1. Jiangsu Institute for Food and Drug Control, Nanjing 210019, China; 2. NMPA Key Laboratory for Impurity Profile of Chemical Drugs, Nanjing 210019, China; 3. Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China
Abstract：OBJECTIVE To establish a high performance liquid chromatography combined with pulsed amperometric detection(HPLC-PAD)method for determination of potency of neomycin sulfate. METHODS An improved HPLC-PAD method from EP method for determination of the content and related substances of neomycin sulfate was established and validated. The study of impurity profile of neomycin sulfate was completed by LC-IT-TOF method with the help of on-line desalination using a suppressor; and the main components in neomycin sulfate were clarified combining the RESULTS of impurity profile and minimum inhibitory concentrations of the main components and impurities. The semi-preparative liquid chromatography-evaporative light scattering detector(ELSD) was self-assembled, highly purified neomycin B and neomycin C were prepared and their structural confirmation was also conducted. The contents of highly purified neomycin B and neomycin C were determined by means of mass balance method. The potencies of highly purified neomycin B and neomycin C were determined by three-dose antibiotic microbial assay and the conversion factors between contents of neomycin B and neomycin C and their potencies were calculated separately and then a formula for the calculation of potency of neomycin sulfate from the content of main components of neomycin B and neomycin C was obtained.At last, a verification experiment for the accuracy of the conversion factor and the formula were designed and a serial of tests were carried out to investigate the interaction and the verification for the actual sample. RESULTS The improved HPLC-PAD method was superior to the European Pharmacopoeia method in the separation ability and stability, and was suitable for accurate quantification of various components of neomycin sulfate and related substance inspection. The successful removal of trifluoroacetic acid in the mobile phase by the technology of desalination on-line using a suppressor broke a new way for the study of impurity profile of aminoglycoside such as neomycin sulfate. Combining the impurity profile with the RESULTS of MIC it was clarified that the main activity components of neomycin sulfate were neomycin B and neomycin C. Highly purified neomycin B and neomycin C were successfully prepared. A conversion factor for the transition from potency to purity of neomycin sulfate was obtained through experiments and calculations and was verified successfully. CONCLUSION It is feasible to replace the microbial assay by HPLC-PAD method for determining the potency of neomycin sulfate.
WANG Y H. Study on the Degradation Products of Amphotericin B and the Unification between HPLC Ptenurity and Microbiological Potency [D]. Peking Union Medical College, 2010.
[13]
CHANG Y, YAO S C, HU C Q. The important change of Pharmacopoeia of the People′s Republic of China 2015 on gentamicin sulfate monograph, the content of gentamicins C from the relative content to the absolute level . Chin J Antibiot (中国抗生素杂志), 2016, 41(1):1-15.
[14]
MOU S F, Y H, CAI Y Z. Analysis of sugars by high performanceanion-exchange chromatography with pulsed amperometric detection. Chin J Chromatogr (色谱), 2009, 27(5):667-674.
[15]
LI R Y, LIANG L N, MOU S F. Application advance in analysis of food carbohydrates with high performance anion exchange chromatography-pulsed amperometricdetection . Food Ferment Ind (食品与发酵工业), 2010, 36(7):97-101.
[16]
WANG Y, YAO S C, WANG M J, et al. Determination of spectinomycin and related substances by HPLC-PAD. Chin J Antibiot(中国抗生素杂志), 2014, 39(6):439-446.
[17]
ROCKLIN R D, CLARKE A, WEITZHANDLER M. Improved long-term reproducibility for pulsed amperometric detection of carbohydrates via a new quadruple-potential waveform. Anal Chem, 1998, 70(8-10):1496-1501.
[18]
CHENG J, LIU Y. Electrochemical detection in high performance liquid chromatography for analysis of pharmceutical drug. Chin Pharma J (中国药学杂志), 2017, 52(20):1753-1771.
[19]
ADAMS E, SCHEPERS R, ROETS E, et al. Determination of neomycin sulfate by liquid chromatography with pulsed electrochemical detection. J Chromatog A, 1996, 741: 233-240.
[20]
YUAN Y Z, ZHANG M, HU C Q. Advances in the components analysis of aminoglycosides . Chin Pharma J(中国药学杂志), 2017, 52(20):1772-1779.
[21]
ZHANG Q, HUANG M W, FANG W, et al. Detection of the content and related substances of neomycin sulfate by high performance liquid chromatography combined with pulsed amperometricdetection. Chin Pharm Univ J (中国药科大学学报), 2019, 50(2):193-199.
[22]
ZHANG T T, LIU L Y, ZHANG Y H, et al. Enhancement of sensitivity for bioanalysis by liquid chromatography-electrospray mass spectrometry with trifluoroacetic acid in mobile phase using a suppressor . Anal Test Tech Instrum (分析测试技术与仪器), 2017, 23(1):18-23.
[23]
WU Y N, ZHAO W, ZHU X Y, et al. Determination of related substances in etimicin sulfate injection and etimicinandsodium chloride injection by LC-PED. Chin J Antibiot (中国抗生素杂志),2016,41(8):618-623.
[24]
YANG D J, XU H Z, CHEN R X. Quantiative determination of ribostamycin and neamine by RP-HPLC. Chin J Antibiot (中国抗生素杂志), 1990,15(5):378-379.
[25]
GOOSSENS A. Neomycin Sulfate[M]. Management of Positive Patch Test Reactions. Springer Berlin Heidelberg, 2003:83-84.
[26]
LI B, ADAMS E, VAN SCHEPDAEL A, et al. Identification of gentamicin impurities by liquid chromatography tandem mass spectrometry. Rapid Commun Mass Spectrom, 2006, (20):393-402.
[27]
YUAN Y Z, ZHANG M, QIAN W, et al. Characterization of related substances in etimicin sulfate sample by high performance liquid chromatography-electrospray ionization-ion trap-mass spectrometry . Chin J Anal Chem(分析化学), 2010,38(6):817-822.
2020, Vol. 55 
