Background: Internal standards are essential in quantitative mass spectrometry (MS) assays to correct for variability in sample extraction and ionization at the source. In liquid chromatography MS assays, analogues of the analyte with several atoms replaced by their stable isotopes, e.g.2 H (D, deuterium) are often used as internal standards.

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Background: The clinical requirements of the users of assay results must be at the centre of assay development. We aimed to develop a single liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for drugs of abuse in urine that would meet the needs of our service users and replace the multiple screening and confirmatory techniques previously in use. Methods: After discussion with our users, it was decided that 13 drugs and metabolites should be measured in our panel: morphine, codeine, norcodeine, dihydrocodeine, 6-monoacetylmorphine, acetyl codeine, methadone and its metabolite, buprenorphine and its metabolite, amphetamine, benzoylecgonine and cotinine. Urine samples were prepared by the addition of internal standard, enzymatic hydrolysis and solid-phase extraction. Chromatography conditions were optimized so that the analytes were separated within a run time of 6 min. Optimal parent to daughter m/z ion transitions were chosen for all drugs and daughter ion ratios were used. Results: The LC-MS/MS assay was successfully validated with acceptable precision and lower limits of quantification for all drugs. No matrix effects were seen. The results produced by the LC-MS/MS assay compared well with the previous combination of techniques in use. Conclusions: We have developed and validated a fit-for-purpose LC-MS/MS assay for 13 drugs of abuse in urine that obviates the need for multiple screening and confirmatory analytical techniques.