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Sample chromatograms are provided from an assay carried out 3/13/00. Included are chromatograms of a solution of standards (A), and samples of plasma from a normal volunteer (B), a patient with MEN-2 and pheochromocytoma (C) and a patient with VHL disease and pheochromocytoma.

These chromatograms provide a reference for results that should be expected using the procedures for measurements of plasma metanephrines outlined in this manual.

(A) The chromatogram showing the standards illustrates the retention times and peak reponses relative to baseline that can be expected for the specified amounts of metanephrines injected.

The above retention times provide optimal chromatographic separation of peaks of interest from occasional interfering substances (e.g., acetaminophen). Retention times are dependent on organic phase concentration, column temperature, and ion pairing reagent concentration. Collapsing the chromatography to provide shorter run times may not provide optimal separation of peaks of interest. Since there is typically a late peak eluting at or soon after 30 minutes run times are aintained at 35 minutes. Sensitivity of the assay (calculated as 3 times baseline noise should be close to 2-4 pg measureable for normetanephrine and metanephrine).

 

(B) The chromatogram for the 2 mL sample of plasma from a normal volunteer illustrate typical peak responses observed in patients in whom pheochromocytoma is excluded or is unlikely. The small size of the normetanephrine and metanephrine peaks serve to illustrate the importance of optimal chromatography for accurate measurements of the low concentrations of free metanephrines typically found in plasma. Plasma levels of free methoxytyramine are normally below 5 pg/mL and often are not detectable.

 

(C) The chromatogram for the 2 mL sample of plasma from the patient with MEN-2 and a pheochromocytoma illustate the elevations of both normetanephrine and metanephrine that are typical of epinephrine-producing pheochromocytomas.

(D) The chromatogram for the 1 mL sample of plasma from the patient with VHL disease and a pheochromocytoma illustate the elevation of plasma normetanephrine and a normal level of metanephrine typical of a solely norepinephrine-producing pheochromocytoma. The elevated methoxytyramine is an unusual finding

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