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Automation and Analytical Techniques |
1 Department of Pathology and Laboratory Medicine, University Medical Center, University of Groningen, Groningen, The Netherlands.
2 Waters Corporation, Manchester, UK.
3 Department of Endocrinology, University Medical Center, University of Groningen, Groningen, The Netherlands.
4 Department of Chemical Endocrinology, University Medical Center, Nijmegen, The Netherlands.
5 Department of Medical Oncology, University Medical Center, University of Groningen, Groningen, The Netherlands.
aAddress correspondence to this author at: Department of Pathology and Laboratory Medicine, University Medical Center Groningen, P.O. Box 30001, 9700 RB Groningen, The Netherlands. Fax 0031503612290; e-mail i.p.kema{at}lc.umcg.nl.
Background: Quantification of plasma free metanephrine (MN) and normetanephrine (NMN) is considered to be the most accurate test for the clinical chemical diagnosis of pheochromocytoma and follow-up of pheochromocytoma patients. Current methods involve laborious, time-consuming, offline sample preparation, coupled with relatively nonspecific detection. Our aim was to develop a rapid, sensitive, and highly selective automated method for plasma free MNs in the nanomole per liter range.
Methods: We used online solid-phase extraction coupled with HPLC-tandem mass spectrometric detection (XLC-MS/MS). Fifty microliters plasma equivalent was prepurified by automated online solid-phase extraction, using weak cation exchange cartridges. Chromatographic separation of the analytes and deuterated analogs was achieved by hydrophilic interaction chromatography. Mass spectrometric detection was performed in the multiple reaction monitoring mode using a quadrupole tandem mass spectrometer in positive electrospray ionization mode.
Results: Total run-time including sample cleanup was 8 min. Intra- and interassay analytical variation (CV) varied from 2.0% to 4.7% and 1.6% to 13.5%, respectively, whereas biological intra- and interday variation ranged from 9.4% to 45.0% and 8.4% to 23.2%. Linearity in the 0 to 20 nmol/L calibration range was excellent (R2 > 0.99). For all compounds, recoveries ranged from 74.5% to 99.6%, and detection limits were <0.10 nmol/L. Reference intervals for 120 healthy adults were 0.07 to 0.33 nmol/L (MN), 0.23 to 1.07 nmol/L (NMN), and <0.17 nmol/L (3-methoxytyramine).
Conclusions: This automated high-throughput XLC-MS/MS method for the measurement of plasma free MNs is precise and linear, with short analysis time and low variable costs. The method is attractive for routine diagnosis of pheochromocytoma because of its high analytical sensitivity, the analytical power of MS/MS, and the high diagnostic accuracy of free MNs.
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