Calculation and Mitigation of Isotopic Interferences in Liquid Chromatography–Mass Spectrometry/Mass Spectrometry Assays and Its Application in Supporting Microdose Absolute Bioavailability Studies

Abstract

A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

Microdosing and drug development: past, present and future

“…The 14 C isotopic label in the intravenously administered drug allows the pharmaco-kinetics of the intravenous dose to be distinguished from that of the oral dose, thereby obtaining both the oral and intravenous pharmacokinetics from the same subject in a single dosing. The intravenous dose is sometimes referred to as a ‘microdose’, for example [27,28], but this is again misleading as the underlying pharmacokinetics of the tracer intravenous dose is very different to a true microdose where the human subjects are only systemically exposed to a maximum of 100 µg irrespective of the route of administration. In the intravenous tracer study, there is no question of extrapolating the pharma-cokinetics from a low ‘microdose’ to a therapeutic dose as the systemic concentrations of drug are mostly driven by the absorbed therapeutic dose given orally.…”

Stable isotope‐labelled intravenous microdose for absolute bioavailability and effect of grapefruit juice on ibrutinib in healthy adults

“…14 C radioisotopes can be measured with high sensitivity using accelerated mass spectrometry (AMS) . Owing to significant improvements that have been made in the sensitivity of triple quadrupole mass spectrometry instrumentation over the past decade, it is now also possible to analyse nonradioactive drugs with high sensitivity, down to the low or sub‐pg ml –1 concentration range . This makes it possible to apply the microdosing approach by administering a SIL drug rather than a 14 C‐labelled drug and to use triple quadrupole mass spectrometry for the quantification of both the SIL‐labelled and unlabelled drug.…”

Combining Isotopic Tracer Techniques to Increase Efficiency of Clinical Pharmacokinetic Trials in Oncology

“…As both unlabeled and stable isotopically labeled drug are present in the circulation at the same time, there is a need to selectively quantify both compounds. The number of labels required to distinguish the intravenous microdose from the unlabeled extravascular dose can easily be determined using isotopic distribution pattern software and the method described by Gu et al [22,23], in combination with the known C max of the extravascular dose and the expected C max of the intravenous microdose. When using an intravenously administered stable isotopically labeled microdose, there is potential that the presence of the isotope alters reaction rates in vivo because the atomic bond strength is dependent on the mass of the bonded isotope.…”