M J Whiting1. 1. Clinical Biochemistry and Pharmacology Laboratory, SA Pathology, Flinders Medical Centre, Bedford Park, South Australia. Malcolm.Whiting@flinders.edu.au
Abstract
BACKGROUND: The measurement of catecholamines and metanephrines in urine is an important diagnostic test in biochemical screening for phaeochromocytoma. Tandem mass spectrometry (MSMS) has the potential to be used in a profiling method for simultaneous assay of these analytes. METHODS: Optimal conditions were established for the MSMS detection of catecholamines (noradrenalin, adrenalin and dopamine) and metanephrines (normetanephrine and metanephrine), including commercially available isotopically labelled compounds for use as internal standards. Chromatographic separation of all five polar biogenic amines was achieved under solvent conditions that were compatible with MSMS and multiple reaction monitoring. Several types of solid-phase extraction cartridge were used to investigate clean-up conditions for urine, and acid-hydrolysates of urine, prior to LC-MSMS. RESULTS: Total catecholamines and metanephrines from acid-hydrolysed urines, or free catecholamines and free metanephrines from native urines, were complexed with diphenyl-boronate and recovered in high yield from polymer cartridges after elution with formic acid. Direct injection of eluates into the LC-MSMS system allowed quantitation of catecholamines and metanephrines with a run time of 6 min per sample. Biogenic amine concentrations for patient urines and quality assurance programme samples, and assay imprecision, were similar to values obtained with high-performance liquid chromatography methods, which used electrochemical detection. In normal urines, the ratio of free to total catecholamines was around three-fold higher than the ratio of free to total metanephrines. CONCLUSION: The assay of urinary catecholamines and metanephrines can be achieved simultaneously using one LC-MSMS method, which is rapid and reduces labour and consumable costs for routine application.
BACKGROUND: The measurement of catecholamines and metanephrines in urine is an important diagnostic test in biochemical screening for phaeochromocytoma. Tandem mass spectrometry (MSMS) has the potential to be used in a profiling method for simultaneous assay of these analytes. METHODS: Optimal conditions were established for the MSMS detection of catecholamines (noradrenalin, adrenalin and dopamine) and metanephrines (normetanephrine and metanephrine), including commercially available isotopically labelled compounds for use as internal standards. Chromatographic separation of all five polar biogenic amines was achieved under solvent conditions that were compatible with MSMS and multiple reaction monitoring. Several types of solid-phase extraction cartridge were used to investigate clean-up conditions for urine, and acid-hydrolysates of urine, prior to LC-MSMS. RESULTS: Total catecholamines and metanephrines from acid-hydrolysed urines, or free catecholamines and free metanephrines from native urines, were complexed with diphenyl-boronate and recovered in high yield from polymer cartridges after elution with formic acid. Direct injection of eluates into the LC-MSMS system allowed quantitation of catecholamines and metanephrines with a run time of 6 min per sample. Biogenic amine concentrations for patient urines and quality assurance programme samples, and assay imprecision, were similar to values obtained with high-performance liquid chromatography methods, which used electrochemical detection. In normal urines, the ratio of free to total catecholamines was around three-fold higher than the ratio of free to total metanephrines. CONCLUSION: The assay of urinary catecholamines and metanephrines can be achieved simultaneously using one LC-MSMS method, which is rapid and reduces labour and consumable costs for routine application.