A sensitive, high-throughput LC-MS/MS method for measuring catecholamines in low volume serum

A robust, sensitive, high-throughput method for the detection and quantification of catecholamines in serum, including dopamine, 5-methoxytryptamine, tyramine, phenylethylamine (PEA), epinephrine (EPI), norepinephrine (NE), metanephrine (MN), and normetanephrine (NMN) is described. It is based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a positive scheduled multiple reaction monitoring (MRM) mode. Key to the success of the method is the inclusion of an amine derivatization step, using phenylisothiocyanate (PITC), prior to liquid chromatographic separation of the targeted analytes on a C18 reversed-phase column. Mass spectrometric conditions, e.g., characteristic fragmentations and quantification transitions were also optimized to obtain maximum sensitivity and specificity. The limits of detection for all the target analytes are in the low nanomolar range. The recovery rates of spiked serum samples with three different concentration levels, i.e., low, medium, and high, are in the range of 93.2%-113% with satisfactory precision values of less than 10.9%. This method was successfully applied to determine the concentrations of dopamine, 5-methoxytryptamine, tyramine, PEA, EPI, NE, MN, and NMN in multiple human serum samples, with results matching closely those reported in the literature. Comparisons to other reported methods for measuring catecholamines indicates this new approach requires 10-20X less volume, making it ideal for targeted metabolomics studies with volume-limited samples. The method has been adapted to a 96-well plate format and has allowed the quantitative determination of catecholamines in more than 800 serum samples on a single instrument in just nine days.