Aqueous normal phase retention of nucleotides on silica hydride-based columns: method development strategies for analytes relevant in clinical analysis.
An aqueous normal phase HPLC method coupled with UV or ESI/MS detection was used for the determination of a wide variety of nucleotides, essential in metabolomics studies. Fifteen nucleotides were tested in clinically relevant mixtures at levels of 100 microg/mL for UV detection and 1 microg/mL for ESI-MS detection. Analysis times for all protocols developed were less than 20 min. The chromatographic conditions were changed to achieve optimized retention and separation of the nucleotides studied. The aqueous normal phase-HPLC methods were developed utilizing two columns, one having a minimally modified hydride surface another having an undecanoic acid moiety on a hydride surface. Volatile, low ionic strength mobile phases were used. Negative ion mode ESI-MS at near neutral pH mobile phase, combined with a TOF detector provided a highly sensitive and specific method, which is equally suitable for quadrupole and ion trap instruments.
Matyska
Maria T
MT
Department of Chemistry, San Jose State University, San Jose, CA 95192, USA. mmatyska@sjsu.edu
Pesek
Joseph J
JJ
Duley
John
J
Zamzami
Mazin
M
Fischer
Steven M
SM
D002851:Chromatography, High Pressure Liquid; D009711:Nucleotides / Q000737:chemistry*; D012015:Reference Standards; D017640:Silicates / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013056:Spectrophotometry, Ultraviolet
D009711:Nucleotides; D017640:Silicates; C487012:silica hydride