Analysis of total and unbound hydromorphone in human plasma by ultrafiltration and LC–MS/MS: Application to clinical trial in patients undergoing open heart surgery

A method for a sensitive and specific analysis of hydromorphone total and unbound drug concentrations in human plasma was developed and validated. Sample preparation was preceded with an ultrafiltration step to separate the unbound drug from the protein bound fraction of hydromorphone. Both the ultrafiltrate and plasma samples were extracted with solid-phase extraction and substituted with stable isotope-labeled hydromorphone that was used as internal standard. Chromatographic separation was performed by gradient elution with UPLC-like system and eluates were analyzed by tandem mass spectrometry equipped with an electrospray ionization source. Sample preparation was optimized for good recovery of hydromorphone and the results were consistent. Calibration curves demonstrated linearity in the concentration range of 78–5000 pg/ml for analysis of both total and unbound concentrations of hydromorphone. The limit of detection was 1 pg and the lower limit of quantification was 78 pg/ml for both total and unbound hydromorphone plasma drug concentrations. Intra- and interassay reproducibility and inaccuracy did not exceed 10%. Hydromorphone was on the average 14% bound to plasma proteins, supporting the previously published unreferenced statements that the protein binding of hydromorphone is low. Method was applied to a clinical trial in patients undergoing open heart surgery to generate a target controlled infusion model for the postoperative patient controlled analgesia with hydromorphone.