Studies On The HPLC/GC Fingerprints And LC-MS Qualitative Analysis For Conjugated Estrogens

Objectives: To establish the HPLC and GC fingerprints of conjugated estrogens (CE) material for the quality control of conjugated estrogens material; To evaluate the pharmaceutical equivalence of conjugated estrogens material by LC-MS method with qualitative analysis.Methods: 1) The HPLC method was used by gradient program with Shimadzu vp-ODS column(250mm×4.6mm); The mobile phases were consisted of 0.1% trifluoroacetic acid solution as mobile phase A and 0.1% trifluoroacetic acid acetonitrile solution as mobile phase B; The flow rate was 1.0mL·min^-1; The UV detection was performed at 215 nm; Estrone sulfate was used as internal reference compound. 2) The GC method was used with Rtx?-225 column(15m×0.25mm× 0.25μm); The detector was a flame-ionization detector (FID); The columu was held at 210℃; The carrier gas was hydrogen flowing at the rate of 2.5 mL·min^-1, The split flow rate is 1 to 3 ml per minute.and linear velocity of 60 mL·min^-1; 3-O-methylestrone was used as internal standard and estrone was used as internal reference compound. 3) The LC-MS method was used with electrospray ionization (ESI) and negative ion mode. The mobile phases were consisted of methanol and 10mM ammonium acetate(50 :50, V/V). The flow rate was 0.5mL·min^-1. Full scan and Seleted Ion Monitoring Mode (SIM) was used for analysis.Results: 1) HPLC fingerprint of CE material consisted of 21 commonpeaks which showed much similarity to the data of FDA. There was much similarity between the fingerprints of CE material and premarin tablets. 2) GC fingerprint of CE material consisted of 20 common peaks which showed much similarity to the data of FDA. There was much similarity between the fingerprints of CE material and premarin tablets. 3) Nine molecular weights were confirmed. 56 AMUs were determined according to the information provided by FDA with the method of LC-MS, in which the SIM chromatograms for 52-54 AMUs were observed to have multiple peaks. The SIM chromatograms for 56 AMUs of CE material showed much similarity comparing to premarin tablets. The SIM chromatograms of 10 components in USP between CE material and premarin tablets were consistent and the SIM chromatograms for 51 AMUs between CE material and premarin tablets showed similarity or minor difference.Conclusions: 1) The method of HPLC and GC fingerprints was accurate, reliable and had good reproducibility. The method was applicable to the determination of CE drug substance, material and tablets, which could be used as a standard for controlling the quality of CE products. 2) CE material contained the components as FDA required. 3) CE material had the similar components to premarin tablets. 4) CE material conformed to the requirement of FDA for pharmaceutical equivalence about conjugated estrogens and was consistent with premarin for pharmaceutical equivalence.