| Gentamicin is a multi-component mixture.When the content of Gentamicin is determined by tube-plate method,there is often a problem of different quality between the standard and the sample.Therefore,it is necessary to establish a new method for the determination of gentamicin and compare it with the tube-plate method.After consulting the literature,it was found that the determination methods of gentamicin in recent years include microbiological assay,polarimetry,spectroscopy,chromatography and so on.Compared with tube-plate method,polarimetry and spectroscopy could also determine all the content of gentamicin,but the sensitivity of polarimetry was too low.Through comparative analysis of spectrophotometry,it was found that ultraviolet-visible spectrophotometry had the advantages of high sensitivity and fast detection,which could be used as the research direction of establishing the determination method of gentamicin content.According to the chemical properties of gentamicin,salicylaldehyde reagents with stable chemical properties were found in this paper.It was found by experiment that the derivatization of Salicylaldehyde and gentamicin took place at room temperature,and the derivatives had the maximum ultraviolet-visible absorption at 400 nm wavelength.The single factor and multi-level design method was used to optimize the reaction conditions.The results showed that salicylaldehyde and gentamicin had the highest absorptivity in phosphate buffer at pH 5.5 after 20 minutes of water bath at 50?,and the experimental conditions were the best.With the best experimental conditions and according to the guiding principles stipulated in the Chinese Pharmacopoeia,the methodological verification of the determination of gentamicin by ultraviolet-visible spectrophotometry was carried out.The results showed that the specificity of the method was good.In the concentration range of 280-519 U/ml,there was a good linear relationship between the absorbance value(A)of derivatives and the concentration value of gentamicin(C).The standard curve A was 0.0007C+0.0186,and the R value was 0.999.The average recovery was 100.09±0.8%,RSD was 0.7%(n=7),and the derivatives were stable in 0-10 minutesMeanwhile,the content of gentamicin was determined by tube-plate method.According to the process of suspension preparation,culture medium preparation,sterilization,test solution preparation,double dish preparation,dripping test solution,culture and measurement of bacteriostasis circle.The measured diameter of bacteriostasis circle was tested by reliability test,reliability limit efficiency and titer calculation according to the statistical method of biological verification.Four independent parallel experiments were conducted.The results showed that the reliability test parameters(F1)of the one of four experiment did not meet the judgment requirement;the reliability limit rate of the one of four experiment was 7.96%,which did not meet the requirements of the standard of gentamicin sulfate injection(no more than 7%);the average content of the results was 101.10%,and RSD was 1.7%(n=4).Finally,the ultraviolet-visible spectrophotometry and tube-plate method were compared.Firstly,the content of gentamicin in gentamicin sulfate injection was determined by ultraviolet-visible spectrophotometry and tube-plate method respectively,and the results were compared with each other.Then,the methodological verification results of ultraviolet-visible spectrophotometry and tube-plate method and the significant differences of the determination results of gentamicin sulfate injection were compared.The results showed that the average content of gentamicin was 100.43%and RSD was 1.2%(n=104)by ultraviolet-visible spectrophotometry.The average content of gentamicin was 101.10%and RSD was 1.7%(n=4)by tube-plate method.The linearity and range of ultraviolet-visible spectrophotometry were 280-519 U/ml,R value was 0.999,recovery is 100.09%±0.8%,RSD was 0.7%.The linearity and range of tube-plate method were 2-12 U/ml,recovery was 100.80%±1.5%/98.7%±1.5%,RSD was 1.7%.According to the t-distribution test,in the confidence interval of P<0.05,t=0.88,less than 1.98[t(0.05,106)].A method for the determination of gentamicin by ultraviolet-visible spectrophotometry was established.The method was rapid and easy to operate.At the same time,the method had good specificity,sensitivity,accuracy and reproducibility,but the stability of the derivatives was not good.Compared with the tube-plate method,the results of the two methods had no significant difference,but the accuracy and reproducibility of ultraviolet-visible spectrophotometry were better.Therefore,ultraviolet-visible spectrophotometry could meet the requirement of constant analysis for the determination of gentamicin content,and provide a new reference basis for controlling its quality,and ensure the safety and effectiveness of gentamicin use. |
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