Qualitative and quantitative analysis of opiate and related metabolites in human urine samples by UPLC-MS/MS

Abstract Although opioids such as morphine are regarded as essential drugs for clinical pain treatment, they are forbidden in athletic events. To maintain the fairness of the competition, rapid detection and quantitative analysis methods are employed. The present study developed a sensitive and robust LC-MS/MS method for the simultaneous quantification of morphine, codeine, and active metabolites, including morphine 3β-glucuronide, morphine 6β-glucuronide, codeine 6β-glucuronide. With a methanol/0.1% formic acid gradient, morphine, codeine, and metabolites were separated on an F5 column. We detected all compounds by multiple reaction mode (MRM) in the positive mode. In addition, the method had a range of reliable concentration-responses of 1.25–100 ng/mL (r2>0.995) for morphine, codeine, and metabolites in urine. The selectivity of the method was established in six different human subjects. For accuracy and precision, they were within 15% of the nominal values. There was no carryover, and the matrix effect was within the allowable range. The average storage stability at Low, Mid, and High QC levels met the requirements, respectively. The assay fulfilled all predefined acceptance criteria. A specific, stable, and precise LC-MS/MS method has been validated for morphine, codeine, and active metabolite quantification in human urine. Moreover, this straightforward method is reproducible and robust to support future morphine, codeine, active metabolite-related pharmacokinetics, bioequivalence, and clinical studies.