Automated mass spectrometric analysis of urinary free catecholamines using on-line solid phase extraction.
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 micromol/mol, 10-50 micromol/mol and 60-225 micromol/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.
de Jong
Wilhelmina H A
WH
Department of Laboratory Medicine, University Medical Center Groningen, University of Groningen, The Netherlands. w.h.a.de.jong@lc.umcg.nl
de Vries
Elisabeth G E
EG
Wolffenbuttel
Bruce H R
BH
Kema
I P
IP
D057205:Automation, Laboratory; D002395:Catecholamines / Q000652:urine*; D004355:Drug Stability; D006801:Humans; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction / Q000379:methods*; D013048:Specimen Handling; D053719:Tandem Mass Spectrometry / Q000379:methods*
D002395:Catecholamines