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In recent years, different methods based on reversed phase liquid chromatography have been developed to detect and quantify these peptides.
The separations were carried out based on reversed phase chromatography or cation exchange chromatography with volatile elution buffer.
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False discovery rates (FDR) of peptide hits (phosphorylated and unphosphorylated) were estimated below 1.5% based on reversed database hits.
The chromatographic separation was based on a reversed phase mechanism carried out under isocratic elution mode for only less than 15-min total run time.
High performance LC (HPLC) was performed by gradient elution using methanol/water/formic acid on reversed phase separation columns.
The plasma of insects was then acidified and fractionated by solid phase extraction on reversed phase Sep-Pak cartridges (Figure 5A).
Pralidoxime was determined by HPLC on reversed phase material with an ion-pairing reagent.
HPLC was performed by gradient elution using methanol/water/formic acid on reversed phase separation columns.
Finally, the products were chromatographically purified on reversed phase chromatography (C18) and their identity verified by MALDI mass spectrometry.
On reversed phase chromatography systems, PGE2 elutes before PGD2, and this elution order is maintained for the glyceryl esters and ethanolamides (unpublished observations).
Hydroxylation of Tyr and Trp and oxidation of Met and Cys to sulfoxide or sulfoacids decrease their hydrophobicity, thus reducing peptide retention on reversed phase (RP) columns.
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