Ingredient detection method of injection for preventing or treating vitamin or calcium deficiency of dairy cow

Abstract

The invention belongs to the technical field of gas chromatography detection, and relates to a gas chromatography method for simultaneously determining the content of ethanol and isopropyl myristate. The set conditions of the gas chromatography are as follows: the temperature of an FID detector and a polyethylene glycol 20M capillary column is 240-260 DEG C, the temperature of the detector is 240-260 DEG C, the carrier gas is nitrogen, the flow rate is 0.9-2.5 mL / min, the sample injection amount is 1 mu L, the column temperature is increased by adopting a program, the initial temperature is 80-90 DEG C, the temperature is increased to 180-190 DEG C at the rate of 9-12 DEG C / min, and the temperature is maintained for at least 10 minutes. The method provided by the invention can realize simultaneous detection of ethanol and isopropyl myristate, after the two substances are dissolved by the N, N-dimethylformamide solvent, the detected spectrogram does not have the phenomena of peak overlapping, trailing and the like, and the method has the advantages of high separation efficiency, high selectivity, high sensitivity, high analysis speed and wide application range.

Description

Technical Field

[0001] This invention belongs to the field of gas chromatography detection technology, and relates to a method for detecting the components of an injection for the prevention or treatment of vitamin or calcium deficiency in dairy cows, and particularly to a gas chromatography method for determining the content of ethanol and isopropyl myristate in vitamin ADE injection compared with other drugs. Background Technology

[0002] Ethanol and isopropyl myristate are commonly used solvents or co-solvents in injection solutions and are frequently used in combination in injection formulations. The 2020 edition of the Chinese Pharmacopoeia records that these two substances can be detected by gas chromatography, but the detection conditions for the two substances are different. In actual testing, the two substances need to be detected separately, which is a cumbersome process. Therefore, developing a gas chromatography method that can simultaneously detect ethanol and isopropyl myristate would be beneficial for the quality control of injection solutions and would save testing time and labor costs. However, due to the significant differences in boiling points and polarities between ethanol and isopropyl myristate, the solvents and chromatographic columns used for the separate detection of the two substances are not feasible. Currently, no method for the simultaneous detection of both has been reported. Summary of the Invention

[0003] This invention addresses the problems existing in the simultaneous detection of ethanol and isopropyl myristate by proposing a novel method for detecting the components of an injection for the prevention or treatment of vitamin or calcium deficiency in dairy cows.

[0004] To achieve the above objectives, the present invention is implemented using the following technical solution: A method for detecting the components of an injectable solution for the prevention or treatment of vitamin or calcium deficiency in dairy cows involves weighing ethanol and nutmeg standards, diluting them separately with organic solvents to obtain a series of ethanol and nutmeg solutions of known concentrations, performing gas chromatography analysis, and obtaining chromatograms of the ethanol and nutmeg standards at various concentrations. An ethanol standard curve is obtained by fitting the ethanol concentration as the x-axis and the peak area as the y-axis; a nutmeg standard curve is obtained by fitting the nutmeg concentration as the x-axis and the peak area as the y-axis. The sample to be tested is diluted with an organic solvent and then subjected to gas chromatography... The gas chromatography was performed under the specified detection conditions to obtain a gas chromatogram. The chromatogram was then quantitatively analyzed. The gas chromatography settings were as follows: FID detector, polyethylene glycol 20M capillary column, injection port temperature 240-260℃, detector temperature 240-260℃, carrier gas nitrogen, flow rate 0.9-2.5 mL / min, injection volume 1 μL, and programmed column temperature ramping: initial temperature 80-90℃, increased to 180-190℃ at a rate of 9-12℃ / min, and maintained for at least 10 min.

[0005] As a preferred option, the gas chromatography settings are as follows: polyethylene glycol 20M capillary column, length 30m, film thickness 0.25μm, injection port temperature 250℃, detector temperature 250℃, carrier gas nitrogen, flow rate 1.8mL / min, injection volume 1μL, and column temperature programmed, with an initial temperature of 85℃, increased to 185℃ at a rate of 10℃ / min, and maintained for 10min.

[0006] Preferably, the organic solvent is N,N-dimethylformamide.

[0007] Compared with the prior art, the advantages and positive effects of the present invention are as follows: 1. The method proposed in this invention can simultaneously detect ethanol and isopropyl myristate. After the two substances are dissolved in N,N-dimethylformamide solvent, the detected spectra do not have peak overlap or tailing, and the accuracy is high. The detection process during sample pretreatment effectively saves detection time and labor costs.

[0008] 2. The method proposed in this invention is suitable for the determination of ethanol and isopropyl myristate content in injection solutions for vitamin or calcium deficiency in dairy cows. It has the advantages of high separation efficiency, high selectivity, high sensitivity, fast analysis speed, and wide applicability. Attached Figure Description

[0009] Figure 1 This is a linear fitting curve of ethanol concentration versus peak area.

[0010] Figure 2 This is a curve showing the fitting relationship between isopropyl myristate concentration and peak area.

[0011] Figure 3 The chromatogram for testing the sample.

[0012] Figure 4 The chromatogram of the sample in Comparative Example 1 is shown. Detailed Implementation

[0013] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described below with reference to specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0014] Numerous specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways than those described herein, and therefore the invention is not limited to the specific embodiments disclosed in the following specification.

[0015] instrument: Swiss Precisa Instrument 290SCS electronic balance; Zhejiang Fuli Analytical Instrument Co., Ltd. FL9720PLUS gas chromatograph.

[0016] Reagents and reagents: Ethanol reference standard, batch number: 130106-202105, content: 100%, purchased from the National Institutes for Food and Drug Control.

[0017] Isopropyl myristate reference standard, batch number: 190181-201701, content: 99.6%, purchased from the National Institutes for Food and Drug Control.

[0018] N,N-Dimethylformamide, batch number: 20240412, Tianjin Fuyu Fine Chemical Co., Ltd.

[0019] One batch of injectable drugs for vitamin or calcium deficiency, batch number: EJP250402, production date: April 3, 2025, shelf life: 1 year, purchased from Hebei Yuanzheng Pharmaceutical Co., Ltd.

[0020] Unless otherwise specified, all other instruments and reagents used are commercially available reagents commonly used in chemical experiments. Unless otherwise specified, all reagents are of analytical grade. Example 1

[0021] (1) Blank solvent: N,N-dimethylformamide.

[0022] (2) Reference solution: Accurately weigh an appropriate amount of ethanol reference standard and dilute it with N,N-dimethylformamide to prepare a solution containing approximately 0.9 mg of solute per 1 mL, thus obtaining the ethanol reference solution. Accurately weigh an appropriate amount of isopropyl myristate reference standard and dilute it with N,N-dimethylformamide to prepare a solution containing approximately 0.9 mg of solute per 1 mL, thus obtaining the isopropyl myristate reference solution.

[0023] (3) Test solution: Accurately measure 1 mL of the sample to be tested (vitamin ADE injection, Hebei Yuanzheng Pharmaceutical Co., Ltd., without isopropyl myristate), place it in a 200 mL volumetric flask, dilute to the mark with N,N-dimethylformamide, shake well, and the solution is ready.

[0024] (4) Detection: The chromatographic conditions in this embodiment are as follows: polyethylene glycol 20M capillary column: length 30m, film thickness 0.25μm, injection port temperature 250℃, detector temperature 250℃, carrier gas is nitrogen, flow rate is 1.8mL / min, injection volume is 1μL, column temperature is programmed, initial temperature is 85℃, increased to 185℃ at a rate of 10℃ / min, and maintained for 10min.

[0025] 1 μL of blank solvent, negative control solution (prepared according to the vitamin ADE injection formula, excluding ethanol and isopropyl myristate), ethanol reference solution, isopropyl myristate reference solution, and test solution were accurately measured using a syringe and injected into the chromatogram under the chromatographic conditions described above. The chromatograms were recorded, and the samples were analyzed. The tests showed that the blank solvent and negative control sample did not interfere with the determination of the two components. The resolution, theoretical plate number, and tailing factor of ethanol and isopropyl myristate all met the requirements. Figure 3 The image shows the chromatogram for the test sample. Note that in this embodiment, the injection needle needs to be rinsed with solvent at least three times after each test, and then rinsed with the test sample at least three times.

[0026] System precision experiment: Following the chromatographic conditions described in this example, the reference solution and the test solution were injected repeatedly, with each sample injected six times. The peak areas (ISD) of both the ethanol peak and the isopropyl myristate peak in the six repeated detection chromatograms of each sample were less than 2%, indicating good system precision under these chromatographic conditions.

[0027] The content of ethanol and isopropyl myristate in this product was calculated based on peak area using the external standard method. Standard curve and its applicable range: Accurately weigh and record appropriate amounts of ethanol and isopropyl myristate reference standards. Prepare a mixture containing approximately 1.8 mg of ethanol and 1.8 mg of isopropyl myristate per 1 mL using N,N-dimethylformamide. Accurately measure 1, 4, 5, and 6 mL of the above reference standard mixture and place them in separate 10 mL volumetric flasks. Dilute to the mark with N,N-dimethylformamide, shake well, and inject 1 μL into the chromatogram under the chromatographic conditions described above. Record the chromatogram. Perform linear regression with sample concentration x (mg / mL) and peak area y. The regression equation for ethanol is Y = 505519x - 8796.4, with a correlation coefficient R0. 2 It is 0.9999, such as Figure 1 The results indicate that ethanol exhibits good linearity within the concentration range of 0.185 mg / ml to 1.850 mg / ml. The regression equation for isopropyl myristate is Y = 707919x - 12131, with a correlation coefficient R0. 2 =1, such as Figure 2 The results indicate that isopropyl myristate exhibits good linearity in the concentration range of 0.193 mg / ml to 1.93 mg / ml.

[0028] (4) Accuracy verification A blank excipient solution without ethanol and isopropyl myristate was prepared according to the formulation of Vitamin ADE injection. Approximately 9g of ethanol reference standard and approximately 9g of isopropyl myristate reference standard were accurately weighed and recorded, and placed in 50mL volumetric flasks. The blank excipient solution was added to the mark, and then 1mL of each was accurately added to a 200mL volumetric flask. N,N-dimethylformamide was added to the mark, and the solution was shaken well and filtered to obtain the sample solution. Three sample solutions were prepared using the same method. Following the above method, the amounts of ethanol and isopropyl myristate were changed to approximately 7.2g and 10.8g, respectively, while keeping the amounts of other substances and the preparation process unchanged. Three sets of sample test solutions containing ethanol and isopropyl myristate were prepared, with three parallel samples in each set (see Table 1, numbers 1-1 to 3-3). Because the blank excipient solution did not contain ethanol and isopropyl myristate, the background value was "0".

[0029] Accurately measure 1 μL of each and inject it into the chromatograph. Test according to the chromatographic conditions of Example 1 and record the chromatogram. Results: The average recoveries of ethanol and isopropyl myristate were between 99% and 100%. The RSDs of ethanol and isopropyl myristate were 0.72% and 0.65%, respectively, which were less than 2%. The results are shown in Table 1 below, indicating that the accuracy of this product is good.

[0030] Table 1. Recovery rates of ethanol and isopropyl myristate in vitamin ADE injection

[0031] (5) Repeatability verification For the repeatability test, 6 test samples were taken and detected according to the chromatographic conditions in Example 1. The contents of ethanol and isopropyl myristate in the test sample solution were determined. The results are shown in Table 2. The RSD of ethanol was less than 2.0%, and isopropyl myristate was not detected, indicating good repeatability.

[0032] Table 2 Repeatability test results

[0033] (6) Sample stability test Take the reference solution and the test solution, and inject 1 μL at 0, 2 and 4 h respectively. Measure the peak areas of ethanol and isopropyl myristate in the test solution. The results are shown in Table 3. It can be seen from Table 3 that the RSD is less than 1%, indicating that the sample is stable within 4 h after preparation.

[0034] Table 3. Results of stability tests on ethanol and isopropyl myristate solutions.

[0035] Comparative Example 1 The solvent was replaced with methanol, and the remaining processing steps were the same as in the previous example to obtain a control sample. The temperature program was as follows: initial temperature 45°C, increased to 185°C at a rate of 10°C per minute, and maintained for 10 minutes; the injection port temperature was 250°C, and the detector temperature was 250°C. With other conditions unchanged, detection showed that the chromatographic peaks of ethanol and solvent could not be effectively separated. Figure 4 As shown.

[0036] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments for application in other fields. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for detecting the components of an injection for the prevention or treatment of vitamin or calcium deficiency in dairy cows, characterized in that ethanol and nutmeg standards are weighed, diluted with organic solvents to obtain a series of ethanol and nutmeg solutions of known concentrations, and analyzed by gas chromatography to obtain chromatograms of ethanol and nutmeg standards at a series of concentrations. An ethanol standard curve is obtained by fitting ethanol concentration as the abscissa and peak area as the ordinate; a nutmeg standard curve is obtained by fitting nutmeg concentration as the abscissa and peak area as the ordinate. The sample to be tested was diluted with an organic solvent and then detected under the detection conditions set by gas chromatography to obtain a gas chromatogram. The chromatogram was then quantitatively analyzed. The gas chromatography settings were as follows: FID detector, polyethylene glycol 20M capillary column, injection port temperature 240-260℃, detector temperature 240-260℃, carrier gas nitrogen, flow rate 0.9-2.5mL / min, injection volume 1μL, and column temperature programmed, with an initial temperature of 80-90℃, increased to 180-190℃ at a rate of 9-12℃ / min, and maintained for at least 10min.

2. The method for detecting the components of an injection for the prevention or treatment of vitamin or calcium deficiency in dairy cows according to claim 1, characterized in that the gas chromatography settings are as follows: a polyethylene glycol 20M capillary column, 30m in length, 0.25μm in film thickness, an injection port temperature of 250℃, a detector temperature of 250℃, nitrogen as the carrier gas, a flow rate of 1.8mL / min, an injection volume of 1μL, and a programmed temperature ramp for the column, with an initial temperature of 85℃, increasing to 185℃ at a rate of 10℃ / min, and maintaining this temperature for 10min.

3. The method for detecting the components of an injection for the prevention or treatment of vitamin or calcium deficiency in dairy cows according to claim 1, wherein the organic solvent is N,N-dimethylformamide.

Images