Strategies on efficient method development of on-line extraction assays for determination of MK-0974 in human plasma and urine using turbulent-flow chromatography and tandem mass spectrometry.

On-line extraction assays using cohesive high-turbulence liquid chromatography (HTLC) coupled with tandem mass spectrometer (MS/MS) have been developed for the determination of MK-0974 in human plasma and urine. In this report, a four-step strategy for efficient method development of an on-line extraction assay was discussed. Several challenges - namely extraction recovery, carryover and analyte loss to urine container - were addressed. The assay procedures included sample preparation on a Packard MultiPROBE II liquid-handling system, direct injection on a Cohesive Flux 2300 system, on-line extraction with a Cohesive C(18) column (0.5mmx50mm, 50microm), HPLC separation on a FluophasePFP column (50mmx3mm, 5microm) under cohesive quick-elution mode and MS detection on a Sciex API4000 in multiple-reaction monitoring (MRM) mode using positive ionization and turbo ion-spray. Since 37-80% analyte loss was observed in urine QCs, 1% BSA was added to bring urine QC accuracy back to approximately 100% of nominal. Because of the nature of BSA, the urine assay was established by adapting the plasma method. Thus, the two assays were able to be validated side-by-side, which reduced the validation time by approximately two-fold. The linear dynamic ranges were 0.5-500 and 1-1000nM for the plasma and urine assays, respectively. Obtained from five standard curves constructed with five lots of human plasma or urine, the intra-day precision (%CV) was <3.14 and <2.62%, and the accuracy was 98.3-101.0 and 99.13-100.64% of nominal for plasma and urine assays, respectively. Both plasma and urine QC samples were stable when kept at room temperature for 4h, at -70 degrees C for 3 weeks, or after three freeze-thaw cycles. Both assays gave reasonable relative recovery (>88.8%) and acceptable matrix effect (<15%). The carryover from the upper limit of quantification (ULOQ) was able to be controlled at <20% of lower limit of quantification (LLOQ).