A method for detecting spermidine purity and related substances

Abstract

The invention discloses a method for detecting the purity of spermidine and its related substances, and the method is a gas chromatography method. The method selects a gas chromatography column suitable for volatile amine compounds, and realizes the detection of the purity of spermidine and its related substances by setting parameters such as a gradient column temperature program and a column flow rate, effectively separates spermidine from its specific impurities, and then accurately detects its purity and related substances. The method has good sensitivity, high accuracy, and is fast, simple, efficient, and practical.

Description

Technical field:

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for detecting the purity of spermidine and its related substances by gas chromatography. Background technology:

[0002] Spermidine is a polyamine widely found in organisms. Spermidine can inhibit neuronal synthases, bind and precipitate DNA; it can also be used to purify DNA-binding proteins and stimulate the activity of T4 polynucleotide kinase. Spermidine was first discovered by separating it from semen, but in fact it is a substance widely found in organisms. Whole-wheat foods, kelp, mushrooms, beans and whole grains are rich in this ingredient.

[0003] On September 1, 2013, German and Austrian scientists conducted a collaborative study and found that spermidine may prevent the onset of Alzheimer's disease. In 2016, Japanese scientist Yoshinori Ohsumi won the Nobel Prize in Physiology and Medicine for his elucidation of the mechanism of cell autophagy. He believes that human cells will gradually age with age, and some protein structures will become dysfunctional and become garbage. If they are not cleaned up in time, it will not only accelerate aging, but also bring about a series of aging diseases. Spermidine can maintain gene stability by enhancing cell autophagy.

[0004] Spermidine also has multiple biological activities such as improving memory, protecting cardiovascular system, regulating nerves, etc. It has good application potential in the prevention and treatment of cardiovascular diseases and Alzheimer's disease, and has potential application value in the fields of food additives, health products and medicine.

[0005] Spermidine is an amine compound without UV absorption. It is easy to produce peak tailing phenomenon with conventional gas chromatographic columns, which makes it difficult to achieve effective separation between it and impurities (see Table 1 for information on spermidine and specific impurities). The purity and related substance detection of amine compounds without UV absorption has always been a difficult point in industry quality control. The detection method of spermidine purity and related substances has not been reported so far. Therefore, providing an efficient and simple detection method for spermidine purity and related substances is a technical problem that needs to be solved in this field.

[0006] Table 1 Basic information of spermidine and its specific impurities

[0007]

[0008] Therefore, the present invention aims to provide a method for quickly determining the purity of spermidine and its related substances. Summary of the invention:

[0009] The object of the present invention is to provide a method for quickly determining the purity of spermidine and its related substances which is simple to operate and easy to use in view of the deficiencies in the prior art.

[0010] The technical scheme adopted by the present invention is as follows: gas chromatography is selected, and the chromatographic conditions are: a special chromatographic column suitable for amine compounds is used, the injection port temperature is 150-180°C, and the column temperature adopts a gradient column temperature program.

[0011] Furthermore, the chromatographic column suitable for amine compounds is a chromatographic column coated with a non-polar stationary phase, which is treated with a multifunctional deactivation technology and has high inertness, and is preferably an Agilent CP-Volamine column.

[0012] Furthermore, the specification of the chromatographic column is 30m*0.32mm.

[0013] Furthermore, the starting temperature of the gradient column temperature program is 40°C, maintained for 8 minutes, then increased to 255°C at 40°C / min, and maintained for 22 to 60 minutes.

[0014] Furthermore, the sample diluent is an alcohol solvent, preferably methanol.

[0015] Furthermore, the concentration of the test solution is 4-20 mg / mL, the injection volume is 5 uL, and the preferred test solution concentration is 4 mg / mL.

[0016] Further, the split ratio was determined to be 10:1.

[0017] Furthermore, the carrier gas is nitrogen or helium, preferably nitrogen.

[0018] Furthermore, the column flow rate is 0.7 to 1.2 mL / min, preferably 1.0 mL / min.

[0019] Furthermore, the injection port temperature is 150-180°C, preferably 150°C.

[0020] Furthermore, the air flow rate is 380-420 mL / min, preferably 420 mL / min.

[0021] Furthermore, the hydrogen fuel gas flow rate is 38-42 mL / min, preferably 40 mL / min.

[0022] Furthermore, the detector selected is a FID detector.

[0023] Furthermore, the detector temperature is 230-250°C, preferably 230°C.

[0024] The beneficial effect of the present invention is that the present invention realizes the detection of the purity and related substances of spermidine by means of gas chromatography, selecting a special chromatographic column suitable for amine compounds, and setting a gradient column temperature program and a column flow rate. The method has good sensitivity, high accuracy, and is fast, simple, efficient, and practical. Description of the drawings:

[0025] Figure 1 Separation degree solution gas chromatogram of Example 1

[0026] Figure 2 Gas chromatogram of the test solution of Example 1

[0027] Figure 3 Gas chromatogram of Example 2

[0028] Figure 4 Gas chromatogram of Example 3

[0029] Figure 5 Gas chromatogram of Example 4

[0030] Figure 6 Gas chromatogram of Example 4 Specific implementation method:

[0031] The technical content of the present invention is further described below in conjunction with specific embodiments, the purpose of which is to provide a better understanding of the content of the present invention, but the protection scope of the present invention is not limited thereto.

[0032] Example 1 Gas chromatography to determine the purity of spermidine and related substances

[0033] This embodiment is a method for determining the purity of spermidine test sample and its related substances of the present invention. The specific implementation method is as follows:

[0034] 1. Chromatographic conditions

[0035] Chromatographic column: Agilent CP-Volamine, 30m*0.32mm;

[0036] Inlet temperature: 150°C;

[0037] Split ratio: 10:1;

[0038] Carrier gas: nitrogen;

[0039] Column flow rate: 1 mL / min;

[0040] Gradient column temperature program: start at 40°C, hold for 8 min, then increase to 255°C at 40°C / min, hold for 22 min;

[0041] Detector temperature: 230°C;

[0042] Air flow rate: 400mL / min;

[0043] Hydrogen gas flow rate: 40mL / min.

[0044] 2. Experimental steps

[0045] Weigh about 20 mg of the impurity N-2-cyanoethylbutyl-1,4-diamine reference substance into a 50 mL volumetric flask, dissolve and dilute to the scale with methanol to use as the impurity stock solution; weigh about 100 mg of the spermidine reference substance into a 25 mL volumetric flask, dissolve and dilute with methanol, then take 250 uL of the impurity stock solution into a volumetric flask, dissolve and dilute to the scale with methanol, shake well, and use as the separation solution (containing 0.1% of the specific impurity N-2-cyanoethylbutyl-1,4-diamine); weigh about 100 mg of the spermidine test sample into a 25 mL volumetric flask, dissolve and dilute to the scale with methanol, shake well, and use as the test sample detection solution; the methanol used for dissolution and dilution is used as the blank solution.

[0046] Take the blank solution, separation solution and test solution and inject them according to the above chromatographic conditions, and record the chromatogram.

[0047] Separation solution gas chromatogram (see Figure 1 ), where the peak with a retention time of about 18.15min is the peak of spermidine, and the peak shape is well symmetrical. The peak with a retention time of about 19.04min is the peak of the impurity N-2-cyanoethylbutyl-1,4-diamine (the relative retention time relative to spermidine is 1.05). The separation between spermidine and the specific impurity peak is 8.0, which fully meets the requirements of baseline separation (baseline separation requires a separation of ≥1.5), and the signal-to-noise ratio of the impurity N-2-cyanoethylbutyl-1,4-diamine peak is 29.6, which meets the signal-to-noise ratio requirements for quantitative detection (quantitative detection requires a signal-to-noise ratio of ≥10), indicating that this method can achieve the sensitivity requirements for 0.1% specific impurity detection. In the gas chromatogram of the test solution (see Figure 2 ), the specific impurity N-2-cyanoethylbutyl-1,4-diamine was not detected, and the maximum single impurity with other unknown structures at a retention time of 16.209 min was 0.16%, with a purity of 99.55%. Baseline separation was achieved between all single impurities and between single impurities and the main peak. This method can achieve the detection of spermidine test sample purity and related substances.

[0048] Example 2 Gas chromatography to determine the purity and related substances of spermidine

[0049] This example investigates the effect of using different types of chromatographic columns on the separation of the method. The same conditions and methods as in Example 1 are used, except that the chromatographic column is replaced with a conventional column Agilent HP-5 (specification

[0050] 30m*0.25mm*0.1um), injection separation solution, spermidine peak symmetry is not good, there is a certain degree of tailing, and can not achieve the separation of spermidine and specific impurity N-2-cyanoethylbutyl-1,4-diamine, the spectrum is shown in Figure 3 .

[0051] Example 3 Gas chromatography to determine the purity of spermidine and related substances

[0052] This example investigates the effect of column flow rate variation on the durability of the method. Using the same conditions and methods as in Example 1, the separation solution was injected, and only the flow rate was changed to 1.2 mL / min. The separation between the main peak and the specific impurity peak was greater than 1.5, which met the requirements for baseline separation. The analysis results are shown in Figure 4 .

[0053] Example 4 Gas Chromatography Determination of Purity and Related Substances of Spermidine

[0054] This example investigates the effect of injection port temperature variation on the durability of the method. A batch of test solutions of the test products are injected using the same conditions and methods as in Example 1. The injection port temperature is raised to 180°C for injection of the test solutions of the test products. Under the two injection port temperature conditions, the deviation of the impurity results is within the specified allowable deviation requirements. The analysis results are shown in Table 2 below. The gas chromatograms at injection port temperatures of 150°C and 180°C are shown in Table 2 below. Figure 5 and Figure 6 .

[0055] Table 2 Analysis of test results at different injection port temperatures

[0056]

Claims

1. A method for detecting the purity of spermidine and its related substances, characterized in that: The detection method is gas chromatography, and the chromatographic conditions are: select a gas chromatography column suitable for amine compounds: Agilent CP-Volamine, the injection port temperature is 150-180°C, the column starting temperature is 40°C, maintained for 8 minutes, then increased to 255°C at 40°C / min, maintained for 22-60 minutes, and an FID detector is selected with a detector temperature of 230°C. The related substance is N-2-cyanoethylbutyl-1,4-diamine.

2. The method according to claim 1, characterized in that The split ratio is 10:

1.

3. The method according to claim 1, characterized in that The carrier gas is nitrogen or helium.

4. The method according to claim 1, characterized in that The column flow rate is 0.7-1.2 mL / min.

5. The method according to claim 1, characterized in that The air flow rate is 380-420 mL / min.

6. The method according to claim 1, characterized in that The hydrogen gas flow rate is 38-42 mL / min.

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