A selective and sensitive assay for urinary metanephrine and normetanephrine using gas chromatography mass spectrometry with selected ion monitoring

Abstract

Vapor phase methodology for quantitative analysis of urine for metanephrine and normetanephrine as the pentafluoropropionyl derivative is described. The use of selected ion monitoring provided sufficient sensitivity and selectivity that milliliter aliquots of urine required only a simple scheme of sample processing; preliminary purification consisted of percolation through a column of XAD-2 followed by solvent extraction. The sample extracts were converted to the pentafluoropropionyl derivatives and analyzed by gas chromatography mass spectrometry with selected ion monitoring. Quantitative analysis was based on alpha[2H2]-beta-[2H1]metanephrine and alpha-[2H2]-beta-[2H1]normetanephrine which were added to the urine sample at the outset. The calibration plots for metanephrine and normetanephrine were linear from 10 ng ml-1 to 2000 ng ml-1 of urine permitting the detection of metanephrine and normetanephrine levels throughout the normal range and well into the subnormal range. Examples of application of the methodology to clinical investigation are described. The overall reproducibility of the sample processing methodology and instrumentation was assessed by replicate analyses of the same urine sample over a period of several weeks; the coefficient of variation was +/- 2.5% (n = 8).

Synthetic calibrators for the analysis of total metanephrines in urine: Revisiting the conditions of hydrolysis

“…Total urinary metanephrines are usually measured after an acid hydrolysis step or more rarely by enzyme treatment with arylsulfatase to cleave the sulfate moiety from the phenolic group [7][8][9][10][11][12][13][14][15][16][17].…”

“…Metanephrines in free form are measured by HPLC with electrochemical or fluorimetric detection, using calibrators prepared inhouse by weighting free synthetic metanephrines or using certified calibrators provided by commercial distributors such as BioRad, Chromsystems or RECIPE [7][8][9][10][11][12][13][14][15][16][17]. It has been reported that most metanephrines present in calibrators and quality controls are free rather than sulfoconjugated forms [18].…”

“…Further adaptations led to routine procedures in clinical laboratories where acidic treatment at pH values ranging from 0.5 to 1.1 and incubation time from 20 to 45 min are commonly applied [7][8][9][10][11][12][13][14][15][16][17]. The problem is that, should shortcomings occur at this step (e.g., insufficient acidification), or should this step be omitted, the commercially available quality control samples will fail to alert the analyst.…”