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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 C(18) column (50 mm x 0.5 mm, 50 mu m), followed by HPLC separation on a FluoPhase RP column (100 mm x 2.1 mm, 5 mu m) and MS/MS detection. The off-line SPE assay used Waters Oasis (R) HLB mu Elution plate to extract the analytes from plasma matrix before injecting on a FluoPhase RP column (150 mm x 2.1 mm, 5 mu m) 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. (C) 2009 Elsevier B.V. All rights reserved.