The widespread use of opioid drugs for nonmedical or recreational purposes has contributed to escalating rates of addiction, drug overdoses, and drug-related deaths. As a result, simple and efficient urine drug testing is critical for clinicians to monitor compliance and to prevent medication diversion. LC-MS/MS provides superior sensitivity and specificity while providing detailed information on the presence of individual drugs and their metabolites. However, the complexities of workflow and instrument operation, along with the requirements of highly trained personnel to troubleshoot and interpret results, present major challenges to clinical laboratories.
In this study, we leveraged upgraded instrumentation to develop and validate a new LC-MS/MS assay allowing us to simplify and improve workflows and data analysis.
Urine specimens were centrifuged and the resulting supernatants were diluted with internal standards by a robotic liquid handler. Sixteen opioids were quantified by using gravimetrically-prepared calibrators, while tramadol and 7 glucuronide-metabolites were detected qualitatively with a single-point cut-off calibrator. The assay was developed and validated simultaneously on two identical Waters Acquity UPLC/Xevo TQXS LC-MS/MS systems. The assay performance was evaluated following CLSI C62-A guidelines and the results were correlated to a previous version of the assay using two Waters Acquity UPLC/Xevo TQMS systems. An in-house developed web-based software application was implemented for quality assurance and for simplifying pathologist review and interpretation.
By modifying the chromatographic gradient, isobaric oxycodone and oxycodone metabolite interferences in the codeine and codeine-6 glucuronide transitions were well-resolved compared to the previous assay. The upgraded mass spectrometers provided better sensitivity and greater dynamic range, which enabled us to expand the analytical measurement ranges with a single injection of one diluted sample. Among all the analytes, intra- and inter-assay imprecision was less than 10% and carryover was less than 0.01%. The lower limit of the measuring interval was less than 5, 0.1, 20, and 200 ng/mL for most opioids, fentanyl, tramadol and opioid glucuronides, and ethyl glucuronide, respectively. The correlation coefficients were >0.95 for quantitative analytes compared to the previous assay. Acceptable limits of relative retention times, ion ratios, and internal standard peak areas were established during validation and included in the web-based software application for quality assurance purposes.
A simplified "dilute-and-shoot" LC-MS/MS opiate assay was developed and validated. The improved analysis maintains clinical requirements while eliminating isobaric interferences, reducing turnaround time, and doubling testing capacity. The combination of analytical and workflow improvements demonstrated in this work lay the groundwork to improve the efficiency of other complexes LC-MS/MS assays in our laboratory.