Sammendrag
In the present work, a 2D-capLC-ESI-TOF-MS method for fractionation and separation of peptides in microdialysates is demonstrated. Both comprehensive and quantitative analysis was conducted. The microdialysates samples (335 μL) were loaded onto a 1 mm I.D. x 5 mm loading column packed with 5 μm Kromasil C18 particles by a carrier solution of 0.1 % formic acid in ACN/H2O (5/95, v/v) at a flow rate of 100 μL/min. Back-flushing onto a fractionating 0.32 mm I.D. x 150 mm ZIC-HILIC column packed with 5 μm particles was performed using a decreasing linear solvent ACN/H2O gradient containing 10 mM ammonium acetate at pH 6.8. Dilution of the effluent was done using a solution
containing 0.1 % formic acid prior to on-line collection of fractions onto multiple 1 mm I.D. x 5 mm Kromasil C18 column packed with 5 μm particles. Back-flushed elution of the fractions onto a 0.3 mm x 150 mm PLRP-S column packed with 3 μm particles was performed using an increasing stepped solvent ACN/H2O gradient containing 0.1 % formic acid. Positive ESI was performed in the m/z range of 200-1500. A post-column standard was introduced to MS detection when performing target compound analysis. The estimated concentration limit of detection (cLOD) was 0.15 ng/mL.