Abstract
Porous graphitic carbon (PGC) is an important tool in a chromatographer’s armory that retains polar compounds with mass spectrometry (MS)-compatible solvents. However, its applicability is severely limited by an unpredictable loss of retention, which can be attributed to contamination. The solutions offered fail to restore the original retention and our observations of retention time shifts of gemcitabine/metabolites on PGC are not consistent with contamination. The mobile phase affects the ionization state of analytes and the polarizable PGC surface that influences the strength of dispersive forces governing retention on the stationary phase. We hypothesized that failure to maintain the same PGC surface before and after running a gradient is a cause of the observed retention loss/variability on PGC. Herein, we optimize the choice of mobile phase solvent in a gradient program with three parts: a preparatory phase, which allows binding of analytes to column; an elution phase, which gives the required separation/peak shape; and a maintenance phase, to preserve the required retention capacity. Via liquid chromatography/tandem mass spectrometry (LC-MS/MS) analysis of gemcitabine and its metabolites extracted from tumor tissue, we demonstrate reproducible chromatography on three PGC columns of different ages. This approach simplifies use of the PGC to the same level as that of a C-18 column, removes the need for column regeneration, and minimizes run times, thus allowing PGC columns to be used to their full potential.
Highlights
Porous graphitic carbon (PGC) is an important tool in a chromatographer’s armory that retains polar compounds with mass spectrometry (MS)-compatible solvents
We report a method for robust maintenance of retention capacity of gemcitabine and its metabolites on porous graphitic carbon (PGC) through careful selection of the mobile phase and sequence in a gradient elution program that obviates the need for washing
We have developed a liquid chromatography method on porous graphitic carbon (PGC) that overcomes a key challenge of variable retention and loss of retention
Summary
Problems with previous methods start when a new column is exposed to mobile phases other than methanol:water ((95:5), hereafter referred to as 95% methanol), which may modify the surface of the stationary phase resulting in altered retention properties. Our previous gradient elution program (Figure S1 in the Supporting Information), which had at least 5% acetonitrile throughout,[6] suffered from the same problems; gemcitabine retention capacity slowly reduced and limited the number of samples that could be analyzed in one batch This meant that after approximately every 50 samples regeneration of the PGC column was required. In order to test how this gradient on PGC compared to the performance of a C-18 column using the same pump, a method was set up on an Acquity T3 C-18 column for gemcitabine and dFdU only (because the phosphorylated metabolites dFdCTP and GdPC are not retained on the C-18 column, eluting in the column void volume) (see Figure S-4 in the Supporting Information). Using acetonitrile in the maintenance step did not work: dFdCTP and GdPC were no longer retained, illustrating the importance of choosing the correct mobile phase to use in a gradient elution program
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