A gas phase analysis method for N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine residue

Abstract

The invention discloses a gas phase analysis method for N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine residue. The method is carried out by dissolving Cinipride in a solvent, shaking it well, and detecting it under the detection conditions set by a gas chromatograph to obtain a gas chromatogram; then the chromatogram of a series of standard products N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine is measured under the same conditions and a standard curve is drawn; the residual amount of N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine is determined by an external standard method. The method of the present invention is simple to operate and method verification is performed, and N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine residue can be analyzed quickly, efficiently and accurately.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a gas phase analysis method for N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine residues. Background Art

[0002] N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine is a compound with the molecular formula C 12 H 22 N2, the core of this compound is the fusion of a cyclohexene ring and a piperidine moiety connected by a methylene bridge. This complex arrangement gives N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine unique chemical properties. Its versatility is further enhanced by the presence of primary amine groups, which can serve as reaction sites for a variety of transformations. It has potential applications in the fields of medicine, pharmaceuticals, chemicals, or other industries that use chemical compounds. In particular, N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine has the potential for drug research and may have properties that make it a candidate for the development of new drugs and new medicines.

[0003] The chemical name of Cinipride is 4-amino-N-[1-(3-cyclohexen-1-ylmethyl)-4-piperidinyl]-2-ethoxy-5-nitrobenzamide, which is a potent 5-H1 and 5-H2 receptor antagonist used for gastrointestinal motility disorders. N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine is an important intermediate in the synthesis of Cinipride. 4-amino-2-ethoxy-5-nitrobenzoic acid is used as a raw material to condense with N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine, and then tartaric acid is used to form a salt to obtain Cinipride tartrate (IN2008MU00401I3, GB1574419). However, after purification, this route still has trace amounts of intermediates remaining. There is no report on the detection method of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine in the existing technology and domestic and foreign literature. Therefore, how to quickly and effectively determine the residual amount of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine in Cinipride and thus better control the quality of Cinipride is an urgent problem to be solved.

[0004] According to the structural formula of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine, it is a fused polar compound in which the cyclohexene ring and the piperidine part are bridged by a methylene group. It has weak ultraviolet absorption, contains a CH bond, and has a boiling point below 300°C (boiling point: 284°C). It can be separated by gas chromatography using a chromatographic column with a medium polarity or polar stationary liquid and detected using a hydrogen flame ionization detector (FID). However, since the boiling point of Cinipride is higher than 300°C and polar chromatographic columns are generally not resistant to high temperatures, when detecting the residue of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine, only the ultra-high inert version, which is also a medium polarity chromatographic column DB-17ht (column temperature range: 40~365°C), can be selected to ensure that Cinipride can be eluted and no residue will interfere with sample detection. The specific physical and chemical properties of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine are shown in Table 1:

[0005] Table 1 Physicochemical properties of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine

[0006] ; Summary of the invention

[0007] The purpose of the present invention is to solve the deficiencies of the prior art and provide a gas phase analysis method for N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine residues to ensure the drug safety of Cinipride.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] A gas phase analysis method for N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine residues comprises the steps of:

[0010] Dissolve Cinipride in a solvent, shake well, and detect under the detection conditions set by a gas chromatograph to obtain a gas chromatogram; then measure the chromatogram of a series of standard products N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine under the same conditions and draw a standard curve; and determine the residual amount of N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine by an external standard method;

[0011] The detection conditions of the gas chromatogram are as follows: the injection port temperature is 280-300°C, the detector (FID) temperature is 300-350°C; the carrier gas is nitrogen, the column flow rate is 1-3 ml / min, the split ratio is 20:1-100:1, and the injection volume is 0.2-1.0 μL;

[0012] Programmed temperature rise: initial temperature 100~150℃, hold for 0~2min, increase the temperature to 280~300℃ at a rate of 10~20℃ / min, hold for 5~10min.

[0013] In the technical solution of the present invention: the chromatographic column is a capillary column using (50%-phenyl)-methylpolysiloxane as a stationary liquid.

[0014] In some preferred technical solutions: the chromatographic column is DB-17ht, with specification parameters of 30m×0.32mm×0.15μm.

[0015] In the technical solution of the present invention: the sample injection volume is 0.2~1.0μL.

[0016] In the technical solution of the present invention: the sample concentration is 0.5~2.0 mg / ml.

[0017] In the technical solution of the present invention: the solvent is methanol.

[0018] In some specific technical solutions: the injection port temperature is 280~300°C, the detector (FID) temperature is 300~350°C; the carrier gas is nitrogen, the column flow rate is 1~3ml / min, the split ratio is 20:1~100:1, and the injection volume is 0.2~1.0μL;

[0019] Programmed temperature rise: initial temperature 100~150℃, hold for 0~2min, increase the temperature to 280~300℃ at a rate of 10~20℃ / min, hold for 5~10min.

[0020] Compared with the prior art, the advantages and positive effects of the present invention are:

[0021] 1. The analytical method of the present invention can effectively detect the residual amount of N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine in Cinipride. When the signal-to-noise ratio is 3.5, the detection limit is 102.41ng. When the quantitative limit signal-to-noise ratio is 10.2, the quantitative limit is 330.20ng; the results show that the method of the present invention has high sensitivity and can effectively quantitatively detect the residual amount of N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine, effectively ensuring the safety of drugs.

[0022] 2. The present invention is simple to operate, has good repeatability and low error, and provides an effective way for quality control of Cinipride.

[0023] 3. The reagents and consumables required for the method of the present invention are cheap and the operating cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the chromatogram of 0.5 μg / ml N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine reference substance.

[0025] Figure 2 This is the chromatogram of Cinipride solution.

[0026] Figure 3 This is the linear relationship diagram of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine.

[0027] Figure 4 This is a comparison chart of specific properties. DETAILED DESCRIPTION

[0028] The above-mentioned technical features of the present invention and the technical features specifically described below (such as implementation cases) can be combined with each other to form new or preferred technical solutions, but the present invention is not limited to these embodiments, and these embodiments do not limit the present invention in any way.

[0029] The experimental methods in the following examples are conventional methods unless otherwise specified. The preparations involved in the following examples are common commercial products unless otherwise specified and can be purchased from the market.

[0030] The present invention is further described in detail below in conjunction with the drawings and embodiments:

[0031] Instruments and reagents.

[0032] Instruments: TRACE 1600 gas chromatograph (Thermo Fisher Scientific, USA); DB-17ht (30 m × 0.32 mm × 0.15 μm; Agilent Technologies, USA) capillary column; XSR105DU electronic balance (Mettler, Switzerland).

[0033] Reagents: methanol (chromatographic grade); N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine reference substance (content 97.2%, Procurenet). Example 1

[0034] This embodiment discloses a gas phase analysis method for N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine residue. The chromatographic conditions are as follows:

[0035] Gas chromatography conditions:

[0036] Chromatographic column: DB-17ht (30m×0.32mm×0.15μm);

[0037] Inlet temperature: 300°C;

[0038] Detector temperature: 350°C;

[0039] Carrier gas: nitrogen;

[0040] Column flow rate: 2.0ml / min;

[0041] Split ratio: 20:1;

[0042] Program temperature rise: initial temperature 100°C, hold for 2 min, increase to 300°C at a rate of 20°C / min, hold for 10 min;

[0043] Test solution: Take an appropriate amount of Cinipride, weigh accurately, dissolve and dilute with methanol to make a solution containing 1 mg per 1 ml.

[0044] Reference solution: Take an appropriate amount of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine reference, accurately weigh it, dissolve it in methanol and dilute it to a solution containing 0.5 μg per 1 ml.

[0045] 1.0 μL of the above solutions were accurately measured and injected into a gas chromatograph. The chromatogram was recorded and the residual amount of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine in Cinipride was calculated by the peak area using the external standard method. Example 2

[0046] This example discloses a test for investigating the linearity and linear range of the detection method of the present invention. Specifically, the method of Example 1 was used to investigate the linearity and linear range. The results are as follows: Figure 3 shown.

[0047] The results showed that the linear correlation coefficient r of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine was 0.9996 in the range of 0.13 μg / ml to 2.12 μg / ml, and the linear equation was y=8.8234x+0.6932, with good linearity. Example 3

[0048] This example discloses the quantitative limit and detection limit test using the method of Example 1, and the results are shown in Table 2:

[0049] Table 2 Detection limit and quantification limit results

[0050] ;

[0051] According to Table 2, when the signal-to-noise ratio is 3.5, the detection limit in the detection method of the present invention is 102.41 ng. When the signal-to-noise ratio of the quantitative limit is 10.2, the quantitative limit is 330.20 ng; the results show that the method of the present invention has high sensitivity and can effectively quantitatively detect the residual amount of N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine, effectively ensuring the safety of drugs. Example 4

[0052] This example discloses a sample recovery test using the method of the present invention to examine the accuracy of the present invention. Specifically, the sample recovery test was conducted using the method of Example 1. The results are shown in Table 3:

[0053] Table 3 N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine recovery results

[0054] ;

[0055] The results show that the recovery rate of the method of the present invention meets the requirements. Example 5

[0056] This example discloses the specificity of the detection method of the present invention. Specifically, the method of Example 1 was used to investigate the specificity.

[0057] The results showed that the blank solvent (methanol) had no interference, and there were no other impurity peaks in the sample that interfered with the peak of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine. The specificity was good. The specificity comparison chart is shown in Figure 4 . Example 6

[0058] This example discloses the repeatability of the detection method of the present invention. Specifically, the method of Example 1 was used to investigate the specificity; the results are shown in Table 4:

[0059] Table 4 Repeatability test results

[0060] ;

[0061] The results showed that N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine was not detected in any of the 6 samples, and the repeatability was good. Example 7

[0062] This embodiment discloses the durability of the detection method of the present invention. The specific durability test range and results are shown in Table 5.

[0063] Table 5 Durability variation range and results

[0064] ;

[0065] The results showed that when the flow rate, column temperature, injection port temperature and detector temperature changed slightly, the blank solvent (methanol) had no interference, and there was no interference from other impurity peaks near the chromatographic peak of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine. The residues of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine could be effectively detected, and the method of the present invention had good specificity. Example 8

[0066] This example discloses the precision of the detection method of the present invention.

[0067] Take an appropriate amount of N-(3-cyclohexen-1-ylmethyl)-4-aminopiperidine reference substance, accurately weigh it, dissolve it in methanol and dilute it to a solution containing 0.5 μg per 1 ml. Take the above solution and repeat the injection 6 times. The specific precision inspection results are shown in Table 6:

[0068] Table 6 Precision results

[0069] ;

[0070] According to the above test results, it can be known that the method of the present invention can effectively detect the residual amount of N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine in Cinipride, providing better quality assurance for the production of Cinipride and ensuring the safety of drug use.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A gas phase analysis method for N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine residue, characterized in that: Includes steps: Dissolve Cinipride in a solvent, shake well, and detect under the detection conditions set by a gas chromatograph to obtain a gas chromatogram; then measure the chromatogram of a series of standard products N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine under the same conditions and draw a standard curve; and determine the residual amount of N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine by an external standard method; The detection conditions of the gas chromatograph are as follows: the chromatographic column is DB-17ht, with specification parameters of 30m×0.32mm×0.15μm; the injection port temperature is 280~300℃, the detector FID temperature is 300~350℃; the carrier gas is nitrogen, the column flow rate is 1~3ml / min, the split ratio is 20:1~100:1, and the injection volume is 0.2~1.0μL; Programmed temperature rise: initial temperature 100~150℃, hold for 0~2min, increase the temperature to 280~300℃ at a rate of 10~20℃ / min, hold for 5~10min.

2. A gas phase analysis method for N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine residue according to claim 1, characterized in that: The sample concentration is 0.5~2.0mg / ml.

3. A gas phase analysis method for N-(3-cyclohexene-1-ylmethyl)-4-aminopiperidine residue according to claim 1, characterized in that: The solvent is methanol.

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