Direct and efficient liquid chromatographic‐tandem mass spectrometric method for opiates in urine drug testing – importance of 6‐acetylmorphine and reduction of analytes

Abstract

Opiates comprise a class of abused drugs that is of primary interest in clinical and forensic urine drug testing. Determination of heroin, codeine, or a multi-drug ingestion is complicated since both heroin and codeine can lead to urinary excretion of free and conjugated morphine. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers advantage over gas chromatography-mass spectrometry by simplifying sample preparation but increases the number of analytes. A method based on direct injection of five-fold diluted urine for confirmation of morphine, morphine-3-glucuronide, morphine-6-glucuronide, codeine, codeine-6-glucuronide and 6-acetylmorphine was validated using LC-MS/MS in positive electrospray mode monitoring two transitions using selected reaction monitoring. The method was applied for the analysis of 3155 unknown urine samples which were positive for opiates in immunochemical screening. A linear response was observed for all compounds in the calibration curves covering more than three orders of magnitude. Cut off was set to 2 ng/ml for 6-acetylmorphine and 150 ng/ml for the other analytes. 6-Acetylmorphine was found to be effective (sensitivity 82%) in detecting samples as heroin intake. Morphine-3-glucuronide and codeine-6-glucuronide was the predominant components of total morphine and codeine, 84% and 93%, respectively. The authors have validated a robust LC-MS/MS method for rapid qualitative and quantitative analysis of opiates in urine. 6-Acetylmorphine has been demonstrated as a sensitive and important parameter for a heroin intake. A possible interpretation strategy to conclude the source of detected analytes was proposed. The method might be further developed by reducing the number of analytes to morphine-3-glucuronide, codeine-6-glucuronide and 6-acetylmorphine without compromising test performance.

Detection of morphine‐3‐sulfate and morphine‐6‐sulfate in human urine and plasma, and formation in liver cytosol

“…Routine urine drug testing samples were used if there was a remaining surplus after analysis. The number of samples selected was 200 and the sample selection was made based on the presence of the heroin metabolite 6-acetylmorphine measured by LC-MS/MS (Andersson et al 2013). This procedure was approved by the local Ethics Committee (Dnr: 2008/1087-32).…”

Evaluation of an on‐site test device for the heroin metabolite 6‐acetylmorphine in urine

“…To reveal a heroin intake the relative proportion of morphine and codeine can be used as basis for interpretation. 5 Modern liquid chromatography-mass spectrometry instruments have made it possible to easily include 6-AM as a parameter in routine confirmation methods. 3 One disadvantage is the low levels of this metabolite in urine and a resulting shorter detection time.…”

“…1,2 Another more reliable, but analytically more challenging approach, is to focus on the specific heroin metabolite 6-acetylmorphine (6-AM). 5,6 In recent time, 6-AM has become a target analyte already in the screening assay. 3,4 It has been demonstrated, however, that the focus on 6-AM in the confirmation method may not lead to any lower detection rate in applied drug testing.…”

“…3,4 It has been demonstrated, however, that the focus on 6-AM in the confirmation method may not lead to any lower detection rate in applied drug testing. 5 Modern liquid chromatography-mass spectrometry instruments have made it possible to easily include 6-AM as a parameter in routine confirmation methods. 5,6 In recent time, 6-AM has become a target analyte already in the screening assay.…”

“…5 Modern liquid chromatography-mass spectrometry instruments have made it possible to easily include 6-AM as a parameter in routine confirmation methods. 5,6 In recent time, 6-AM has become a target analyte already in the screening assay. [7][8][9] Because of the low concentrations and the structure similarities to other morphine alkaloids and metabolites many of the 6-AM immunoassays suffer from unwanted crossreactivity.…”