Development of LC/MS/MS Methods for Cocktail Dosed Caco-2 Samples Using Atmospheric Pressure Photoionization and Electrospray Ionization

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Abstract
Good reliability of Caco-2 permeability studies requires competent sampling and analytical methods to ensure the comparability of day-to-day experiments. In this work, two n-in-one LC/MS/MS methods based on two different ionization techniques were developed and validated for a group of reference compounds; eight of them are recommended by the Food and Drug Administration (FDA) for the evaluation of oral drug permeability. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), as an interface for quantitative LC/MS analysis was evaluated in comparison to the electrospray ionization (ESI). Generally, the validation parameters, including sensitivity, accuracy, and repeatability, were comparable for the APPI and ESI methods. The main difference was that the linear quantitative range of APPI was 3−4 orders of magnitude (r2 ≥ 0.998) whereas in ESI it was typically 2−3 orders of magnitude (r2 ≥ 0.990). By the APPI and ESI methods, the simultaneous analysis of nine highly heterogeneous compounds was achieved within 5.5−7 min, which leads to significant savings in time and cost of the analyses. The successful validation data indicate the usefulness of both the methods for the rapid and sensitive (LOD values typically ≤5 nM) testing of drug transport across the Caco-2 monolayers, and after case-specific modification, the methods enable the permeability screening of new drug candidates. The applicability and reliability of both methods was demonstrated with a real batch of Caco-2 samples, each providing similar values of apparent permeability (Papp). In addition, the individual permeabilities of the compounds in the cocktail experiments correlated closely to the ones obtained from single-compound experiments.

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