Elimination of diastereomer interference to determine Telcagepant (MK-0974) in human plasma using on-line turbulent-flow technology and off-line solid-phase extraction coupled with liquid chromatography/tandem mass spectrometry.
To eliminate the diastereomer interference on Telcagepant (MK-0974) determination during clinical study support, on-line high turbulent-flow liquid chromatography (HTLC) methods, HTLC-A and HTLC-B that covered dynamic range of 0.5-500 nM and 5-5000 nM, respectively, were developed. To meet the requirement of rapid assay transfer among multiple laboratories and analysts, a solid-phase extraction (SPE) assay was derived from the existing HTLC-B assay under the same dynamic range. The on-line HTLC assays were achieved through direct injection of plasma samples, extraction of analyte with a Cohesive C18 column (50 mm x 0.5 mm, 50 microm), followed by HPLC separation on a FluoPhase RP column (100 mm x 2.1 mm, 5 microm) and MS/MS detection. The off-line SPE assay used Waters Oasis HLB microElution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm x 2.1 mm, 5 microm) for LC-MS/MS analysis. Under both on-line and off-line assay conditions, the diastereomer 1c was chromatographically separated from MK-0974. Cross-validation with the pooled samples demonstrated that both on-line and off-line assays provided comparable data with a difference of < 2.6%. The assays were proved to be specific, accurate and reliable, and have been used to support multiple clinical studies. The pros and cons of on-line and off-line assays with regard to man power involved in sample preparation, total analysis time, carryover, cost efficiency, and the requirement for assay transfer are discussed.
Xu
Yang
Y
Merck Research Laboratories, Clinical PK/PD, Department of Drug Metabolism, WP75A-303, West Point, PA 19486, USA. yang_xu@merck.com
Willson
Kenneth J
KJ
Anderson
Melanie D G
MD
Musson
Donald G
DG
Miller-Stein
Cynthia M
CM
Woolf
Eric J
EJ
D001381:Azepines / Q000097:blood* / Q000627:therapeutic use; D002853:Chromatography, Liquid / Q000379:methods*; D017022:Flow Injection Analysis / Q000379:methods*; D006801:Humans; D007093:Imidazoles / Q000097:blood* / Q000627:therapeutic use; D013058:Mass Spectrometry / Q000379:methods*; D008881:Migraine Disorders / Q000188:drug therapy; D052616:Solid Phase Extraction / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods*
D001381:Azepines; D007093:Imidazoles; C525458:telcagepant