Suppression effects of carbonate on the interaction between stainless steel and phosphate groups of phosphate compounds in high-performance liquid chromatography and electrospray ionization mass spectrometry

We examined the suppression methodology of the interaction between phosphate compounds, such as nucleotides, and the stainless steel surfaces of high-performance liquid chromatography and electrospray ionization mass spectrometry (HPLC/ESI-MS) equipment in an effort to prevent the tailing of peaks seen in HPLC chromatograms of phosphate compounds. Addition of carbonate (CO32−) to mobile phase was highly effective in suppressing the interaction of phosphate compounds derived from a complexation between phosphate groups and metal ions that exist on a stainless steel surface in a mechanism similar to Fe(III)- and Cr(III)-immobilized metal affinity chromatography (IMAC). Addition of ammonium hydrogen carbonate to mobile phase achieved a simple and reliable HPLC/ESI-MS analysis of mono-, di-, and triphosphate compounds (six nucleotides) without peak tailing due to the interaction between stainless steel surfaces and phosphate groups. Moreover, ammonium hydrogen carbonate buffer, a volatile buffer with good buffering capacity at neutral pH, does not compromise the stability of silica-based HPLC columns, decreases in sensitivity, ion source pollution, clogging of the ESI interface, and/or ion suppression in HPLC/ESI-MS.