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Editorials |
1 Department of Laboratory Medicine, and Pathology, Mayo Clinic, Rochester, MN
2 Departments of Clinical Chemistry, and Medicine, University of Dresden, Dresden, Germany
aAddress correspondence to this author at: Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 1st St. SW, Rochester, MN 55905. Fax 507-284-9758; e-mail Singh.Ravinder{at}mayo.edu.
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 sulfate-conjugated 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 high-throughput 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 labor-intensive 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 blood. Commercial automated immunoassays have revolutionized clinical diagnostics, because large numbers of patient samples can be tested in a cost-effective and timely manner. Compared to such immunoassays, the equipment expense, labor-intensive and complex sample preparation, and chromatographic separation steps are limitations to the use of HPLC or gold standard MS technology in clinical diagnostic laboratories. Thus, immunoassays hold advantages for laboratories for which convenience is a priority and sample throughput is too low to justify a large capital expenditure on equipment.
Relatively convenient RIAs and enzymatic immunoassays based on microtiter plate technology have been developed for the rapid and quantitative determination of plasma and urinary normetanephrine and metanephrine (7)(8). Reports that immunoassays for plasma free metanephrines provide more accurate diagnosis of pheochromocytoma than measurements of catecholamines further encourage use of these assays (9). Immunoassays are, however, susceptible to artifacts caused by nonspecific binding. They may also be subject to cross-reactivity and other analytical interferences due to drugs often present in specimens from patients with hypertension. Subsequently there is often very poor agreement between the results obtained by different immunoassays, even from the same manufacturer, making patient follow-up over time or between laboratories, as well as longitudinal studies, extremely difficult.
In many US laboratories, testing of metanephrines is performed with methods involving HPLC with electrochemical detection. Although their analytical sensitivity and precision are acceptable, these methods are labor-intensive and require relatively long chromatographic run times, limiting sample throughput. Occasionally there are substances that coelute with the analytes, further complicating interpretation of data. Standardization among various laboratories performing testing for metanephrines has been a major challenge independent of the measurement method. For example, we recently reported that values assigned to the commonly used commercial calibrators for metanephrines were 24%–33% lower than their assigned values (10).
Assays based on GC-MS address many of the shortcomings of automated immunoassays and HPLC with electrochemical detection. However, functional sensitivity can be limiting, and the length of preparation steps and run times also limits sample throughput. LC-MS/MS has proven superior to GC-MS in many scenarios, in terms of both sensitivity and sample throughput. In combination with isotope dilution, LC-MS/MS currently provides the most appropriate reference methodology.
The LC-MS/MS assay described by de Jong et al. (1) is sensitive and offers automated online extraction with high-throughput processing of samples. The additional measurement of the dopamine metabolite, methoxytyramine, provides the assay with added utility for detection of dopamine-producing paragangliomas, which may be a currently underdiagnosed clinical entity (11). The capability to detect all 3 O-methylated metabolites in as little as 50 µL of plasma may furthermore make the assay suitable for diagnosis of other neuroendocrine tumors, particularly neuroblastomas, for which blood sample volume is a limiting factor and currently used urinary tests offer suboptimal diagnostic sensitivity, allowing detection of only 73% of these often deadly pediatric neoplasms (12).
Although the capital costs of LC-MS/MS equipment require an economy of scale that is best achieved by laboratories that process thousands of samples a year, these costs are coming down. Advances in analytical technologies enabling accurate and increasingly efficient analysis of metanephrines provide promise for improved diagnosis of a deadly tumor that is still missed more often than detected. Such advances, combined with adherence to recommendations for appropriate sampling conditions (13), interpretation, and follow-up of test results (2)(14), may make it more cost-effective and appropriate to use these tests for screening purposes in larger populations of patients than dictated by current standard clinical practice.
Acknowledgments
Grant/funding support: None declared.
Financial disclosures: None declared.
References
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