Electrospray LC-MS Method with Solid-Phase Extraction for Accurate Determination of Morphine-, Codeine-, and Ethylmorphine-Glucuronides and 6-Acetylmorphine in Urine

A method for the identification and quantification of morphine-3-glucuronide, codeine-6-glucuronide, ethylmorphine-6-glucuronide, and 6-acetylmorphine in human urine based on solid-phase extraction (SPE) and electrospray ionization liquid chromatography-mass spectrometry (LC-MS) was validated for use as a confirmation procedure in combination with immunochemical screening for opiates. Three deuterium-labelled analogues were used as internal standards: morphine-3-glucuronide-d3, codeine-d3, and 6-acetylmorphine-d3. Fifty-microliter aliquots of urine were prepared by SPE using 30-mg Oasis HLB cartridges. The chromatographic system consisted of a 2.0 x 100-mm C18 column and the gradient elution buffers used acetonitrile and 25 mmol/L formic acid. The protonated molecular ions were monitored in the selected ion monitoring mode together with one qualifier ion for each analyte. The interassay variability was less than 10% at the reporting limit 30 ng/mL for 6-acetylmorphine and 300 ng/mL for the other analytes. The method was validated by comparison with a reference gas chromatographic (GC)-MS method using authentic urine samples. The two methods agreed completely regarding identified analytes, and for the quantitative results there were slightly lower levels when measuring glucuronides directly as compared to total determination after hydrolysis by GC-MS. This result was to be expected because the free compounds are not measured with the LC-MS method. This study concludes that the presented LC-MS method is robust and reliable, and suitable for use as a confirmation method in clinical urine drug testing for opiates.