Urinary metanephrines by liquid chromatography tandem mass spectrometry: using multiple quantification methods to...

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• Accurate determination of urinary metanephrines is crucial for diagnosing and monitoring pheochromocytoma.
• Existing assays for metanephrines are frequently hindered by interferences, impacting reliability.
• A need exists for a robust, high-throughput method for urinary metanephrine and normetanephrine measurement.

Purpose of the Study:

• To develop and validate a reliable, high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
• To measure metanephrine and normetanephrine concentrations in urine samples.
• To address and overcome interferences common in existing assays.

Main Methods:

• Urine samples underwent acid hydrolysis followed by solid-phase extraction using a mixed-mode cation exchange sorbent in a 96-well format.
• Separation was achieved using a Restek perfluorophenyl column with an isocratic mobile phase and gradient cleanout.
• Detection was performed on an API 3200 triple quadrupole mass spectrometer using electrospray ionization in positive mode and multiple reaction monitoring.

Main Results:

• The method demonstrated linearity up to 15,000 nmol/L with limits of detection and quantification at 2.5 and 10 nmol/L, respectively.
• Imprecision values (within-run, between-day, total) were consistently low, at or below 2.7% for both analytes.
• The LC-MS/MS method showed excellent correlation with the established GC-MS method, featuring a 6-minute injection-to-injection time.

Conclusions:

• The developed LC-MS/MS method is a reliable and high-throughput assay for quantifying urinary metanephrine and normetanephrine.
• This validated method effectively overcomes interferences, providing accurate results for pheochromocytoma diagnosis.
• The method has been successfully implemented in clinical service since August 2010, demonstrating exceptional performance.