Effect of mobile phase pH, aqueous-organic ratio, and buffer concentration on electrospray ionization tandem mass spectrometric fragmentation patterns: implications in liquid chromatography/tandem mass spectrometric bioanalysis.
Liquid chromatography/tandem mass spectrometry (LC/MS/MS) based on selected reaction monitoring (SRM) is the standard methodology in quantitative analysis of administered xenobiotics in biological samples. Utilizing two SRM channels during positive electrospray ionization (ESI) LC/MS/MS method development for a drug compound containing two basic functional groups, we found that the response ratio (SRM1/SRM2) obtained using an acidic mobile phase was dramatically different from that obtained using a basic mobile phase. This observation is different from the well-established phenomenon of mobile phase affecting the [M+H](+) response, which is directly related to the amount of the [M+H](+) ions produced during the ionization. Results from follow-up work reported herein revealed that the MS/MS fragmentation patterns of four drug or drug-like compounds are affected not only by the pH, but also by the aqueous-organic ratio of the mobile phase and the buffer concentration at a given apparent pH. The observed phenomenon can be explained by invoking that a mixture of [M+H](+) ions of the same m/z value for the analyte is produced that is composed of two or more species which differ only in the site of the proton attachment, which in turn affects their MS/MS fragmentation pattern. The ratio of the different protonated species changes depending on the pH, aqueous-organic ratio, or ionic strength of the mobile phase used. The awareness of the mobile phase dependency of the MS/MS fragmentation pattern of precursor ions of identical m/z value will influence LC/MS/MS-based bioanalytical method development strategies. Specifically, we are recommending that multiple SRM transitions be monitored during mobile phase screening, with the MS/MS parameters used for each SRM optimized for the composition of the mobile phase (pH, organic percentage, and ionic strength) in which the analyte elutes.
Wang
Jian
J
Bristol-Myers Squibb, Research & Development, Analytical Research and Development, Princeton, NJ 08543, USA. jian.wang@bms.com
Aubry
Anne
A
Bolgar
Mark S
MS
Gu
Huidong
H
Olah
Timothy V
TV
Arnold
Mark
M
Jemal
Mohammed
M
D002853:Chromatography, Liquid; D024841:Fluoroquinolones / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009930:Organic Chemicals / Q000737:chemistry; D009994:Osmolar Concentration; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D053719:Tandem Mass Spectrometry; D014867:Water / Q000737:chemistry
D024841:Fluoroquinolones; D009930:Organic Chemicals; D014867:Water