Method for determining amino acid in cordyceps sinensis and small water turtle oral liquid

Abstract

The invention provides a method for determining amino acid in cordyceps sinensis and small water turtle oral liquid. The method comprises the following steps: preparing a mixed reference substance, preparing a test solution, and detecting amino acid by adopting high performance liquid chromatography. By optimizing the sample preparation method and detection conditions, the problem of chromatographic peak pattern forking caused by solvent effect in the process of detecting each amino acid of the cordyceps sinensis and cuora trifasciata oral liquid is solved, the accuracy of medicine detection is improved, the method can be applied to quality control of the cordyceps sinensis and cuora trifasciata oral liquid, and the safety and reliability of clinical medication are ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of medical detection, and in particular relates to a method for determining amino acids in a cordyceps and golden coin tortoise oral liquid. Background Art

[0002] There are many types of amino acids contained in Cordyceps and Golden Tortoise Oral Liquid. In the existing methods for determining the content of amino acids, solvent effects are often produced due to the influence of processes such as derivatization and the solvents used, resulting in bifurcation of the chromatographic peaks of each amino acid during the detection process, thereby affecting the determination of the target peak, reducing the accuracy and sensitivity of the detection, and being unfavorable for the quality control of Cordyceps and Golden Tortoise Oral Liquid. It can be seen that exploring a method for determining amino acids in Cordyceps and Golden Tortoise Oral Liquid to overcome the influence of solvent effects in the detection process of each amino acid is a technical problem that needs to be solved. Summary of the invention

[0003] The present invention aims to provide a method for determining amino acids in Cordyceps sinensis and Golden Turtle oral liquid, and solves the problem of bifurcation of chromatographic peaks in amino acid detection by optimizing sample preparation methods and detection conditions, thereby improving detection accuracy.

[0004] The technical solution of the present invention is a method for determining amino acids in Cordyceps sinensis and Golden Turtle oral liquid, characterized in that the method comprises the following steps: 1. Preparation of mixed reference substances: Weigh appropriate amounts of each amino acid reference substance, place them in 10 ml volumetric flasks, dissolve them in 0.1 mol / L hydrochloric acid solution and dilute to the mark, shake well, and obtain the stock solution of each amino acid reference substance. Take 1 ml of each, place them in the same 50 ml volumetric flask, add 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mol / L triethylamine acetonitrile solution, shake well, leave at room temperature for 1 h, add 50% acetonitrile to the mark, shake well. Take 50 ml, add 50 ml of n-hexane, shake, leave for 10 min, take the lower layer solution, filter, and dilute the lower layer solution to obtain the mixed reference substance solution.

[0005] 2. Preparation of test solution: Accurately pipette 5.0 ml of Cordyceps Golden Turtle Oral Liquid into a 25 ml volumetric flask, dilute with 0.1 mol / L hydrochloric acid solution and dilute to the mark, shake well. Accurately pipette 2.0 ml of the above solution into a headspace injection bottle, add 10 ml of 6 mol / L hydrochloric acid solution, press the bottle cap, hydrolyze in a 120 ℃ electric blast drying oven for 6 hours, cool, transfer to a glass evaporating dish, wash the headspace injection bottle with 15 ml of water several times, combine the washing liquid into the glass evaporating dish and place it in a water bath to evaporate until almost dry, dissolve with 0.1 mol / L hydrochloric acid and transfer to a 25 ml volumetric flask, dilute to the mark, shake well to obtain the test stock solution. Take 10 ml of the test sample stock solution, place it in a 25 ml volumetric flask, add 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mol / L triethylamine acetonitrile solution respectively, leave it at room temperature for 1 hour, add 50% acetonitrile-water solution to dilute and make up to the mark, shake well. Take 10 ml to a separatory funnel, add 10 ml of n-hexane, shake, leave it for 10 min, take the lower layer solution, and the lower layer solution is diluted to obtain the test sample solution.

[0006] 3. High performance liquid chromatography conditions: chromatographic column: Welch Ultimate Amino Acid (4.6×250mm, 5 μm); flow rate: 1.0 ml / min; column temperature: 40°C; detection wavelength: 254 nm; injection volume: 10 μl; gradient elution program: (ratios are by volume):

[0007] Preferably, the addition of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mol / L triethylamine acetonitrile solution in step 1 and step 2 of the above method are both in equal volume ratios.

[0008] Preferably, the lower layer solutions in step 1 and step 2 of the above method are diluted 5 times with a 2% acetonitrile-water solution.

[0009] The beneficial technical effects of the present invention are: solving the problem of chromatographic peak shape bifurcation occurring during the detection of amino acids in the Cordyceps and Golden Turtle Oral Liquid, overcoming the influence of the solvent effect, ensuring the separation and determination of 16 kinds of amino acids in the Cordyceps and Golden Turtle Oral Liquid, improving the accuracy of the detection, and can be used for the quality control of the Cordyceps and Golden Turtle Oral Liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a comparison chart of different injection volumes of the mixed reference substance before dilution.

[0011] Figure 2 This is a comparison chart of different injection volumes of the mixed reference substance after being diluted 5 times with 50% acetonitrile-water solution.

[0012] Figure 3 For the mixed reference substance, a 2% acetonitrile-water solution was diluted 5 times and compared with the undiluted solution. Implementation

[0013] The following is further described with reference to examples, but the present invention is not limited to these examples.

[0014] Example: A mixed reference solution was prepared for testing, and the factors affecting the solvent effect were studied.

[0015] 1. Preparation of mixed reference substances: Take appropriate amount of each amino acid reference substance, place them in 10 ml volumetric flasks, add 0.1 mol / L hydrochloric acid solution to dissolve and dilute to the mark, shake well to obtain the stock solution of each amino acid reference substance. Take 1 ml of each, place it in the same 50 ml volumetric flask, add 8 ml of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 8 ml of 1 mol / L triethylamine acetonitrile solution, shake well, leave at room temperature for 1 h, add 50% acetonitrile-water solution to the mark, shake well. Take 50 ml, add 50 ml of n-hexane, shake, leave for 10 min, take the lower layer solution, filter, and dilute the lower layer solution to obtain the mixed reference substance solution. The sample weight and concentration of each amino acid reference substance are shown in Table 1.

[0016]

[0017] 2. HPLC detection conditions: Chromatographic column: Welch Ultimate Amino Acid (4.6×250 mm, 5μm), SN: 60190901240; flow rate 1.0 ml / min; column temperature 40 ℃; detection wavelength 254 nm; injection volume 10 μl; elution program see Table 2.

[0018]

[0019] 3. Study on factors affecting solvent effect: (1) Comparison of different injection volumes of mixed reference substance before dilution Depend on Figure 1 It can be seen that the smaller the injection volume, the more the bifurcation peak disappears. It is speculated that: ① the concentration of amino acids has an effect on the bifurcation peak; ② the generation of solvent effect has an effect on the bifurcation peak. The next step is to dilute the mixed reference substance 5 times and then use different injection volumes to determine the influencing factors.

[0020] (2) Comparison of different injection volumes of mixed reference substance after dilution 5 times with 50% acetonitrile-water solution Depend on Figure 2It can be seen that the mixed reference substance diluted with 50% acetonitrile-water solution still has bifurcation peaks. It is also found that the bifurcation peak gradually disappears only as the injection volume decreases. It is confirmed that the existence of the bifurcation peak is caused by the generation of solvent effect. The next step is to use 2% acetonitrile-water solution to dilute and compare the mixed reference substance with undiluted state to eliminate the solvent effect.

[0021] (3) Comparison of mixed reference sample diluted 5 times with 2% acetonitrile-water solution and undiluted solution Depend on Figure 3 It can be seen that after the sample was diluted 5 times with 2% acetonitrile-water solution (injection volume 10 μl), the bifurcation of the chromatographic peaks of each amino acid disappeared. By studying the peak shape of the chromatographic peaks after injection of different injection volumes, it was found that the solvent effect of the chromatographic peaks was eliminated. Therefore, it was determined that in the preparation step of the mixed reference substance, the lower layer solution obtained after adding n-hexane and shaking should be diluted 5 times with 2% acetonitrile-water solution, thereby solving the problem of peak bifurcation, overcoming the influence of the solvent effect, and improving the accuracy of the detection.

[0022] Although the present invention has been described in detail above by general description, specific embodiments and tests, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, these modifications or improvements made on the basis of not departing from the spirit of the present invention belong to the scope of protection claimed by the present invention.

Claims

1. A method for determining amino acids in Cordyceps sinensis and Golden Turtle oral liquid, characterized in that: The following steps are involved: (1) Preparation of mixed reference substances: Weigh an appropriate amount of each amino acid reference substance, place them in a 10 ml volumetric flask, dissolve them in 0.1 mol / L hydrochloric acid solution and dilute to the mark, shake well, and obtain the stock solution of each amino acid reference substance. Take 1 ml of each, place them in the same 50 ml volumetric flask, add 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mol / L triethylamine acetonitrile solution, shake well, leave at room temperature for 1 h, add 50% acetonitrile to the mark, and shake well. Take 50 ml, add 50 ml of n-hexane, shake, leave for 10 min, take the lower layer of solution, filter, and dilute the lower layer of solution to obtain the mixed reference substance solution; (2) Preparation of test solution: Accurately pipette 5.0 ml of Cordyceps sinensis oral solution into a 25 ml volumetric flask, dilute with 0.1 mol / L hydrochloric acid solution and dilute to the mark, shake well. Accurately pipette 2.0 ml of the above solution into a headspace injection bottle, add 10 ml of 6 mol / L hydrochloric acid solution, tighten the bottle cap, hydrolyze in a 120 ℃ electric blast drying oven for 6 h, cool, transfer to a glass evaporating dish, wash the headspace injection bottle with 15 ml of water several times, combine the washing liquid into the glass evaporating dish and place it in a water bath to evaporate until almost dry, dissolve with 0.1 mol / L hydrochloric acid and transfer to a 25 ml volumetric flask, dilute to the mark, shake well to obtain the test stock solution; Take 10 ml of the test sample stock solution, place it in a 25 ml volumetric flask, add 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mol / L triethylamine acetonitrile solution respectively, leave it at room temperature for 1 h, add 50% acetonitrile-water solution to dilute and make up to the mark, shake well. Take 10 ml to a separatory funnel, add 10 ml of n-hexane, shake, leave it for 10 min, take the lower layer solution, and the lower layer solution is diluted to obtain the test sample solution; (3) HPLC conditions: Welch Ultimate Amino Acid column (4.6×250 mm, 5 μm); flow rate 1.0 ml / min; column temperature 40 °C; detection wavelength 254 nm; injection volume 10 μl; gradient elution program as follows (ratios are by volume):

2. The method according to claim 1, characterized in that The addition of 0.1 mol / L phenyl isothiocyanate acetonitrile solution and 1 mol / L triethylamine acetonitrile solution in step (1) and step (2) are both in equal volume ratios.

3. The method according to claim 1, characterized in that The lower layer solutions in step (1) and step (2) are both diluted 5 times with a 2% acetonitrile-water solution.

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