and Mass Spectrometry

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An Optimized Stationary Phase for the HPLC/ESI/MS Analysis

presented by

Shane Needham
Alturas Analyticals, Inc.

September 14, 2000

The Scripps Research Institute, W.M. Keck Foundation Amphitheater


Shane Needham, recently of the Analytical group at Pfizer Central Research, is now running Alturas Analytics, Inc., located in Moscow, ID. Shane joined Pfizer in 1993 after graduating from Washington State University with a B.S. in Chemistry. While an undergraduate at Washington State University, Shane conducted research with Professor Herb Hill. With Hill's Group, he developed extraction and analysis methods using SFE, SFC/FID and GC/FID. Shane earned his Ph.D. in Analytical Chemistry under Professor Phyllis Brown at the University of Rhode Island. His area of academic research included the investigation of optimized stationary phases for the analysis of pharmaceuticals by HPLC/ESI/MS. At Pfizer, Shane evaluated and implemented new analytical technologies to improve current HPLC/MS and HPLC/MS/MS methods for qualitative and quantitative analysis. Shane developed high-throughput, sensitive HPLC/MS and HPLC/MS/MS assays for the quantitation of drugs and metabolites in biological fluids, and also performed in vivo and in vitro structure elucidation studies of drugs using HPLC/MS/MS and HPLC/MSn instrumentation.


The use of a pentafluorophenylpropyl (PFPP) stationary phase to provide optimum signal and peak shape for the high performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS) of basic pharmaceutical compounds is presented. A PFPP stationary phase gives retention factors (k) greater than 4 for various basic drugs with the use of 90% acetonitrile mobile phase. The PFPP phase also provides good peak shape (asymmetry factor = 1) for a variety of basic pharmaceuticals. C18 and other phenyl ring phases require less than 40% acetonitrile in the mobile phase to provide similar retention of the basic drugs. The increased concentration of acetonitrile in the mobile phase with the PFPP stationary phase provides better desolvation in the ESI interface and leads to a >10x increase in the MS signal when compared to a C18 phase. The PFPP phase was successful in the HPLC/ESI/MS analysis of many different classes of basic drugs with the use of one mobile phase composition. The drugs ranged in pKa (7.2-14.2), polarity and molecular weight (227.7-385.5 Daltons). To demonstrate the broad applicability of the PFPP stationary phase in the pharmaceutical industry, this phase was used for the validation of cocaine and its metabolite, ecgonine methyl ester assay in human urine by HPLC/ESI/MS/MS. The assay was accurate and precise. In addition, due to the better peak shape and use of 90% acetonitrile mobile phase, the limit of detection was 4 times lower than that obtained on a C18 stationary phase which could use only 12% acetonitrile mobile phase to provide a retention factor greater than 4.

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