Ultra‐fast gradient vs. fast isocratic chromatography in bioanalytical quantification by liquid chromatography/tandem mass spectrometry

Abstract

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods developed for quantification using rapid ('ballistic') gradients on narrow bore, short HPLC columns have been previously described by this laboratory. This paper compares the fast gradient approach with the more traditional high-organic isocratic LC/MS/MS methods. The comparison is based on an analysis of the effectiveness of the chromatographic separations when using the two approaches (i.e. k', N, and W). The data presented herein are derived from actual biological samples analyzed as part of the drug discovery process.

Fast analysis in liquid chromatography using small particle size and high pressure

“…Method validation was performed successfully with this short column packed with 5 lm particles coupled with ESI/MS/MS. Other applications reported in the literature with short columns used gradient LC methods coupled with MS or MS/MS [5][6][7][8][9][10][11][12][13][14].…”

Principles and Applications of LC-MS/MS for the Quantitative Bioanalysis of Analytes in Various Biological Samples

“…Today, two driving forces continue to test the limits of HPLC. One is the need for faster separations, such as analyses of either simple samples or a few constituents in a complex sample (Romanyshyn, 2001). The second is the desire to achieve greater separation power to quantify or identify all the constituents of a complex sample or to compare the contents of complex samples with each other (van der Horst & Schoenmakers, 2003;Liu et al 2002;Plumb et al 2003).…”

LC‐MS in Drug Metabolism and Pharmacokinetics: A Pharmaceutical Industry Perspective

“…Bioanalytical scientists that support ADME screens are constantly searching for ways to improve LC -MS analysis speed. High -throughput LC -MS bioanalytical approaches such as high speed separation on short columns packed with small particles [52] , ultrahigh pressure liquid chromatography ( UHPLC ) using sub -2 μ m [53,54] or other high effi ciency columns such as fused core columns [55] , are all very much applicable in ADME This process of obtaining an optimal SRM transition and corresponding lens voltages is a prerequisite for achieving the best sensitivity and specifi city for quantitative bioanalysis, and has to be performed for each individual test compound of interest. Usually, SRM optimization is conducted manually by infusing a compound solution into the instrument while the mass spectrometer is scanning, and ramping individual parameters to obtain their optimum values, at a throughput of about 5 -10 min per compound.…”