Separation of Diastereomeric Flubatine Metabolites using SelexION Technology

Key Challenges of Metabolite Separation 1. Metabolite analysis features complex matrices. Samples from plasma and urine are common, but also from in vitro metabolite studies using liver microsomes, possibly causing elevated background signals or isobaric interferences. 2. Enzymatic metabolization often results in a set of isobaric compounds. MS/MS fragmentation can differentiate constitutional differences of an isomeric analyte pair. However, to achieve separation of stereoisomers, additional techniques, e.g. chromatography or ion mobility separation are needed. (DMS, Figure 1 & 2) features an additional dimension of selectivity and is easily combined with other common separation techniques e.g. chromatography or MS/MS. 2. DMS allows distinguish of constitutional isomers and diastereomers. 3. A general background and interference elimination for MRM base quantification is possible. 1. Interference-free quantification of constitutional isomers and diastereomers. 2. Mapping of compensation voltage, a compound specific DMS parameter, generates a unique fingerprint for future comparison of metabolite samples. 3. DMS based isomer separation allows for shortened LC gradients, resulting in an increased throughput. Figure 1: SelexION ® technology ion path components: source extension ring (1), heated curtain plate (2), DMS cell (3).. Figure 2: A DMS mobility cell is composed of 2 flat plates that are parallel and define a mobility region. RF voltages (separation voltages) are applied across the ion transport channel, perpendicular to the direction of the transport gas flow. Due to the difference between high and low field ion mobility coefficients, ions will migrate toward the walls and leave the flight path unless their trajectory is corrected by a counterbalancing voltage, a DC potential referred to as compensation voltage. The compensation voltage is scanned serially to pass ions according to their differential mobility, or set to a fixed value to pass only the ion species with a particular differential mobility.