Determination of the content of flumazenil

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Determination of flumazenil content

sex antagonists are clinically used to terminate the general anesthesia induced and maintained by benzodiazepines, as well as the first aid for poisoning by such drugs

the intermediate products and related impurities produced by flumazenil and its synthesis in the process of promoting the development of new ceramic aluminum material industry in Huaibei City were determined by HPLC

1. The instrument and test drug

use octadecylsilane bonded

as the filler, acetonitrile water (V ∶ v=22 ∶ 78) as the mobile phase, and fluorohydroxime as the internal standard to determine the content of flumazenil. Under this chromatographic condition, it can be well separated from the main peak and impurity peak, with short separation time and good repeatability. The content determination result is satisfactory. Instrument and reagent Shimadzu lc-10a liquid chromatograph, class-lc10 workstation, spd-m10a diode array detector, cto-10a column incubator, carloerba1106 element

instrument made in Italy; Acetonitrile is chromatographically pure and water is secondary re distilled water; The reference materials and samples of flumazenil and fluorohydroxime were provided by the synthesis room of Shenyang Pharmaceutical University

2. Chromatographic conditions

2.1 selection of detection wavelength determine the UV spectra of the sample and the internal standard respectively. The maximum absorption wavelength of flumazenil is 243nm, and the maximum absorption wavelength of fluorohydroxime is 275nm. The maximum absorption wavelengths of the two are far away. Therefore, the wavelength with strong absorption of both is selected as the detection wavelength

2.2 purity test of reference substance weigh the sample in a tin foil container and put it into the sampler. The sample is catalytically burned in a 1050 ℃ combustion tube, and the stable nitrogen flow is used as the carrier gas. C, h and N are converted into CO2, H2O and N2 respectively. The mixed component enters the chromatographic column to separate N2, CO2 and H2O. Each component flows through the thermal conductivity cell together with the carrier gas to generate an electrical signal. After being processed by the integrator and computer, the relevant elemental analysis data are shown in the table. Under the above chromatographic conditions, the purity of flumazenil reference substance is 99.7% and RSD is 0.96% (n=5)

3. Methods and results

3.1 linear range investigation: weigh about 100mg of flumazenil reference substance. If the precision is only reduced but not increased, weigh it, place it in a 100ml volumetric flask, dissolve it with methanol and dilute it to the scale to prepare flumazenil reference solution Weigh about 100mg of fluorohydroxime reference substance, accurately weigh it, put it into a 100ml volumetric flask, dissolve it with methanol and dilute it to the scale to prepare the internal standard solution Accurately suck 1, 2, 3, 4 and 5ml of flumazenil reference solution into 10ml volumetric flask, add 3ml of internal standard solution to each volumetric flask, dissolve with methanol, dilute to the scale, and shake well Inject 20 l into the liquid chromatograph, measure the peak area (AI) of the reference substance and the peak area (as) of the internal standard substance respectively, take the injection volume of the reference substance as the abscissa, and the ratio of the peak area of the reference substance of flumazenil to the peak area of the internal standard substance as the ordinate, draw the standard curve, and obtain the regression equation through linear regression: c=0.00931a+3.18, and the correlation coefficient r=0.9999 It can be seen that the linearity of flumazenil 5. Starting the oil pump motor is good within the range of 2 ~ 10 G

3.2 preparation of test solution take about 100mg of flumazenil sample, accurately weigh it, put it into a 100ml volumetric flask, dissolve it with methanol, dilute it to the scale, and shake it well Accurately take 3ml of the solution, put it into a 10ml measuring bottle, dilute it with methanol to the scale, shake it well, and use it as the test sample solution

3.3 stability evaluation precisely suck the same test solution, and determine the peak area (AI) and internal standard peak area (as) values of the test solution at 0, 2, 4, 6, 8 and 10h intervals. The experimental results show that the chromatographic peak area does not change significantly within 10h after the preparation of the test solution, indicating that the chemical properties of the test sample are stable after determination within this time

3.4 reproducibility study prepare 6 test sample solutions of the same batch number and determine them respectively according to the above chromatographic conditions

4. Discussion

4.1 separation of intermediate products in the process of flumazenil synthesis, four important intermediate products are produced: fluorohydroxime, 5-fluoroindirubin, 6-fluoroindirubin anhydride and heterodiketone Through a large number of experiments, the mobile phase acetonitrile water (V ∶ v=22 ∶ 78) can completely separate the five substances

4.2 selection of internal standard flumazenil mother liquor was used for system adaptability test. It was found that the retention time of 17 min was flumazenil, and the retention time of 4 min and 9 min were the peaks of related substances The peak of the internal standard may be inserted between 9min and 17min or after 17min The retention time of fluorohydroxime was 13min, and the resolution of the peak was very good. Therefore, fluorohydroxime was used as the internal standard of flumazenil

4.3 qualitative and quantitative analysis of related substances in the sample. In the HPLC chromatogram of flumazenil, the impurity peak with a retention time of 4min was compared with the retention time of the intermediate. It was speculated that it was a heterodiketone By adding an appropriate amount of heterodiketone reference substance, it was observed that the chromatographic peak increased significantly after 4min application for a period of time, which confirmed that the impurity was indeed heterodiketone The content of the impurity is less than 0.5% by external standard method

the results showed that there was a good linear relationship in the range of 2 ~ 10 g for the injection amount of the reference substance, the separation time was short, the repeatability was good, and the content determination results were satisfactory

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