A severe peak tailing of phosphate compounds caused by interaction with stainless steel used for liquid chromatography and electrospray mass spectrometry

Abstract

A severe peak tailing was observed for adenosine 5'-monophosphate in flow injection analysis with stainless steel tubing and water/methanol mixture (1:1, v/v) as carrier. The cause of the peak tailing was investigated by focusing on the chemical structure of the analytes, the material used for the analytical systems and the composition of the carrier. We clarified that the peak tailing was caused by the interaction between phosphate residues in the analytes and stainless steel. The severe peak tailing did not occur with stainless steel tubing when the phosphate compounds were analyzed with carrier containing phosphoric acid or phosphate buffer. The findings indicate that such ill peak profiles are usually not considerable in conventional HPLC separation because phosphoric acid or phosphate buffer is quite commonly used in eluents. In LC-MS, however, the use of phosphoric acid and phosphate buffer is usually avoided because of their non-volatility; therefore this interaction between stainless steel and phosphate compound becomes predominant and results in severe peak tailings. We also found an effective method for avoiding the interaction. When stainless parts, such as LC tubing and ESI spray capillary, were treated with phosphoric acid prior to analysis, the peak profiles of the phosphate compounds were dramatically improved, even when non-phosphate buffer is used as carrier.

Fully validated assay for the quantification of endogenous nucleoside mono- and triphosphates using online extraction coupled with liquid chromatography–tandem mass spectrometry

“…The reduction of peak tailing by DEA was previously observed for nucleoside di-and triphosphates [9]. According to the literature, peak tailing results from interactions between phosphate compounds and stainless steel [22][23][24]. Therefore, triphosphate compounds are more affected than diphosphates and monophosphates [9].…”

Some factors that can lead to poor peak shape in hydrophilic interaction chromatography, and possibilities for their remediation

“…By analogy, the same interaction is known to take place with nucleotides [31]. The analysis of nucleotides under typical RPLC conditions can result in severely tailed peaks, which is a result of the metal components contained within the wetted parts of chromatographic and electrospray systems [33][34][35]. In this study, adenosine (nucleoside), adenosine-monophosphate (AMP), adenosine-diphosphate (ADP) and adenosinetriphosphate (ATP) were chosen as model test compounds to probe the severity of these interactions in HILIC.…”

Mass spectrometry in the quantitative analysis of therapeutic intracellular nucleotide analogs

“…Two groups extensively reported on the effect and concluded that it could, at least in part, be due to adsorption of phosphate groups to metal surfaces (Wakamatsu et al, 2005;Tuytten et al, 2006). Wakamatsu et al (2005) noticed extensive peak tailing when analyzing adenosine MP (AMP).…”

“…Wakamatsu et al (2005) noticed extensive peak tailing when analyzing adenosine MP (AMP). The same effect was seen for guanosine MP (GMP) and uridine MP (UMP), but not for adenosine, guanosine, or uridine.…”