title: Assay methodology for the quantitation of unbound ertapenem, a new carbapenem antibiotic, in human plasma.
abstract: Ertapenem is a new once-a-day antibiotic with excellent coverage of common community gram negative and gram positive aerobes and anaerobes. It demonstrates nonlinear protein binding in human plasma (about 94% bound). An assay for unbound drug was developed to study the pharmacokinetics of unbound ertapenem in plasma. Unbound drug is separated from plasma samples (1.0 ml) by ultrafiltration using a Centrifree((R)) centrifugal filter device. Ertapenem (vulnerable to hydrolysis of the beta-lactam moiety) is stabilized in the filtrate by adding an equal volume of 0.1 M MES buffer, pH 6.5 and then is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection (300 nm). Non-specific binding to the Centrifree((R)) device is <3%. A suitable internal standard is not available. The assay is specific and linear over the concentration range of 0.25 to 100 microgram/ml in plasma filtrate. The lower limit of quantitation (LLOQ) is 0.25 microgram/ml. Intra-day precision is C.V.<10% and accuracy ranges from 97 to 101% of nominal concentration. Inter-day precision and accuracy were determined using quality control samples (QCs) prepared in plasma ultrafiltrate at 0.5, 12 and 80 microgram/ml and stored at -70 degrees C with stabilizer. Inter-day assay accuracy and precision ranged from 100 to 111% of nominal concentration and 1.8 to 5.3% C.V. (n=40), respectively. The assay has been used to analyze plasma samples from subjects receiving 500 and 2000 mg i.v. doses of ertapenem (30 min infusion).
authors.affiliation: Merck Research Laboratories, WP75A-303, Sumneytown Pike, 19486, West Point, PA, USA
authors.forename: Donald G
authors.initials: DG
authors.lastname: Musson
authors.forename: Kimberly L
authors.initials: KL
authors.lastname: Birk
authors.forename: Chester J
authors.initials: CJ
authors.lastname: Kitchen
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
authors.forename: John Y K
authors.initials: JY
authors.lastname: Hsieh
authors.forename: Wei
authors.initials: W
authors.lastname: Fang
authors.forename: Anup K
authors.initials: AK
authors.lastname: Majumdar
authors.forename: John D
authors.initials: JD
authors.lastname: Rogers
mesh_terms: D000900:Anti-Bacterial Agents / Q000097:blood* / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D000077727:Ertapenem; D006801:Humans; D007769:Lactams; D012015:Reference Standards; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D014462:Ultrafiltration; D047090:beta-Lactams
chemical_list: D000900:Anti-Bacterial Agents; D007769:Lactams; D047090:beta-Lactams; D000077727:Ertapenem