Automated mass spectrometric analysis of urinary free catecholamines using on-line solid phase extraction |
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Authors: | Wilhelmina HA de Jong Elisabeth GE de Vries Bruce HR Wolffenbuttel IP Kema |
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Institution: | 1. Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, The Netherlands;2. Department of Medical Oncology, University Medical Center Groningen, University of Groningen, The Netherlands;3. Department of Endocrinology, University Medical Center Groningen, University of Groningen, The Netherlands |
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Abstract: | Analysis of catecholamines (epinephrine, norepinephrine and dopamine) in plasma and urine is used for diagnosis and treatment of catecholamine-producing tumors. Current analytical techniques for catecholamine quantification are laborious, time-consuming and technically demanding. Our aim was to develop an automated on-line solid phase extraction method coupled to high performance liquid chromatography–tandem mass spectrometry (XLC–MS/MS) for the quantification of free catecholamines in urine. Five microlitre urine equivalent was pre-purified by automated on-line solid phase extraction, using phenylboronic acid complexation. Reversed phase (pentafluorophenylpropyl column) chromatography was applied. Mass spectrometric detection was operated in multiple reaction monitoring mode using a quadrupole tandem mass spectrometer with positive electrospray ionization. Urinary reference intervals were set in 24-h urine collections of 120 healthy subjects. XLC–MS/MS was compared with liquid chromatography with electrochemical detection (HPLC–ECD). Total run-time was 14 min. Intra- and inter-assay analytical variations were <10%. Linearity was excellent (R2 > 0.99). Quantification limits were 1.47 nmol/L, 15.8 nmol/L and 11.7 nmol/L for epinephrine, norepinephrine and dopamine, respectively. XLC–MS/MS correlated well with HPLC–ECD (correlation coefficient >0.98). Reference intervals were 1–10 μmol/mol, 10–50 μmol/mol and 60–225 μmol/mol creatinine for epinephrine, norepinephrine and dopamine, respectively. Advantages of the XLC–MS/MS catecholamine method include its high analytical performance by selective PBA affinity and high specificity and sensitivity by unique MS/MS fragmentation. |
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Keywords: | HPLC high performance liquid chromatography LC&ndash MS/MS liquid chromatography with tandem mass spectrometric detection PBA phenylboronic acid SPE solid phase extraction ACE automated cartridge exchanger |
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