title: Development and validation of a method for the purity determination of (3beta,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol(FF-MAS) in pharmaceutical products containing recombinant human albumin.
abstract: A simple and sensitive method has been developed and validated for purity determination of FF-MAS (also known as (3beta,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol an endogenous substance usually present in the pre-ovulatory follicular fluid) at very low concentrations (200 ng per unit) in pharmaceutical formulations containing RECOMBUMIN (recombinant human albumin) as the matrix. The paper focuses on development of the sample preparation for the product containing recombinant human albumin. After removal of recombinant human albumin by precipitation using a mixture of water and ethanol, the FF-MAS was concentrated by evaporation using a vacuum centrifuge and the prepared sample was analyzed. The purity method was based on a reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet absorption detection at 250 nm. The method was validated according to ICH guidelines. The method indicated a significant degree of specificity with good selectivity and no significant effect from the matrix. The limit of detection was found to be 0.3-0.8% (depending on the impurity) corresponding to 1.9-5.1 ng. The limit of quantification was found to be 0.8-2.5% (depending on the impurity) corresponding to 5.2-16 ng. The recovery was found to be between 90 and 101% for the FF-MAS, and 100-129% for the six known impurities. The tested range for FF-MAS was from 320 to 960 ng corresponding to 50-150% of the nominal concentration (640 ng, injection volume is 100 microl). The linearity of each compound (FF-MAS and the six impurities) was investigated. The squared correlation coefficient (r(2)) was 0.999 for FF-MAS (50-150% level) and 0.977-0.998 for the six known impurities (at four levels: 0.20, 0.50, 1.00, 2.00%). The R.S.D. in the repeatability study was found to be 9.2% for the total amount of impurities, and 10.4% for single impurities. The R.S.D. in the intermediate precision study was found to be 10.9% for total impurities, and 12.0% for single impurities. The validation results showed that the method was suitable for the purity analysis. The validated method was then ready for use for samples analysis of phase II clinical studies and the stability investigations of the pharmaceutical product.
authors.affiliation: Department of Analytical Development, CMC Development, Global Development, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Maaloev, Denmark. bjam@novonordisk.com
authors.forename: Babak
authors.initials: B
authors.lastname: Jamali
authors.forename: Steen G
authors.initials: SG
authors.lastname: Andersen
authors.forename: Helle
authors.initials: H
authors.lastname: Brødholt
authors.forename: Lene
authors.initials: L
authors.lastname: Wendel
authors.forename: Vibeke
authors.initials: V
authors.lastname: Bødstrup
mesh_terms: D000418:Albumins / Q000737:chemistry*; D002782:Cholestenes / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D004340:Drug Contamination; D006801:Humans; D007202:Indicators and Reagents; D011786:Quality Control; D011994:Recombinant Proteins / Q000737:chemistry; D012015:Reference Standards; D015203:Reproducibility of Results; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000418:Albumins; D002782:Cholestenes; D007202:Indicators and Reagents; D011994:Recombinant Proteins; D012996:Solutions; C100324:4,4-dimethylcholesta-8,14,24-trienol