High-throughput, automated, and accurate biochemical screening for pheochromocytoma: are we there yet?

Biochemical testing for pheochromocytoma is a diagnostic challenge due both to the highly variable nature of this rare catecholamine-producing tumor and the technical demands required for accurate and reliable laboratory tests of catecholamine excess. In this issue of Clinical Chemistry, de Jong et al. (1 ) describe a high-throughput automated liquid–chromatography-tandem mass spectrometry (LC-MS/MS) method enabling simultaneous extraction, concentration, separation, and mass-selective detection of plasma free normetanephrine and metanephrine, the respective O-methylated metabolites of norepinephrine, and epinephrine. This method for measurements of plasma free metanephrines (Note: the term “metanephrines” refers to both normetanephrine and metanephrine) also allows quantification of methoxytyramine, the O-methylated derivative of dopamine and a metabolite normally present in plasma at lower concentrations ( 0.1 nmol/L) than free normetanephrine ( 0.6 nmol/L) or metanephrine ( 0.3 nmol/L). Recognition that catecholamines are metabolized to free metanephrines within pheochromocytoma tumor cells, and that this process is independent of catecholamine release, provides a rationale for use of these metabolites in the diagnosis of pheochromocytoma (2 ). The diagnostic superiority of metanephrines over catecholamines has led to the recommendation that initial testing for pheochromocytoma should always include measurements of metanephrines in plasma or urine or both (3 ). Metanephrines in urine are usually measured after an acid hydrolysis step that converts the high concentrations of sulfateconjugated metabolites into free metanephrines. In part because of this step, concentrations of metanephrines in urine are 2–3 orders of magnitude higher than those for the free metanephrines in plasma. These higher concentrations, along with the simpler matrix, make urinary metanephrines easier to measure than plasma free metanephrines. However, the enzyme responsible for sulfate conjugation is present mainly in gastrointestinal tissues (2 ). Thus, the free metanephrines produced within tumor cells should provide the most direct and accurate test for diagnosis of pheochromocytoma. Consequently, the development of new and improved methods to measure plasma free metanephrines has received considerable attention. A method developed at the Mayo Medical Laboratories that uses LC-MS/MS for measurement of plasma free metanephrines (4 ) has been successfully used for highthroughput analysis of 20 000 samples per year (5 ). Currently most of the LC-MS/MS methods for polar compounds require offline extraction to clean and concentrate samples before injection into the mass spectrometer. The use of turboflow technology has alleviated this laborintensive step for nonpolar steroids by allowing online extraction. For polar compounds, such as plasma free metanephrines, a 4-fold improvement in sample throughput has been achieved through application of a multiplexing parallel LC system before MS/MS detection, resulting in a considerable reduction in instrument cycle time (4 ). The method described by de Jong et al. (1 ) involves online extraction without any multiplexing. The total run time of 11 min for this method is longer than that described for the method at the Mayo Medical Laboratories (4 ), but nevertheless the online extraction cuts down considerably on time spent in sample preparation. Until turboflow extraction can be validated for the analysis of free metanephrines, the approach by de Jong et al. (1 ) may provide the optimal method for automated extraction. The analytical sensitivity of MS instrumentation is currently limited, so that direct injection of unextracted samples for the analysis of metanephrines is not possible. Improvements in analytical and functional sensitivity may be achieved by decreasing the noise resulting from solvents and matrix components, thus effectively increasing the signal-to-noise ratio. The highly polar nature of the plasma matrix makes an offline or online extraction step for LC-MS/MS determination of metanephrines mandatory to minimize ionization effects at the MS source from endogeneous plasma constituents (6 ). Without such a step, ionization-related suppression of the signal generated by the metanephrines leads to considerable reduction in achievable analytical sensitivity. Medications can also present problems. In particular the drug regimens used in the treatment of hypertension cover a diverse range of agents, some of which have similar physicochemical properties to catecholamines and metanephrines, with the resulting potential for analytical interference. The choice of selective buffers and solid phases to enhance the recovery of analytes, such as free metanephrine present in plasma at low concentrations, requires substantial effort for optimization of the extraction protocols. Although devices involving solid-phase extraction cartridges are available for semiautomated offline extraction, until now these cartridges have not been incorporated into any automated system for measurements of plasma free metanephrines. The assay described by de Jong et al. (1 ) uses an online solid-phase extraction system with a high-pressure dispenser to provide solid-phase extraction cartridges with solvents for conditioning, equilibration, sample application, and cleanup. This online extraction approach has considerable potential for application to other clinically relevant polar analytes. An automated LC-MS/MS method for measurements of plasma free metanephrines clearly provides an important advance in terms of sample throughput and minimal time spent in sample processing, but what are the relative advantages and disadvantages of such a method compared to other available assays? Traditionally immunoassays have dominated the field for analysis of steroids, proteins, and drug metabolites in serum, plasma, or Editorial