The application of small porous particles, high temperatures, and high pressures to generate very high resolution LC and LC/MS separations.

The effect of combining sub-2 microm porous particles with elevated operating temperatures on chromatographic performance has been investigated in terms of chromatographic efficiency, productivity, peak elution order, and observed operating pressure. The use of elevated temperature in LC does not increase the obtainable performance but allows the same performance to be obtained in less time. Increasing the column temperature did allow the use of longer columns, generating column efficiencies in excess of 100,000 plates and gradient peak capacities approaching 1000. Raising the temperature increased the optimal mobile phase linear velocity, negating somewhat the pressure benefits observed by reducing the solvent viscosity. When operating at higher temperature the analyte retention is not only reduced, but the order of elution will also often change. High temperature separations allowed exotic organic modifiers such as isopropanol to be exploited for alternative selectivity and faster analysis. Finally, care must be taken when using high temperature separations to ensure that the narrow peak widths produced do not compromise the quality of data obtained from detectors such as high resolution mass spectrometers.

A study of ion suppression effects in electrospray ionization from mobile phase additives and solid-phase extracts.

Since the wide adoption of liquid chromatography/tandem mass spectrometry (LC/MS/MS), the ion suppression/enhancement phenomenon is the latest barrier to high-throughput analysis. This consequence of a nonoptimized analytical method can lead to adverse effects during quantitation (i.e. poor accuracy and precision). Previous papers have reported that ion suppression is a direct result of endogenous material present in biological samples. However, in the case of a solid-phase liquid chromatography/tandem mass spectrometry (SPE/LC/MS/MS) system, the measured result is the combination of several operating conditions and parameters. Little has been done to effectively monitor and/or choose optimized conditions for the complete sequence of extraction, clean up, separation and analysis. This paper describes a simple setup for quantification of ion suppression/enhancement. Several mobile phase additives, ion-pairing agents and SPE extracts were measured and compared against a standard reference. The results demonstrated that a clean up of plasma extracts based on ion exchange leads to minimal ion suppression/enhancement for the compounds that were investigated.

Analysis of a basic drug by on-line solid-phase extraction liquid chromatography/tandem mass spectrometry using a mixed mode sorbent.

Two on-line configurations using multiple 6- and 10-port valves were investigated for high-flow on-line extraction of a basic drug in rat plasma and human urine using a reversed-phase and a cation-exchange SPE sorbent. The first configuration studied was a single reversed-phase extraction cartridge (2.1 x 20 mm, 25 microm) using an optimized washing protocol. The results showed that up to 1.5 mL of sample (urine or plasma diluted 1:1) can be injected with limits of quantification (LOQs) as low as 100 pg/mL. The second configuration studied was the combination of a cation exchanger and a reversed-phase cartridge using at-column dilution. The results showed better LOQs than those obtained with the single cartridge at 10 pg/mL with the same injection volume. The mass spectrometer was operated in the multiple reaction monitoring (MRM) mode. All calibration curves gave an average of 5% relative standard deviation (RSD).