Dilute, derivatise and shoot: Measurement of urinary free metanephrines and catecholamines as ethyl derivatives by LC-MSMS

Background

The measurement of catecholamines and their metabolites in either urine or plasma is an important diagnostic test used to exclude the presence of neuroendocrine tumours. Because of weak chromatographic retention and potential ion-suppression, reverse-phase LC-MSMS is not ideal for analysis of these polar molecules. Here, we investigate derivatisation by ethylation as an alternative approach.

Methods

A simple and rapid method involving acetaldehyde and a reducing agent was used to convert urine free metanephrines and catecholamines, and their deuterated analogues as internal standards, to mono-ethyl or diethyl- derivatives. Using an Agilent 6460 triple-quadrupole mass spectrometer, precursor and product ion mass spectra were recorded to allow comparison of multiple reaction monitoring methods for both derivatised and non-derivatised analytes under reverse-phase LC-MSMS conditions with positive electrospray ionization.

Results

Conversion of biogenic amines to less polar ethyl derivatives increased their mass and enhanced the intensity of their molecular ions and fragments. Ethylation also improved the chromatographic properties of the amines, with greater retention and elution from reverse-phase HPLC columns with a methanol or acetonitrile gradient. The signal response of tandem mass spectrometric detection was increased up to 50-fold for ethyl metanephrines compared to non-derivatised compounds. This increase allowed for the omission of solid-phase extraction of urine as a clean-up step prior to analysis. The ‘dilute-derivatise-shoot’ method maintained analytical performance with respect to between-run imprecision (CV < 6%) and accuracy in an external quality assurance program. Gender-related ranges for free metanephrines in early-morning spot urines, collected from adult patients, were similar using either derivatised or non-derivatised samples.

Conclusions

The LC-MSMS detection of free urine biogenic amines can be greatly enhanced by ethyl derivatisation, which is easy and rapid to perform. Advantages include improved chromatography and lower limits of quantitation, that negate the requirement for solid-phase clean-up of urine prior to analysis. A disadvantage is the potential toxicity of the derivatising agents used if they are not handled appropriately.