Differential adsorption of analyte and deuterated analogue onto the rotor seal of the injector in high-performance liquid chromatography/electrospray ionisation mass spectrometry.

Rationale Quantitation of analytes by liquid chromatography/mass spectrometry (LC/MS) is now very widely used, usually with an appropriate internal standard, but this does not guarantee problem-free analyses. Investigation of a problem that arose during method development is described, where it was surmised that differential adsorption onto the high-performance liquid chromatography (HPLC) injector of the analyte (Rac GTPase inhibitor NSC23766) and its deuterated analogue, as internal standard, was taking place. Methods Samples were injected onto the HPLC system via either a Valco C14W.1 internal sample injector or a Rheodyne 7125 injector, both with dissimilar Vespel rotor seals, and linked to a quadrupole time-of-flight (QTOF) mass spectrometer. Alternatively, samples were infused directly into the mass spectrometer via a syringe-driver. Electrospray ionisation in positive mode was used in both cases. Results Experiments demonstrated significant differential adsorption of the analyte (RAC inhibitor, NSC23766) and its tri-deuterated internal standard onto the Vespel rotor seal, with the latter seeming to be preferentially adsorbed. The deuterated analogue also showed lower electrospray sensitivity, as demonstrated by syringe infusion of a mixture of the compounds, compared with their separate infusion at the same concentration. Conclusions This study has demonstrated the problem of differential adsorption onto HPLC Vespel rotor seals, and that the use of a stable-isotope-labelled analogue as internal standard does not guarantee the constancy of the analyte/internal response ratio in quantitative methods. The partial solution was to work at much higher concentration where adsorption, whilst still apparent, was relatively insignificant.