A gas phase analysis method for detecting side chain residues in pivmecillin hydrochloride

Abstract

The invention belongs to the technical field of drug analysis, and relates to a gas phase analysis method for detecting the residual amount of side chain N-dimethoxymethyl azepane in pivmecillin hydrochloride, pivmecillin hydrochloride is dissolved in a solvent, shaken, and detected under the detection conditions set by a gas chromatograph to obtain a gas chromatogram; then the chromatogram of series standard N-dimethoxymethyl azepane is measured under the same conditions and a standard curve is drawn; the residual amount of side chain (N-dimethoxymethyl azepane) in pivmecillin hydrochloride is determined by external standard method. The inventive method is simple to operate and method verification is performed, and the residual amount of side chain (N-dimethoxymethyl azepane) in pivmecillin hydrochloride can be analyzed quickly, efficiently and accurately.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis and relates to a gas phase analysis method for detecting side chain (N-dimethoxymethylazepane) residues in pivmecillin hydrochloride. Background Art

[0002] Pivimecillin hydrochloride is a prodrug of mecillin. After oral absorption, it is rapidly hydrolyzed into mecillin in the body, binds to penicillin-binding protein-2 (PBP-2), interferes with bacterial cell wall biosynthesis, causes bacterial morphology changes and cell lysis, and thus exerts an antibacterial effect. Pivimecillin is more active against Gram-negative bacteria than Gram-positive bacteria, and has strong activity against Escherichia coli that produces extended-spectrum β-lactamases (ESBLs). The drug has been on the market overseas for decades. It is safe and effective, has a low resistance rate, and has pharmacoeconomic advantages.

[0003] At present, the synthesis process of pivmecillin hydrochloride is mainly prepared from the side chain (N-dimethoxymethylazacycloheptane) and 6-aminopenicillanic acid (6-APA), among which 6-APA is widely used in pharmaceutical intermediates and is the core structure of penicillin. It is obtained from the fermentation broth of Penicillium and is used as the main starting block for the preparation of many semi-synthetic penicillins. There are many reports on the research and detection of 6-APA. However, there is no report on the detection research of the side chain (N-dimethoxymethylazacycloheptane) in domestic and foreign literature. Therefore, how to quickly and effectively determine the residual amount of the side chain (N-dimethoxymethylazacycloheptane) in pivmecillin hydrochloride, and then better control the quality of pivmecillin hydrochloride is an urgent problem to be solved.

[0004] Classical synthetic route of pivmecillin hydrochloride:

[0005]

[0006] According to the side chain chemical name N-dimethoxymethylazepane, according to its structural formula, it has weak ultraviolet absorption, contains CH bonds, is a polar compound, and has a boiling point below 300°C (boiling point: 203.2°C). It can be separated by gas chromatography using a chromatographic column with a medium polarity or polar stationary liquid and detected by a hydrogen flame ionization detector (FID). However, since the boiling point of pivmecillin hydrochloride is higher than 300°C and the polar chromatographic column is not resistant to high temperatures, when detecting the residue of N-dimethoxymethylazepane in pivmecillin hydrochloride, only the ultra-high inert version, which is also a medium polarity chromatographic column DB-624UI (column temperature range: -20~260°C), can be selected to ensure that pivmecillin hydrochloride will not have residual interference in sample detection. The specific physical and chemical properties of N-dimethoxymethylazepane are shown in Table 1.

[0007] Table 1 Physical and chemical properties of N-dimethoxymethylazepane

[0008] . Summary of the invention

[0009] The purpose of the present invention is to solve the deficiencies of the prior art and provide a gas phase analysis method for detecting the residual amount of the side chain (N-dimethoxymethylazepane) in pivmecillin hydrochloride to ensure the safety of the drug.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] A gas chromatography analysis method for detecting the residual amount of side chain N-dimethoxymethylazepane in pivmecillin hydrochloride comprises the steps of:

[0012] Dissolve pivmecillin hydrochloride 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-dimethoxymethylazepane under the same conditions and draw a standard curve; and determine the residual amount of the side chain (N-dimethoxymethylazepane) in pivmecillin hydrochloride by an external standard method;

[0013] The detection conditions of the gas chromatogram are as follows: the injection port temperature is 220-255°C, the detector (FID) temperature is 280-305°C; the carrier gas is nitrogen, the column flow rate is 1-3 ml / min, and the split ratio is 10:1-50:1;

[0014] Programmed temperature rise: initial temperature 80-120°C, hold for 2-10 min, increase the temperature to 220-240°C at a rate of 5-10°C / min, hold for 5-10 min.

[0015] In the technical solution of the present invention, the chromatographic column is a capillary column using 6% cyanopropyl / phenyl-94% polydimethylsiloxane as a stationary liquid.

[0016] In some preferred technical solutions: the chromatographic column is DB-624UI, with specification parameters of 30m×0.32mm×1.8μm.

[0017] In the technical solution of the present invention: the sample injection volume is 0.2-1.0 μL.

[0018] In the technical solution of the present invention: the sample concentration is 0.5-2.0 mg / ml.

[0019] In the technical solution of the present invention: the solvent is methanol.

[0020] In some specific technical schemes: the injection port temperature is 245-255°C, the detector temperature is 295-305°C; the carrier gas is nitrogen, the column flow rate is 1.0-3.0 ml / min, and the split ratio is 20-50:1; the temperature is increased according to the following program: initial temperature 80-105°C, maintained for 2-5 minutes, increased to 240°C at a rate of 5-10°C / min, and maintained for 5-10 minutes.

[0021] Compared with the prior art, the advantages and positive effects of the present invention are:

[0022] 1. The analytical method of the present invention can effectively detect the residual amount of the side chain (N-dimethoxymethylazepane) in pivmecillin hydrochloride.

[0023] 2. The present invention is simple to operate, has good repeatability and low error, and provides an effective approach for the quality control of pivmecillinam hydrochloride.

[0024] 3. The reagents and consumables required for this method are cheap and the operating cost is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the chromatogram of 0.5 μg / ml N-dimethoxymethylazepane reference substance.

[0026] Figure 2 This is the chromatogram of pivmecillin hydrochloride solution.

[0027] Figure 3 This is a comparison chart of specific properties.

[0028] Figure 4 is a linear curve graph of the side chain. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] The present invention is further described in detail below in conjunction with the drawings and embodiments:

[0032] Instruments and reagents

[0033] Instrument: TRACE 1600 gas chromatograph (Thermo Fisher Scientific, USA); DB-624UI

[0034] (30m×0.32mm×1.8μm; Agilent, USA) capillary column; XSR105DU electronic balance (Mettler, Switzerland)

[0035] Reagents: methanol (chromatographic grade); N-dimethoxymethylazepane reference substance (content 99.5%, TRC).

[0036] Example 1

[0037] This embodiment discloses a method for determining the residual amount of the side chain (N-dimethoxymethylazepane) in pivmecillin hydrochloride by gas chromatography. The chromatographic conditions are as follows:

[0038] Gas chromatography conditions:

[0039] Chromatographic column: DB-624UI (30m×0.32mm×1.8μm);

[0040] Inlet temperature: 250°C;

[0041] Detector (FID) temperature: 300°C;

[0042] Carrier gas: nitrogen;

[0043] Column flow rate: 2.0ml / min;

[0044] Split ratio: 10:1;

[0045] Program temperature rise: initial temperature 100°C, hold for 2 min, increase to 240°C at a rate of 10°C / min, hold for 10 min;

[0046] Test solution: Take an appropriate amount of pivmecillin hydrochloride, weigh accurately, dissolve and dilute with methanol to make a solution containing 1 mg per 1 ml.

[0047] Reference solution: Take an appropriate amount of N-dimethoxymethylazepane reference, weigh accurately, dissolve in methanol and dilute to a solution containing 0.5 μg per 1 ml.

[0048] Accurately measure 1.0 μL of the above solution, inject it into the gas chromatograph, and record the chromatogram (such as Figures 1-2 ), and the content of the side chain (N-dimethoxymethylazepane) in pivmecillin hydrochloride was calculated by the peak area according to the external standard method.

[0049] Example 2

[0050] 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.

[0051] like Figure 4The results showed that the linear correlation coefficient r was 0.9999 and the linear equation was A=4.4767C-0.0935 in the range of 0.05μg / ml to 5.02μg / ml of the side chain, indicating good linearity.

[0052] Example 3

[0053] This example discloses the quantitative limit and detection limit test using the method of Example 1. The results are shown in Table 2.

[0054] Table 2 Detection limit and quantification limit results

[0055] project result Detection limit (ng) 212.31 Detection limit to noise ratio 3.4 Limit of quantification (ng) 700.60 Limit of quantitation noise ratio 11.2

[0056] According to Table 2, when the signal-to-noise ratio is 3.4, the detection limit in the detection method of the present invention is 212.31 ng. When the signal-to-noise ratio of the quantitative limit is 11.2, the quantitative limit is 700.60 ng; the results show that the method of the present invention has high sensitivity and can effectively quantitatively detect the residual amount of the side chain in the raw material of pivmecillin hydrochloride, effectively ensuring the safety of the drug.

[0057] Example 4

[0058] 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

[0059] Table 3 Recovery results

[0060]

[0061] The results show that the recovery rate of the method of the present invention meets the requirements.

[0062] Example 5

[0063] 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.

[0064] 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 side chain peaks. The specificity was good. The specificity comparison chart is shown in Figure 3 .

[0065] Example 6

[0066] 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.

[0067] Table 4 Repeatability test results

[0068] project result Repeatability -1 Not detected Repeatability -2 Not detected Repeatability - 3 Not detected Repeatability - 4 Not detected Repeatability -5 Not detected Repeatability - 6 Not detected

[0069] The results showed that no side chains were detected in the 6 samples, and the repeatability was good.

[0070] Example 7

[0071] 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.

[0072] Table 5 Durability variation range and results

[0073] project Range of variation result Column flow 2.0±0.2ml / min There was no interference in the blank, and no interference from other impurity peaks near the side chain chromatographic peak. Initial column temperature 100±5℃ There was no interference in the blank, and no interference from other impurity peaks near the side chain chromatographic peak. Inlet temperature 250±5℃ There was no interference in the blank, and no interference from other impurity peaks near the side chain chromatographic peak. Detector temperature 300±5℃ There was no interference in the blank, and no interference from other impurity peaks near the side chain chromatographic peak.

[0074] 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 side chain chromatographic peak, and the side chains could be effectively detected. The method of the present invention has good specificity.

[0075] Example 8

[0076] This example discloses the precision of the detection method of the present invention.

[0077] Take an appropriate amount of N-dimethoxymethylazepane reference substance, weigh it accurately, dissolve it with 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 investigation results are shown in Table 6.

[0078] Table 6 Precision results

[0079]

[0080] According to the above test results, it can be known that the method of the present invention can effectively detect the residual amount of the side chain (N-dimethoxymethylazepane) in pivmecillin hydrochloride, providing better quality assurance for the production of pivmecillin hydrochloride and ensuring the safety of drug use.

[0081] 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 chromatography analysis method for detecting the residual amount of side chain N-dimethoxymethylazepane in pivmecillin hydrochloride, characterized in that: Includes steps: Dissolve pivmecillin hydrochloride 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-dimethoxymethylazepane under the same conditions and draw a standard curve; and determine the residual amount of the side chain (N-dimethoxymethylazepane) in pivmecillin hydrochloride by an external standard method; The detection conditions of the gas chromatogram are as follows: the injection port temperature is 220-255°C, the detector (FID) temperature is 280-305°C; the carrier gas is nitrogen, the column flow rate is 1-3 ml / min, and the split ratio is 10:1-50:1; Programmed temperature rise: initial temperature 80-120°C, hold for 2-10 min, increase the temperature to 220-240°C at a rate of 5-10°C / min, hold for 5-10 min.

2. The gas chromatography analysis method for detecting the residual amount of side chain N-dimethoxymethylazepane in pivmecillin hydrochloride according to claim 1, characterized in that: The chromatographic column is a capillary column using 6% cyanopropyl / phenyl-94% polydimethylsiloxane as a stationary liquid.

3. The gas chromatography analysis method for detecting the residual amount of side chain N-dimethoxymethylazepane in pivmecillin hydrochloride according to claim 2, characterized in that: The chromatographic column is DB-624UI, with specification parameters of 30m×0.32mm×1.8μm.

4. The gas chromatography analysis method for detecting the residual amount of side chain N-dimethoxymethylazepane in pivmecillin hydrochloride according to claim 1, characterized in that: The sample injection volume is 0.2-1.0 μL.

5. The gas chromatography analysis method for detecting the residual amount of side chain N-dimethoxymethylazepane in pivmecillin hydrochloride according to claim 1, characterized in that: The sample concentration is 0.5-2.0 mg / ml.

6. The gas chromatography analysis method for detecting the residual amount of side chain N-dimethoxymethylazepane in pivmecillin hydrochloride according to claim 1, characterized in that: The solvent is methanol.

7. The gas chromatography analysis method for detecting the residual amount of side chain N-dimethoxymethylazepane in pivmecillin hydrochloride according to claim 1, characterized in that: The injection port temperature is 245-255°C, the detector temperature is 295-305°C; the carrier gas is nitrogen, the column flow rate is 1.0-3.0 ml / min, and the split ratio is 20-50:1; The temperature was raised according to the following procedure: initial temperature 80-105°C, maintained for 2-5 min, then raised to 240°C at a rate of 5-10°C / min, and maintained for 5-10 min.

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