A method for detecting the purity of spermidine hydrochloride and its related substances

Abstract

The invention discloses a method for detecting the purity of spermidine hydrochloride and its related substances, and the method is a gas chromatography method. The method firstly releases high-boiling-point spermidine hydrochloride with alkali to obtain spermidine, and then uses gas chromatography to detect the purity and related substances of relatively low-boiling-point spermidine, and determines the purity and related substances of spermidine hydrochloride by an indirect method. The method uses a gas chromatography column suitable for amine compounds, and realizes the detection of the purity of spermidine hydrochloride and its related substances by setting parameters such as 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

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 hydrochloride and related substances thereof 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] At present, domestic spermidine is difficult to store and transport due to problems such as low melting point, and is generally prepared in the form of spermidine trihydrochloride. Spermidine hydrochloride is an amine hydrochloride compound with a high boiling point and no ultraviolet absorption. The purity and related substance detection of amine hydrochloride compounds without ultraviolet absorption have always been a difficult point in industry quality control, and the detection method of spermidine hydrochloride purity and related substances has not been reported so far. Therefore, providing an efficient and simple detection method for spermidine hydrochloride purity and its related substances (spermidine hydrochloride and specific impurity information are shown in Table 1) is a technical problem that is urgently needed to be solved in this field.

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

[0007]

[0008] Therefore, the present invention aims to provide a method for quickly determining the purity of spermidine hydrochloride 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 hydrochloride 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: firstly, spermidine hydrochloride is freed with alkali, the method firstly frees high-boiling-point spermidine hydrochloride with alkali to obtain spermidine, then uses gas chromatography to detect the purity and related substances of relatively low-boiling-point spermidine, and then uses an indirect method to determine the purity and related substances of spermidine hydrochloride. Gas chromatography is selected, a special chromatographic column suitable for amine compounds is used, the injection port temperature is 150-180°C, and the column temperature uses a gradient column temperature program.

[0011] Furthermore, the base is an organic base, preferably triethylamine.

[0012] 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.

[0013] Furthermore, the specification of the chromatographic column is 30m*0.32mm.

[0014] 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, maintained for 22 to 60 minutes, preferably maintained for 22 minutes.

[0015] Furthermore, the sample diluent is an alcohol solvent, preferably methanol.

[0016] Furthermore, the concentration of the test solution is 7-35 mg / mL, the injection volume is 1-5 uL, and the preferred test solution concentration is 7 mg / mL.

[0017] Further, the split ratio was determined to be 10:1.

[0018] Furthermore, the carrier gas is nitrogen or helium, preferably nitrogen.

[0019] Furthermore, the column flow rate is 0.7 to 1.2 mL / min, preferably 1.0 mL / min.

[0020] Furthermore, the injection port temperature is 150-180°C, preferably 150°C.

[0021] Furthermore, the air flow rate is 380-420 mL / min, preferably 420 mL / min.

[0022] Furthermore, the hydrogen fuel gas flow rate is 38-42 mL / min, preferably 40 mL / min.

[0023] Furthermore, the detector selected is a FID detector.

[0024] Furthermore, the detector temperature is 230-250°C, preferably 230°C.

[0025] The invention has the beneficial effects that, in the invention, spermidine hydrochloride with a high boiling point is first freed with an alkali to obtain spermidine, and then the purity and related substances of spermidine with a relatively low boiling point are detected by gas chromatography, and the purity and related substances of spermidine hydrochloride are determined by an indirect method. The method uses a special gas chromatography column suitable for amine compounds, and realizes the detection of the purity of spermidine hydrochloride and its related substances by setting parameters such as 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:

[0026] Figure 1 Separation degree solution gas chromatogram of Example 1

[0027] Figure 2 Gas chromatogram of the test solution of Example 1

[0028] Figure 3 Gas chromatogram of Example 2

[0029] Figure 4 Gas chromatogram of Example 3

[0030] Figure 5 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 Determination of Purity and Related Substances of Spermidine Hydrochloride

[0033] This embodiment is a method for determining the purity of spermidine hydrochloride test sample and its related substances of the present invention.

[0034] The specific implementation is as follows:

[0035] 1. Chromatographic conditions

[0036] Chromatographic column: Agilent CP-Volamine, 30m*0.32mm;

[0037] Inlet temperature: 150°C;

[0038] Split ratio: 10:1;

[0039] Carrier gas: nitrogen;

[0040] Column flow rate: 1 mL / min;

[0041] 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;

[0042] Detector temperature: 230°C;

[0043] Air flow rate: 400mL / min;

[0044] Hydrogen gas flow rate: 40mL / min.

[0045] 2. Experimental steps

[0046] Weigh about 35 mg of the impurity N-2-cyanoethylbutyl-1,4-diamine reference substance into a 50 mL volumetric flask, dissolve it with methanol and dilute to the mark as the impurity stock solution; weigh about 35 mg of spermidine hydrochloride reference substance into a 5 mL volumetric flask, dissolve it with 1.5 mL of water, add 0.3 mL of triethylamine and mix well, add 1 mL of water, then take 50 uL of the impurity stock solution to a volumetric flask, dilute to the mark with methanol, shake well, and use it as the separation solution (containing 0.1% of the specific impurity N-2-cyanoethylbutyl-1,4-diamine); prepare the blank solution and the test solution for the test sample in the same way (no impurities and samples are added when the blank solution is prepared; the test solution is prepared with the test sample without adding impurities).

[0047] Take the blank solution, separation solution and test solution and inject them according to the above chromatographic conditions, and record the chromatogram.

[0048] Separation degree solution gas chromatogram (see Figure 1 ), where the peak with a retention time of about 18.15min is the peak of free spermidine, and the peak shape is well symmetrical. The peak with a retention time of about 19.03min 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 the main peak and the specific impurity peak is 7.6, 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 30.0, 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 0.28%, the largest single impurity with unknown structure at 19.476min was 0.26%, the purity was 99.27%, and baseline separation was achieved between all single impurities and between single impurities and spermidine peak. This method can realize the detection of spermidine hydrochloride test sample purity and related substances.

[0049] Example 2 Gas Chromatography Determination of Purity and Related Substances of Spermidine Hydrochloride

[0050] This example investigates the effect of different types of free alkali (inorganic alkali) on the method. The same conditions and methods as in Example 1 were used for detection, except that triethylamine was replaced with 80 mg / mL sodium hydroxide solution. Since inorganic sodium chloride salts that are difficult to vaporize are generated during the process, it is easy to cause contamination of the chromatographic column port, and the contaminated baseline blank (see Figure 3 ) interferes with the detection of impurities in the sample, making it impossible to accurately determine the purity of this product and its related substances.

[0051] Example 3 Gas Chromatography Determination of Purity and Related Substances of Spermidine Hydrochloride

[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 Hydrochloride

[0054] This example investigates the effect of injection port temperature variation on the durability of the method. The same conditions and methods as in Example 1 are used, only the injection port temperature is increased to 180°C, and the batch of test solutions in Example 1 are injected. 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 chromatogram obtained at an injection port temperature of 150°C is shown in Figure 2 The gas chromatogram obtained at the injection temperature of 180°C is shown in Figure 5 .

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

[0056]

Claims

1. A method for detecting the purity of spermidine hydrochloride and its related substances, characterized in that: The method firstly liberates spermidine hydrochloride into spermidine using triethylamine, and then uses gas chromatography to detect the purity of spermidine. The chromatographic conditions are: using a gas chromatography column suitable for amine compounds, the injection port temperature is 150-180°C, the column temperature is 40°C at the start, maintained for 8 minutes, and then increased to 255°C at 40°C / min, maintained for 22-60 minutes. The gas chromatography column for amine compounds is a chromatographic column coated with a non-polar stationary phase. The detection uses an FID detector for detection, and the related substance is N-2-cyanoethylbutyl-1,4-diamine.

2. The method according to claim 1, characterized in that The concentration of the test solution is 7-35 mg / mL, and the injection volume is 1-5 uL.

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

1.

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

5. The method according to claim 1, characterized in that The flow rate is 0.7~1.2mL / min.

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

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

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