Abstract
Pharmacokinetic (PK) studies on small animals are challenging as only small volumes of samples are available, in which the analyte is present at low concentration in a complex matrix. In this context, the use of miniaturized analytical techniques may provide undeniable advantages in terms of sensitivity, sample and solvent consumption compared to the reference UHPLC–MS/MS methods In this study, we present the development of a nanofluidic-LC–MS/MS method to analyze two model analytes of therapeutic interest, namely estradiol (E2) and estetrol (E4) after microsampling with volumetric absorptive microsampling (VAMS) devices, an innovative sampling technique to collect small volumes of whole blood. The nanofluidic LC–MS/MS method was developed using an experimental design to find the optimal conditions to analyze both E2 and E4 with the highest sensitivity. Subsequently, the optimized method was validated according to ICH guidelines and compared to a previously developed UHPLC–MS/MS method. A limit of quantitation of 50pg/ml was reached with the LC-chip method, which is 50 times better than UHPLC–MS/MS. Both methods were then critically evaluated from the analytical and operational points of view. Finally, the quantitation of estrogens after whole blood microsampling was compared with the results obtained with the corresponding plasma samples.
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