Simultaneous Determination of Catecholamines and Metanephrines in Urine by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry: Successful Clinical Application

Abstract

Determination of urinary catecholamines and metanephrines is important for diagnosis of pheochromocytoma. Liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for the determination of catecholamines and metanephrines in human urine using deuterated internal standards (IS). In this method, 1000 µL of urine samples were treated by liquid-liquid extraction using ethyl acetate and subjected to LC-MS/MS analysis using positive electrospray ionization (ESI+). A BDS HYPERSILTM C18 column (125 mm × 3 mm, 3 µm) was used and mobile phase was water:methanol (98:2, v/v) with 0.25% of formic acid at 200 µ L min −1 . The method has a chromatographic running time of approximately 10 min. The average of recovery was 92.9-106.1% for epinephrine, 94.6-107.7% for norepinephrine, 98.0-108.8% the dopamine, 94.2-105.1% the metanephrine, 97.0-106.0% for normetanephrine. The precision was lower than 7.0% for all analytes. Furthermore, this method has been implemented successfully in routine laboratory due to its easy execution and excellent precision. ]]>

Pre-analytical and analytical validations and clinical applications of a miniaturized, simple and cost-effective solid phase extraction combined with LC-MS/MS for the simultaneous determination of catecholamines and metanephrines in spot urine samples

“…Available assays often suffered low sensitivity for detecting trace amount of E [19,21,25,26] and narrow linearity ranges for monitoring high levels of DA and metanephrines [23]. Consequently, additional time and cost were needed to quantify these analytes.…”

“…Available sample preparation involving an essential pH adjustment made the process cumbersome and time-consuming [19][20][21][22][23][24][25][26]32]. This was attributed to the substantial pH variation in urine and large sample volume (typically over 500 μL).…”

“…An effective sample cleanup for the complex urine matrix prior to analysis is crucial. Liquid-liquid extraction [19,25] and solid phase extraction (SPE) [17,[20][21][22][23][24] have become the popular choices among other techniques [12,30]. It is noteworthy that the pH of the pre-treated urine sample was crucial for extraction recovery in the reported assays.…”

“…Hence, to achieve a reliable extraction, it was cumbersome to adjust pH of each sample to a narrow pH range. For example, a liquid-liquid extraction needed a pH of 9.5 prior to LC-MS/MS analysis [19,25]; SPE using Oasis WCX, the most common cleanup of these analytes, required a narrow pH range of 6-7 [23,26,32].…”

“…Due to difficult collection of tissues, and supine rest constraint of blood draw for plasma, urine has been recommended as an alternative specimen for the analysis owing to its non-invasive and convenient collection [10,[14][15][16]. Among a wide range of methodologies with different detectors applied for the measurement of urinary catecholamines and metanephrines [6,12,17,18], liquid chromatography-tandem mass spectrometry (LC-MS/MS) has gradually taken the center stage due to its inherent selectivity and sensitivity [19][20][21][22][23][24][25][26].…”