and a gradient elution program is adopted.
[0007] The chromatograph used is an Agilent 1260 liquid chromatograph, which is equipped with a VWD ultraviolet detector.
[0008] The size of the chromatographic column is 4.6 mm × 150 mm, 3.5 μm.
[0009] The gradient elution program is as follows: from 0 to 12 min, the volume ratio of mobile phase B ranges from 35% to 75%; from 12 to 12.1 min, the volume ratio of mobile phase B returns from 75% to the initial ratio of 35%; from 12.1 to 20 min, the volume ratio of mobile phase B ranges from 35% to equilibrate the system.
[0010] The present invention has the following beneficial effects: The present invention provides a method for detecting the purity of Z-phenylalanyltryptophan, which mainly solves the problem that in production, water-soluble polypeptide amino acids utilize the characteristics of petroleum ether, ethyl acetate, etc. that are immiscible with water, and then use the characteristics of saturated brine to wash the by-products and organic impurities generated. That is, in the purification process, saturated brine is used to increase the density of the aqueous phase, which helps to prevent the emulsification phenomenon between water and organic solvents, and at the same time can effectively remove a large amount of water from the organic layer. Through multiple extractions with petroleum ether and ethyl acetate, the by-products and organic impurities are extracted from the aqueous solution. At the same time, due to the volatility of ethyl acetate, it can be recovered pure by distillation after use; then, with the precise quantification of the automatic sampler of the high-performance liquid chromatograph, the VWD ultraviolet detector can have a good response value for Z-phenylalanyltryptophan, so as to efficiently detect the purity of the product, achieving the effects of simple operation, rapidity, accuracy, and good stability. Description of the Drawings
[0011] Figure 1 HPLC chromatogram of product purification. Detailed Embodiments
[0012] Main Instruments: Agilent 1260 high-performance liquid chromatograph, equipped with a VWD ultraviolet detector, Waters Sun Fire C18 (4.6 mm × 150 mm, 3.5 μm), USA; ultrasonic extractor (KQ5200E, Zhengzhou Ketai Experimental Equipment Co., Ltd.), 0.22 μm polypropylene organic filter membrane (Shanghai Anpu Scientific Instruments Co., Ltd.), electronic analytical balance (CPA225D, Sartorius Scientific Instruments (Shanghai) Co., Ltd.) Main Reagents: The reagents for measurement include deionized water; acetonitrile (preparation grade, Titan Chemicals Co., Ltd.), formic acid (chromatographic grade, Shanghai Guoyao Co., Ltd.); deionized water is conventional.
[0013] Pretreatment of the sample: Weigh a certain amount of the test sample and place it in a beaker. Add water and stir to dissolve at room temperature. Take an appropriate amount of petroleum ether for extraction. Adjust the pH of the aqueous phase to 5 with a citric acid aqueous solution, and then extract twice with ethyl acetate. Wash the extraction solution twice with water and once with saturated brine. Rotavapor to dry the organic phase to obtain a dry product. The dry product is then stirred with a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 3:1 for 20 minutes, and finally filtered to obtain a wet product. After drying in a forced-air oven at 45 °C for 60 minutes, the final finished product is obtained. Take an appropriate amount of the finished product, add acetonitrile and dissolve it by ultrasonic wave at room temperature, then add deionized water. Finally, filter the test sample with a polypropylene organic filter membrane for detection.
[0014] Chromatographic conditions: The temperature of the sample chamber is room temperature, the injection volume of the auto-sampler is 10 µL, Waters Sun Fire C18, column temperature: 35 °C, flow rate: 1.0 mL / min, mobile phase A is an aqueous solution of formic acid with a molar concentration of 0.05%; mobile phase B is an acetonitrile solution with a mass percentage concentration of 100%. A gradient elution program is adopted. The gradient elution program is as follows: from 0 to 12 min, the volume ratio of mobile phase B increases from 35% to 75%, from 12 to 12.1 min, the volume ratio of mobile phase B decreases from 75% to the initial ratio of 35%, and from 12.1 to 20 min, the volume ratio of mobile phase B increases from 35% to balance the system.
[0015] Selection and optimization of the sample pretreatment method: Currently, Z-phenylalanyltryptophan on the market is a powdery solid, and the sample determined in this invention is a product produced by Gil Biochemical (Shanghai) Co., Ltd.
[0016] In this invention, the temperature of the sample chamber is set to room temperature, which is beneficial to the preservation of the acetonitrile solvent in the sample chamber. The temperature of the column oven is set to 40 °C in this invention.
[0017] The HPLC chromatogram after product purification is as Figure 1As shown, the Z-phenylalanyltryptophan product was purified by this method. The results showed that after purification of the Z-phenylalanyltryptophan product by this method, the detection results indicated that this method was rapid, accurate, highly sensitive, with a symmetrical main peak shape, baseline separation from multiple by-product impurity peaks, good stability, and had practical application value. Since the product is synthesized into an alkaline sodium salt during production, first, petroleum ether is used for a conventional extraction of organic by-products once, and then the characteristics of weak acid of citric acid are utilized to adjust the product to be in a slightly acidic state and then extracted with a solvent again; the product enters the organic phase, and then the crude product is obtained by distillation. The most crucial thing is the ratio of the final mixed solution of petroleum ether and ethyl acetate. The product is slightly soluble in ethyl acetate in the solid state, while petroleum ether is a non-polar solvent and the product is insoluble in petroleum ether. By using the characteristics of polarity to ensure the yield after purification, in a large number of experiments, it was found that mixing petroleum ether and ethyl acetate in a ratio of 3:1 for pulping to remove impurities was the most effective, which could not only remove impurities well but also ensure the yield of the product.
Claims
1. A method for purifying Z-phenylalanyltryptophan; characterized in that: The following steps are involved: Step 1: Prepare the reagents used: acetonitrile-preparative grade, deionized water, chromatographic grade-formic acid, Step 2, sample processing: weigh the sample and place it in a beaker, add water and stir at room temperature to dissolve, extract with petroleum ether, adjust the pH of the aqueous phase to acidic with citric acid aqueous solution, extract with ethyl acetate, wash the extract with water, wash once with saturated brine, and dry the organic phase with rotary evaporation to obtain a dry product; the dry product is then stirred with a mixture of petroleum ether and ethyl acetate, and finally filtered to obtain a wet product, which is dried in a blast oven to obtain a final product, take an appropriate amount of the finished product, add acetonitrile and dissolve it with ultrasound at room temperature, then add deionized water, and finally filter the sample with a polypropylene organic filter membrane for detection; Step three, perform high performance liquid chromatography determination: the chromatographic conditions are that the sample chamber temperature is room temperature, automatic sample injector, chromatographic column: the chromatographic column uses Waters Sun Fire C18, column temperature: 35 °C, flow rate: 1.0 mL / min, mobile phase A is a molar concentration of 0.05% formic acid aqueous solution; mobile phase B is a mass percentage concentration of 100% acetonitrile solution, and a gradient elution program is used.
2. A method for purifying Z-phenylalanyltryptophan according to claim 1, characterized in that: Step 2: The aqueous phase is adjusted to pH 5 with citric acid aqueous solution.
3. A method for purifying Z-phenylalanyltryptophan according to claim 1, characterized in that: Step 2: The dried product was stirred with a mixture of petroleum ether and ethyl acetate in a volume ratio of 3:1 for 20 minutes.
4. A method for purifying Z-phenylalanyltryptophan according to claim 1, characterized in that: The chromatograph used in the liquid chromatography determination is an Agilent 1260 liquid chromatograph, which uses a VWD ultraviolet detector.
5. A method for purifying Z-phenylalanyltryptophan according to claim 1, characterized in that: The size of the chromatographic column is 4.6 mm × 150 mm, 3.5 μm.
6. A method for purifying Z-phenylalanyltryptophan according to claim 1, characterized in that: The gradient elution program is as follows: 0-12 min, the volume ratio of mobile phase B increases from 35% to 75%, 12-12.1 min, the volume ratio of mobile phase B decreases from 75% to an initial ratio of 35%, and 12.1-20 min, the volume ratio of mobile phase B decreases from 35% to a balanced system.