Serum testosterone quantitation by liquid chromatography‐tandem mass spectrometry: Interference from blood collection tubes

During the development of a testosterone assay by LC-MS/MS, we encountered significant assay interference introduced by blood collection tubes. We examined a number of commonly used blood collection tubes for the presence of interference and its impact on testosterone quantitation. A number of commonly used blood collection tubes were examined by incubation of zero, low and high testosterone concentration samples with them over time, followed by sample preparation using liquid–liquid extraction and analysis by LC-MS/MS. Source of interference was identified by separately incubating blood collection tube coating, stopper and separator gel in clean glass tubes containing zero calibrator. Significant interference was found in some blood collection tubes, with the separator gel identified as the main source. The magnitude of the interference increases over time and mainly affected one of the two testosterone mass transitions used in the quantitation, making it readily detected by the discrepant results obtained by each of the two testosterone mass transitions. We were unable to eliminate the interference by adjustment of the sample preparation procedure, and by changing LC or MS parameters. Accurate quantitation of testosterone is possible when the problematic tubes are avoided, and blood collection tubes free of interference are used instead. Significant LC-MS/MS testosterone assay interference that originated from certain type of blood collection tubes hampered testosterone analysis. Examination of blood collection tube and any other laboratory test tubes for interference should therefore be an integral part of the development and validation of any LC-MS/MS assay used in a clinical diagnostic laboratory.