Daniel Mueller (Presenter)
University Hospital Zurich
Authorship: Stefanie Mueller (1), Ursula Gutteck (1), Daniel Mueller (1)
Institute for Clinical Chemistry, University Hospital Zurich, Zurich, Switzerland
| Short Abstract To enable diagnosis of of pheochromocytomas and paragangliomas, we developed two LC-MS/MS methods: method A for the quantification of catecholamines, and method B for the quantification of free metanephrines. After semi-automated solid phase extraction, chromatography was performed under acidic conditions on a Hypersil Gold C8 column. As mass spectrometer, an AB Sciex QTrap 6500 was used. A sufficient dynamic range could be achieved for all substances. Imprecision was <9.9% for all substances, accuracy between 100-102%. Sufficiently low lower limits of quantification could be achieved, and matrix effects were tested as well. Sufficient sample stability could be shown. All substances tested could be shown as non-interfering. The described methods were successfully introduced into our routine laboratory practice. |
Long Abstract
Introduction: Catecholamines and free metanephrines in plasma are important biomarkers for the diagnosis of pheochromocytomas and paragangliomas (PPGLs), tumors from adrenal and extraadrenal chromaffin cells. PPGLs may be a cause of endocrine hypertension and may be potentially lethal. To enable early diagnosis of these tumors, we developed two LC-MS/MS methods with semi-automated sample preparation: method A for the quantification of the catecholamines epinephrine, norepinephrine, and dopamine in plasma; and method B for the quantification of free metanephrine, normetanephrine, and 3-methoxytyramine in plasma. Even tough LC-MS/MS is much more specific than techniques used before for the quantification of these compounds (e.g., HPLC with electrochemical detection), also this modern technique is not forearmed against interferences. Therefore, careful elimination of interferences was done during method development.
Methods: After addition of internal standards (epinephrine-d3, norepinephrine-6 and dopamine-d4 for method A, metanephrine-d3, normetanephrine-d3, 3-methoxytyramine-d4 for method B), 1 ml of sample volume was submitted to solid-phase extraction. Semi-automated solid phase extraction was performed for both methods on a GX-274 Aspec liquid handler. Evolute WCX-50 solid-phase extraction cartridges were used. Both methods were developed on an AB Sciex QTrap 6500, equipped with an electrospray ionization source operated in positive mode. The chromatographic system consisted of a Hypersil Gold C8 column (100x3 mm, 3µm particles), and water + 0.5% formic acid plus water/acetonitrile 10/90 v/v + 0.5% formic acid as mobile phases.
The following substances were tested and could be shown as non-interfering with the method: homovanillic acid, vanillylmandelic acid, 5-hydroxyindole-3-acetic acid, 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxymandelic acid, serotonin, O-methyl DOPA, 3,4-methylenedioxy-methamphetamine, 3,4-methylenedioxyamphetamine, methamphetamine, amphetamine, acetylsalicylic acid, acetaminophen, and ephedrine.
A method comparison with a reference laboratory could be done only for metanephrine, normetanephrine, and 3-methoxytyramine.
Results: The calibrated range was for epinephrine 0.110-2.73 nmol/L, for norepinephrine 0.590-14.8 nmol/L, for dopamine 0.130-3.26 nmol/L, and for metanephrine 0.140-7.18 nmol/L, for normetanephrine 0.196-9.96 nmol/L, for 3-methoxytyramine 0.108-8.19 nmol/L. Imprecision was for epinephrine <4.9%, for norepinephrine <2.1%, for dopamine <5.7%, and for metanephrine <9.9%, for normetanephrine <6.5%, for 3-methoxytyramine <8.1%. Accuracy was 100% for epinephrine, 102 % for norepinephrine, 101% for dopamine, and 101% for metanephrine, 100% for normetanephrine, 100% for 3-methoxytyramine.
The following lower limits of quantification could be achieved: 0.10 nmol/L for epinephrine, 0.59 nmol/L for norepinephrine, 0.13 nmol/L for dopamine, and 0.05 nmol/L for metanephrine and normetanephrine, 0.03 nmol/L for 3-methoxytyramine.
Matrix effects were 97.1% for epinephrine, 96.5% for norepinephrine, 93.5% for dopamine, 105% for metanephrine, 99.0% for normetanephrine, 105% for 3-methoxytyramine.
Sample stability could be shown for 4h benchtop at room temperature, two weeks at -20°C, 24h post processing at 10°C and for two freeze-thaw cycles for metanephrine, normetanephrine and 3-methoxytyramine in heparinized plasma. For epinephrine, norepinephrine and dopamine, the following stabilities could be shown: 4h benchtop in heparinized plasma, stabilized with EGTA/glutathione, one week at -80°C, 24h post processing at 10°C, and for two freeze-thaw cycles.
All substances tested could be shown as non-interfering.
For metanephrine, the following equation according to Passing-Bablok could be determined for our values vs. the values from the reference laboratory: for metanephrine 1.03x+0.004; for normetanephrine: 0.992 + 0.014; and for 3-methoxytyramine: 0.914 - 0.008.
Conclusions: The described methods were successfully introduced into our routine laboratory practice. Since the introduction of our newly developed methods, we measured several hundred samples and did never observe interferences. Critical from the point of view of a clinical laboratory is the fact that there are no proficiency testing schemes for metanephrine, normetanephrine, and 3-methoxytyramine in central Europe, at least not to the best of our knowledge.
References & Acknowledgements:
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