Quantitative aspects of analyzing small molecules - monitoring singly or doubly charged ions? A case study of ximelagatran.

Precision, reproducibility and lower limit of quantitation (LLOQ) are important characteristics of a quantitative method. We have investigated these properties for Ximelagatran (Xi), which has a high tendency to form doubly charged ions in electrospray ionization (ESI), by studying the percentage of doubly charged species formed when varying the formic acid (FA) concentration, analyte concentration, amount of organic modifier and flow rate. It was found that the percentage of [Xi + 2H](2+) can be controlled to be more than 90% or less than 10% by varying the amount of FA present, and that the change between these values is dramatic. Furthermore, the percentage of [Xi + 2H](2+) formed decreases with increased analyte concentration and increased flow rate. No apparent relationship with the amount of organic modifier was found. The results have the implication that, by carefully controlling the selected parameters, the LLOQ, precision and reproducibility can be improved. We have compared the fragmentation of the singly and doubly charged species and concluded that the [Xi + 2H](2+) ion is more inclined to undergo fragmentation than [Xi + H](+). As a consequence, unusual instrumental settings had to be used for the experiments. The fragmentation patterns are to a great extent similar, but the doubly charged species is more inclined to generate low-mass product ions.