Trace determination of peptides in water samples using large volume injection column switching temperature programmed packed capillary liquid chromatography with UV and MS detection

Abstract

A packed capillary column switching system has been developed, suitable for solute focusing of large sample volumes on a short precolumn prior to temperature-programmed elution on an analytical column. The peptides angiotensin II, bradykinin, bombesin, corazonin, neurotensin and substance P were used as model compounds, and a mobile phase composition of acetonitrile:water:trifluoroacetic acid (22:77.9:0.1, v/v %) served as the eluting mobile phase. 3 cm 320 µm I.D. columns were used as precolumns, and 15 cm 320 µm I.D. as analytical column. For the analytical column and precolumn, the stationary phases used was 3.5 µm Kromasil C18 and 10 µm Kromasil C18 particles, respectively. Sample volumes up to 1.0 mL were focused on the precolumn, prior to column switching and temperature programming. The low back pressure of the precolumn allowed a sample loading flow rate of 250 µL/min. The mass limit of detection of the method was on average 1.3 ng. This corresponds to a concentration limit of detection (cLOD) of 1.3 µg/L when using an injection volume of 1.0 mL using UV detection at 210 nm. The within assay precision and between assay precision for the method for peak areas were on average 5.9 % and 4.9 %, respectively. The method was applied for the determination of peptides in different spiked water samples. In spiked tap water peptides containing methionine, tryptophan and cystine were oxidized by hypochlorite. The oxidation products were further investigated and tentatively identified by packed capilary liquid chromatography with electrospray ionization mass spectrometer detection in the positive mode. Finally, the combined used of temperature gradient superimposed on a mobile phase gradient was shown to provide better selectivity for separation of the selected model peptides.