Individual Abstract Info - Session: LC/MS
Solution Parameters Effecting Electrospray Signal Response and Quantitation in Gradient Reverse Phase Liquid Chromatography Electrospray Mass Spectrometry
David Welkie; Thomas P. White; Craig M. Whitehouse
Analytica of Branford, Inc., Branford, CT
Generating fine resolution Electrospray response curves for gradient LC/ES/MS operating conditions by conducting Electrospray current scans using Electrospray membrane probes.
Changes in organic solvent concentration causes change solution conductivity for a given electrolyte concentration during a reverse phase liquid chromatography (LC) gradient resulting in changes in Electrospray (ES) total current and sample signal response. Electrospray signal response for a given compound will change with acid, base or buffer (electrolyte) composition even when Electrospray is run at the same total current. In addition, the relative Electrospray signal response for different sample concentrations can change disproportionately at different Electrospray total currents. Consequently, analyte signal response in gradient LC Electrospray ionization can vary significantly depending on where the analyte elutes along the LC gradient. These differences in LC/ES/MS signal response along the LC gradient can directly affect quantitation linearity, accuracy and reproducibility.
A study was conducted to map the Electrospray signal response versus Electrospray total current over typical reverse phase LC solvent gradient compositions. A test set of peptides, each having different pKa values and molecular weights, and a test set of lower molecular weight drug compounds were Electrosprayed with pneumatic nebulization assist. A standard Electrospray inlet probe and an Electrospray membrane probe interfaced to an Analytica of Branford Time-Of-Flight mass spectrometer were used with both ES probes operating at ground potential. Electrospray total current scans were conducted by running gradients of acetic acid, formic acid, hydrochloric acid and triflouroacetic acid through the Electrospray membrane probe second solution flow path. Sample solution compositions were varied to simulate reverse phase LC gradients.
Electrospray electrical current versus organic solvent concentration in aqueous solutions was measured for different concentrations of acids added directly to the sample solution and for acids added only to the Electrospray membrane probe second solution. Similar Electrospray total current curves were generated using standard ES probes and ES membrane probes operating at ground potential for the complete range of solvent compositions tested. This allowed fine resolution mapping of Electrospray signal response over the ES total current and solvent composition space with direct correlation to standard Electrospray performance during gradient LC/ES/MS runs. Electrospray total current decreases with increasing organic solvent concentration when holding the sample solution electrolyte (acid) concentration constant. Non polar solvent acetonitrile concentration gradients in aqueous solutions were run first. Polar solvent Methanol concentration gradients were run second to provide a direct comparison of Electrospray signal response. Non polar Acetonitrile reduces Electrospray signal response for polar compounds such as peptides when compared to ES signal response using a polar methanol solvent at all organic solvent concentrations measured. Inorganic Acids (HCL and TFA) further reduce ES ion signal when compared with the signal response when using organic acids (acetic and formic). ES signal response for peptides can vary widely with changes in a polar (methanol) organic solvent concentrations in aqueous solutions. ES Signal response for peptides shows less variation with changes in non polar organic solvent (acetonitrile) concentrations ranging from 10% to 50% although the overall signal response is reduced compared with ES response running a methanol sample solution. ES Signal generally decreased with increasing ES current above 300 nA and for increasing concentrations of acetonitrile above 60%. Electrospray signal response curves will be presented for the full range of organic solvent compositions and gradients, Electrospray total current, sample species, electrolyte types and electrolyte concentrations tested. Presentation Poster