The separation of intact proteins is inherently more complex than that of small molecules using reversed-phase liquid chromatography. The goal of this work was to determine a reasonable set of operational parameters (a recommended starting point for other analysts) for the separation of intact proteins and their detection by triple quadrupole mass spectrometry. Although protein separations have been studied for many years, the direct detection of intact proteins with mass spectrometry requires special considerations of mobile phase additives to achieve efficient separation and sensitive detection. Myoglobin, cytochrome c, lactalbumin, lysozyme, and ubiquitin were used as model analytes to investigate chromatographic method development using a triple quadrupole mass spectrometer and detection by multiple reaction monitoring. Chromatographic parameters including the concentration of trifluoroacetic acid, flow rate, gradient slope, temperature, mobile phase composition, and stationary phase chemistry were evaluated. Protein charge state profiles were also monitored for temperature and modifier effects. An optimized method using 0.2 mL/min flow rate, 15% gradient slope, and 75°C with a combined trifluoroacetic acid and formic acid modified mobile phase was developed.

Nine cigarette smokers ingested an average daily 0.032 mg/kg dose of sodium warfarin for 2 wk while continuing to smoke and for an additional 2 wk after having abstained from cigarette smoking for a month washout phase. Steady-state plasma levels of warfarin, clearances, t 1/2, apparent volumes of distribution, steady-state prothrombin times, and plasma thiocyanate levels were measured during both the smoking and nonsmoking phases. During the nonsmoking phase there was a 13% increase in average steady-state warfarin level and a 13% decrease in warfarin clearance rate. There also was a 23% increase in warfarin t 1/2 and an 11% increase in the apparent volume of distribution. Prothrombin time did not change. Thiocyanate levels were 3 to 4 times as high during the smoking than the nonsmoking phase. It appears that cigarette smoking does affect warfarin clearance, t 1/2, and apparent volume of distribution, though the net effect on warfarin's pharmacodynamic activity is negligible, at least at doses which are ineffective therapeutically.

There are various reversed-phase stationary phases that offer significant differences in selectivity and retention. To investigate different reversed-phase stationary phases (aqueous stable C18 , biphenyl, pentafluorophenyl propyl, and polar-embedded alkyl) in an automated fashion, commercial software and associated hardware for mobile phase and column selection were used in conjunction with liquid chromatography and a triple quadrupole mass spectrometer detector. A model analyte mixture was prepared using a combination of standards from varying classes of analytes (including drugs, drugs of abuse, amino acids, nicotine, and nicotine-like compounds). Chromatographic results revealed diverse variations in selectivity and peak shape. Differences in the elution order of analytes on the polar-embedded alkyl phase for several analytes showed distinct selectivity differences compared to the aqueous C18 phase. The electron-rich pentafluorophenyl propyl phase showed unique selectivity toward protonated amines. The biphenyl phase provided further changes in selectivity relative to C18 with a methanolic phase, but it behaved very similarly to a C18 when an acetonitrile-based mobile phase was evaluated. This study shows the value of rapid column screening as an alternative to excessive mobile phase variation to obtain suitable chromatographic settings for analyte separation.

No abstract