Validation of a quantitative liquid chromatography/hybrid quadrupole-linear ion trap MS/MS method for carfentanil and...

Carfentanil, an opioid 10K times more potent than morphine, has no licit clinical use. A powerful CNS depressant, it has been identified increasingly as the cause of overdose death in the United States. Because it is highly lipophilic, the law enforcement and medical communities have been concerned that responding personnel could be percutaneously exposed and that exposure could be enhanced with the use of alcohol-containing hand sanitizers. An LC-MS/MS method was developed to evaluate solvent effects on percutaneous absorption of carfentanil in a live human epidermal model.

In this study, a quantitative liquid chromatography/hybrid triple quadrupole-linear ion trap method was developed for carfentanil and for caffeine, a molecule routinely used to monitor epidermal cell culture viability. The method employed reverse-phase liquid chromatography coupled with positive electrospray ionization and multiple reaction monitoring (MRM) to quantify carfentanil and caffeine against calibration curves formulated from authentic standards. Limits of detection (LOD) for the two compounds were determined using 10:1 signal-to-noise requirements for all product ions with relative peak areas within ±20% of those observed for a mid-level calibrator. Precision and accuracy were determined by analyzing positive controls formulated in quintuplicate, by a different analyst, at three concentrations bracketing the method dynamic range. Inter-day precision was evaluated using data collected from three separate days of analyses.

Calibration curves for seven formulated replicates of the two compounds met linearity requirements over at least four orders of magnitude concentration range. The accuracy of measured concentration results was within ±20% of the actual, precision across results (%CV) was ≤15%, curve coefficients of determination (r) were ≥0.980 (correlation coefficient r > 0.990), and relative ion ratios of all qualifier ions were within ±20% of those for a mid-level standard. Limits of quantification (LOQs) for carfentanil and caffeine were 230 pg/ml and 12 ng/ml, respectively. Intra-day accuracies (mean concentrations) for carfentanil and caffeine ranged from 90.1% to 100.8% and from 87.1% to 108.9%, respectively; inter-day accuracies ranged from 98.7% to 100.4% and from 97.5% to 101.7%, respectively. Intra-day precision (%CV) over the dynamic range ranged from 1.31 to 8.88 and from 1.49 to 6.72 for carfentanil and caffeine, respectively. Inter-day precision ranged from 4.7 to 9.9 %CV for carfentanil and 7.6-121 %CV for caffeine.

The method was used to evaluate the percutaneous absorption kinetics of carfentanil in solution as a function of solvent composition using an in vitro, live human epidermis model. Counterintuitively, as previously reported, the addition of organic solvents to the formulations decreased rather than increased the percutaneous absorption rate of the ultra-potent opioid, carfentanil.