Development of a Small Volume Sampling Technique and LC-MS Orbitrap Assay for Pediatric Pharmacokinetic Studies of Fentanyl and its Metabolites
Mon 11:30 AM - Track 1: Small Molecule Analysis I
Uwe Christians
University of Colorado Denver
James J. Thomas, Keith Hoffman, Claudia Clavijo, Jeffrey Galinkin, Uwe Christians.

Clinical Research and Development, Department of Anesthesiology, University of Colorado Denver
Term and preterm neonates are considered “therapeutic orphans” for almost all drugs currently available. Without appropriate analytic techniques to effectively examine pharmacokinetic levels of parent drugs and metabolites it is impossible to make meaningful conclusions about drug distribution and potential toxicities short of clinical observation. Pharmacokinetic studies in infants, especially neonates, have been extremely difficult to realize to date because of (A) the difficulties with enrolling an adequate number of subjects, (B) the lack of validated low-volume bioanalytical assays, and (C) the ethical and patient safety issues with frequent sample collection of relatively large blood volumes without direct therapeutic benefit, especially in smaller, fragile preterm and sick infants. It was our goal to design and validate a platform strategy based on blood spot sampling on filter paper and LC-MS analysis using a Thermo Fisher Exactive orbitrap instrument. The challenge of filter sampling strategies is that the drugs and their metabolites have to be analyzed in whole blood, a much more complex matrix than plasma or serum typically used for the analysis of adult pharmacokinetics samples and that the blood volumes are small (20 µL volume). In addition we wanted to ensure that unknown metabolites were not missed, a common problem with triple stage quadrupole instruments. Thus, we used a highly sensitive Exactive orbitrap mass spectrometer in high resolution full scan mode.

Approximately 20-50 µL of human blood was spotted onto filter paper (Protein Saver 903 Snap Apart cards, by Whatman). The blood spot was allowed to dry and 5 mm punch was removed from the dried blood spot. The punched sample was placed into a 1.7 mL microcentrifuge tube along with 50 ìL of HPLC grade water. The sample was vortexed for 5 minutes and 300 µL of a solution containing 30% 0.2 M ZNSO4 / 70% methanol and deuterated fentanyl as internal standard. The samples were centrifuged for 10 minutes and the eluent was transferred to an injection vial. A 0,1 % formic acid / methanol gradient was run at a flow rate of 1.0 mL / min on an Agilent XDB C8 column (4.6 x 150 mm) with a 10 ìL injection volume. The assay was also able to detect and quantify fentanyl metabolites in the dried blood spots from pediatric patients. A lower limit of quantitation (LLOQ) of 200 pg/mL was achieved with a correlation coefficient of 0.998. Inter-assay variability was below 15% and inter-day accuracy was between 85-115% (except at the LLOQ). Dried fentanyl blood spots were stable on filter paper at room temperature for 5 days.

This work was supported by the National Institutes of Health, grant UL1RR025780.