Mitigating analyte to stable isotope labelled internal standard cross-signal contribution in quantitative liquid chromatography-tandem mass spectrometry.

Background: Utilising stable isotope labelled internal standards (SIL-IS) in quantitative LC-MS/MS drug analysis is the most widely used approach to normalise for variability during sample quantification processes. However, compounds containing atoms such as Sulphur, Chlorine or Bromine, could potentially cause cross-signal contribution to the SIL-IS from the naturally occurring isotopes, resulting in non-linear calibration curves. A simple, novel method of mitigating the effect is presented here. It entails monitoring of a less abundant SIL-IS isotope, as the precursor ion, of a mass that has no/minimal isotopic contribution from the analyte isotopes. Methods: Experiments were conducted on two LC-MS/MS analysers: Waters Xevo TQ-S and Shimadzu 8050. Flucloxacillin (FLX) was used as an example. Two transitions were selected for FLX (m/z 454 â†’ 160 â†’ 295) and one for each of the SIL-IS isotopes (m/z 458 â†’ 160 for the isotope 457 g/mol and m/z 460 â†’ 160 for the isotope 459 g/mol). Assay biases were assessed at three SIL-IS concentrations: 0.7, 7 and 14 mg/L for each isotope. Results: When using the SIL-IS isotope m/z 458 â†’ 160 at a concentration of 0.7 mg/L, biases were up to 36.9 % on both instruments. Increasing the SIL-IS concentration to 14 mg/L, reduced the bias to 5.8 %. Using the less abundant isotope, m/z 460 â†’ 160, resulted in biases of 13.9 % at an SIL-IS concentration of 0.7 mg/L. Conclusions: Applying this method will mitigate cross-signal contribution from the analyte isotopes to the corresponding SIL-IS, minimise the use of SIL-IS, and, thereby, reduce overall cost.