Direct-injection HPLC assay for the determination of a new carbapenem antibiotic in human plasma and urine.
Reversed-phase high-performance liquid chromatography (RP-HPLC) assays using ultraviolet (UV) absorbance detection have been developed for the determination of a new carbapenem antibiotic I in human plasma and urine. A column-switching technique is employed in the HPLC methods to perform on-line extraction and separation for each sample. Each plasma sample is thawed, centrifuged, stabilized, and then injected onto an in-line reversed-phase extraction column using a methanol (8%)/phosphate buffer, pH 6.5. After 3 min, the analytes are back-flushed off the extraction column with a mixture of acetonitrile (5.5%) and methanol (10%)/phosphate buffer (pH 6.5) for 3 min onto a BDS Hypersil 3 microm C18 (100 x 4.6 mm i.d.) analytical column. The sample preparation and HPLC conditions for the urine assay are similar to the plasma assay, except that a CN extraction column is used. Both assays are specific with respect to endogenous material and the major metabolite II, and both are linear over the concentration range of 0.25-50, and 2-200 microg/ml, respectively. The assays were successfully applied to a clinical dose-ranging study. One limitation of the on-line extraction method is that the extraction column needs to be replaced regularly every 100-150 plasma samples and every 200-300 urine samples. Subsequently, the urine method was modified to an ion-pair HPLC assay for the simultaneous determination of both the antibiotic I and its metabolite II.
Zhang
Jin
J
Merck Research Laboratories, WP75A-303, Sumneytown Pike, West Point, PA 19486, USA. jin_zhang@merck.com
Musson
Donald G
DG
Birk
Kimberly L
KL
Cairns
Ann M
AM
Fisher
Alison L
AL
Neway
William
W
Rogers
John D
JD
D015780:Carbapenems / Q000097:blood* / Q000737:chemistry / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D006801:Humans
D015780:Carbapenems