Studies On The Method For Determination Of The Content And Related Substances Of Amikacin Sulfate And Study On The Optimization Of Its Prescription

Amikacin is the third generation of aminoglycoside antibiotics, its antimicrobial spectrum is broad, it is stable to the majority of aminoglycoside-modifying enzyme(AME)and can be used for the treatment of infections caused by gram-negative bacteria. ototoxicity and nephrotoxicity are the main adverse reactions of it. In this study, reference to the International Pharmacopoeia(IP)and the 7.5 version of EP, A HPLC-UV method and a HPLC-pulsed electrochemical detection(PED) method were established to determine Amikacin’s content and related substances respectively, and the content and related substances of amikacin sulfate injection of 35 pharmaceutical companies were evaluated. At the same time according as the p H limit of amikacin sulfate injection in Chinese pharmacopoeia(Edition 2010) was too broad and the security hidden trouble of the sterilizing condition in most companies existed, a research was studied. The p H, AE value and osmotic pressure of 194 batches of amikacin sulfate injection were determined. Part one Determination of Amikacin Sulfate by HPLC-UV endabsorption methodObjective: To develop a sensitive and fast HPLC-UV method for the analysis of the content of amikacin sulfate, and the material amount in amikacin sulfate injection of different pharmaceutical companies were evaluated.Methods: The content of amikacin sulfate injection of 35 pharmaceutical companies were determined by HPLC-UV method. The column was Phenomenex-C18(150mm × 4.6mm, 5μm). The mobile phase was sodium 1-octanesulphonate 1.8g, anhydrous sodium sulfate 20 g, dissolved with 50 m L phosphate buffer of p H 3.0(0.2mol/L), diluted with water to 1000 m L-acetonitrile(92:8). The detection wavelength was 200 nm. The mobile phase flow rate was set at 1.0m L/min, the injection volume was 10μL.Results: The linear relationship, linearity range, precision, stability, LOD and LOQ of the method were good for amikacin sulfate. The recovery was found to be 100.7% and the precision in terms of RSD was 0.3%. Amikacin sulfate injection of 35 pharmaceutical companies were determined. The results demonstrated that the content of amikacin sulfate injection was in the rang of 91.7%~107.7%, they were all in accord with the National Standard, It demonstrated that the pharmaceutical companies basically feed according to the 100% feeding. The content of amikacin sulfate injection of 8 pharmaceutical companies were analyzed with the single factor analysis of variance, There were significant differences between different companies(P<0.05).Conclusion: The method was sensitive and suitable for the analysis of the content of amikacin sulfate, the results demonstrated that there were significant differences between different companies, at the same time it was suggested that we shall strengthen control of products homogenization. Part two Determination of the related substances of amikacin sulfateinjection by high-performance liquid chromatography withpulsed electrochemical detectionObjective: To develop a reversed-phase liquid chromatographic(LC) method combined with pulsed electrochemical detection(PED) for the analysis of the related substances of amikacin sulfate injection, and the source of the related substances was investigated.Methods: The related substances of amikacin raw materials of 3 pharmaceutical companies and amikacin sulfate injection of 25 pharmaceutical companies were determined by HPLC-PED method. The column was Boston Green ODS-C18(250 mm × 4.6 mm, 5μm). The mobile phase was composed of an aqueous solution containing 1.8g/L sodium 1-octanesulphonate, 14 m L/L tetrahydrofuran, 50 m L/L of phosphate buffer(p H 3.0) and sodium sulphate, which was 20g/L in mobile phase A and 28g/L in mobile phase B, gradient eluents, pulsed electrochemical detection detect potential was E1=0.10V、E2=-2.00V、E3=0.60 V and E4=-0.10 V, t1、t2、t3 and t4 was respectively 0-0.40s、0.41-0.42s、0.43 s and 0.44-0.50 s, integral interval was 0.20-0.40 s. The post-column solution was sodium hydroxide, flow rate of post-column solution was 0.3m L/min.Results: The results demonstrated that the main peak and every impurity peak were separated well. Amikacin raw materials of 3 pharmaceutical companies and amikacin sulfate injection of 35 pharmaceutical companies were determined, and 9 impurities in amikacin raw materials and 10 impurities in amikacin sulfate injection have been detected. Under high temperature and oxidative condition, the number of impurities and the amount of impurity D increased, the result showed that the impurity D was raw material and degraded product.Conclusion: This method was sensitive and can be applied for the related substances of amikacin, it provided that the technology was for better analysis of domestic amikacin sulfate injection, while achieving the detection of the impurity H, it could be an important indicator of quality evaluation. At the same time the quality of raw materials were investigated so that we could make the main impurities of the injection and raw materials to establish a correspondence. Part three Evaluation and optimization of different prescription ofamikacin sulfate injection of different manufacturersObjective: To establish the reasonable range of the p H value of amikacin sulfate injection, the influence of p H value on the antibacterial activity was studied. The adjuvants were evaluated by osmotic pressure, at the same time we selected a reasonable sterile condition to ensure sterility.Methods: 1 The osmotic pressure of 194 batches of amikacin sulfate injection were determined by cryoscopy 2 The p H value of 194 batches of amikacin sulfate injection were determined, we divided them into three groups according to different p H range(4.2 ~ 5.0, 4.2 ~ 5.8, 5.9 ~ 6.5) and measured their AE value. At the same time the total impurities of the samples in the three groups were compared(the raw materials of the samples from the same manufacturer and sterilization conditions are the same). 3 The raw materials from the three production enterprises were respectively made into aqueous solution of different p H value range, the antibiotics’ potencies were determined and the antibacterial activity of amikacin aqueous solution were evaluated. 4 The color, p H value, related substances and content of the three prescriptions were studied before and after the 4 kinds of sterilization conditions(100℃15min, 100℃30min, 115℃30min, 121℃15min), and this product tolerance of the sterilization conditions was evaluated.Results: 1 The osmotic pressure results show: the osmotic pressure of 125 batches of samples was less than 285 m Osmol/kg, the osmotic pressure of 33 batches of samples was between 285 m Osmol/kg and 310 m Osmol/kg, the osmotic pressure of 36 batches of samples was greater than 310 m Osmol/kg. 2 The p H value of 194 batches injection was between 4.2 and 6.5, the p H comparison results of the 3 groups showed that the samples of p H value in the range of 4.2 to 5 had the lowest AE values and total impurities. 3 The results of 3 groups of amikacin solution potencies showed that the sample of p H value of 3.9~ 5 range had the strongest antibacterial activity, followed by 3~ 3.5, the difference was 6 ~ 6.5. 4 The appearance of samples and p H value did not obviously change before and after the 4 kinds of sterilization conditions, the related substances and content ware changed, but still could reach the standard of provision.Conclusion: 1 64% of the samples’ osmotic pressure were lower than that of normal human’s, it could cause hemolysis in low permeability. The result of osmotic pressure suggested that the prescription of individual enterprise was not scientific. 2 The investigation about p H value showed that the current standard for p H limit(4.0~7.0) was too broad. Then it determined that the p H limit(3.5~5.5) in USP35 and BP2012 was reasonable, which provided the basis for the revision of the standard and quality control. 3 Through the investigation about Sterilization condition, we suggested that enterprises adopted moist heat sterilization(121℃ 15min)in order to ensure aseptic level.