Analyte adduct formation is well known in LC-MS n [1,2]; however, in comparison to the most important means of ionization in ESI MS such as analyte protonation (positive ion mode) and deprotonation (negative ion mode), its application can be defined as rather limited. This is not really a surprise when considered that adduct formation was sidelined as a pitfall in LC-MS n analysis equivalent to other sources of inaccuracy [3,4]. Nevertheless, the latest research demonstrated that this strategy of analyte ionization/fragmentation is a promising alternative for solving different problems concerning the analysis of small molecules relevant in clinical/forensic toxicology, with possibilities exceeding standard applications focused on higher sensitivities in comparison to protonation/deprotonation often observed and used for quantitative analysis [5,6]. In this article, different strategies of LC-MS n analyses of problematic small-molecule drugs based on analyte adduct formation interesting for the field of bioanalysis were discussed on the basis of the latest literature. In focus are both small drugs with poor fragmentation, where the detection of small ion fragments is necessary when the conventional LC-MS 2 strategy is applied, and small drugs producing no stable ion fragments, where the product ion spectrum has no signals applicable for drug detection. Further, the application of analyte adducts in the LC-MS n analysis of small-molecule drugs with problematic ionization based on protonation/deprotonation was also pointed out.
Generation of bigger ion fragments of small-molecule drugs with poor fragmentation for LC-MS2 applications To avoid the detection of small ion fragments, an analyte adduct formation/fragmentation strategy was developed for the generation of bigger ion fragments that could be used for LC-MS 2 analysis of γ-hydroxybutyrate (GHB), which was used as a model drug [7]. A GHB sodium acetate adduct ion was described as a target, of which the fragmentation was the basis for LC-MS 2 analysis to avoid the detection of ions smaller than 85 Da as described previous [8]. Sample preparation based on a protein precipitation performed with the mobile phase, together with a short analysis time of 3 min and a calculated LOD/LOQ of 1 μg/ml, directly warranted a fast and sensitive analysis. Under conditions used with a mobile phase consisting of 10% A (H 2 O/methanol = 95/5, vv) and 90% B (H 2 O/methanol = 9/97, vv) both with 10 mM ammonium acetate and 0.1% acetic acid corresponding adducts were identified for GHB-D6 applied as internal standard for appropriate signal compensation.
Realization of LC-MS3 analyzes for small-molecule drugs with poor ion fragmentation The same mobile phase conditions were used to develop a protein precipitation based sample preparation for GHB analysis, with a three-stage m/z separation strategy that was applied for drug detection by LC-MS 3 . The application of the GHB sodium acetate adduct ion enabled the optimization of a two-step fragmentation (185→103→85), which resulted in an ...