Clinician Feedback- and Laboratory Auditing-Based Addition of Glucuronide Metabolites to a Multiplex Urine UPLC-MS/MS Assay for Use in Chronic Pain Management

Abstract Background We previously described the clinical validation of a second-generation rapid dilute-and-shoot ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay to quantify and semi-quantify over 30 prescription drugs, drugs of abuse, and related glucuronides and other metabolites in human urine. Unlike our institution’s first-generation format, this second-generation assay does not include enzymatic glucuronide hydrolysis. Based on clinician feedback and an analysis of 12 months of immunoassay (IA) and first- and second-generation UPLC-MS/MS clinical pain management testing at our institution, three additional drug metabolites (hydromorphone, lorazepam, and oxazepam glucuronides) were added to the UPLC-MS/MS pain management assay while maintaining facile sample preparation and acceptable analytical performance. Methods Clinical testing results from 6 months of IA plus our first-generation UPLC-MS/MS assay were compared to test results from the first 6 months of the recently validated second-generation UPLC-MS/MS assay. Along with soliciting clinician feedback, data were analyzed for clinical performance, including positivity rates, to determine the clinical need for additional analyte testing. Human urine samples were diluted with water and spiked with deuterated internal standards without any proceeding enzymatic hydrolysis, analyte extraction, or sample purification. Analytes were separated by reverse-phase UPLC and then quantified by positive-mode electrospray ionization and collision-induced dissociation MS. Assay performance was validated per FDA bioanalytical guidelines. Results Analysis of clinical testing between the first- and second-generation UPLC-MS/MS pain management assays showed appreciable decreases in positivity rates for diazepam (and metabolites such as oxazepam), hydromorphone, and lorazepam, presumably due to the fact that the second-generation assay did not include analysis of the glucuronide metabolites. Three glucuronide metabolites were added to the newest version of the UPLC-MS/MS assay for pain management. Total analytical runtime was maintained at 5 minutes. All analytes, including the additional glucuronide metabolites, demonstrated acceptable linearity, analytical limits of detection, accuracy, precision, stability, and matrix effects, based on FDA bioanalytical guidelines and clinical specifications. Conclusions The additional glucuronide metabolites were successfully incorporated into an