Investigation of optimized stationary phases for the high performance liquid chromatography electrospray ionization mass spectrometric analysis of basic pharmaceuticals
The use of optimized stationary phases to improve the signal for the high performance liquid chromatography/electrospray ionization/mass spectrometry (HPLC/ESI/MS) of basic pharmaceutical compounds is presented. A cyanopropyl (CN) and pentafluorphenylpropyl (PFPP) stationary phase give retention factors (k) greater than 4 for various basic (pKa's > 7.0) drugs with the use of 90% acetonitrile mobile phase. The CN and PFPP phases also provide good peak shape (asymmetry factor ≈ 1) for a variety of basic pharmaceuticals. Hydrophobic chain phases (C18, C8 and C4) 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 CN and 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 CN and PFPP phases were 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). The CN and PFPP provided separations that were rapid with most of the analyses taking under seven minutes with the use of 30 x 2.1 mm I.D. columns. Also, the PFPP stationary phase was useful for the assay validation of cocaine and its metabolite, ecgonine methyl ester assay in human urine. The assay was accurate, precise, and rugged and the limit of detection was 4 times lower than that obtained on a C18 stationary phase. ^ Studies of the PFPP phase showed that the propyl chain connecting the pentafluorophenyl ring to the silica backbone was necessary to provide retention (k > 4) and good peak shape for the HPLC/ESI/MS analysis of basic drugs with the use of 90% acetonitrile. Additional research showed that the fluorine groups on the phenylpropyl moiety were necessary to provide the PFPP peak shape and retention characteristics. Investigation of the CN stationary phase showed that replacing the CN group with a methyl group diminished the peak shape of the basic solutes and retention of the solutes with 90% acetonitrile in the mobile phase was not possible. A hydroxypropyl phase gave retention of the basic solutes with the use of 90% acetonitrile; however, the peak shape of the solutes was not good enough to investigate this stationary phase further. Both the CN and PFPP phase will improve the analysis of pharmaceuticals by providing lower HPLC/ESI/MS detection limits than with the popular C18 phase. In addition to the enhanced MS signal when compared to a C18 phase, the CN and PFPP phases will also expedite the HPL method development process since one mobile phase composition has been shown to be successful for the HPLC/ESI/MS analysis of many basic drugs. ^
Shane Roy Needham,
"Investigation of optimized stationary phases for the high performance liquid chromatography electrospray ionization mass spectrometric analysis of basic pharmaceuticals"
Dissertations and Master's Theses (Campus Access).