Automated Dispersive Pipette Extraction of Catecholamines and Metabolites from Urine Using Hamilton Robotics and LC-MS/MS
Extraction < 15 minutes
The catecholamines, epinephrine, norepinephrine, and dopamine, are bioamines that play an integral role as neurotransmitters in the central and peripheral nervous system. Screening for catecholamines and their O-methylated metabolites, metanephrine and normetanephrine, is a widely accepted approach for diagnosis of catecholamine-secreting tumors, such as pheochromocytomas, neuroblastomas, and paragangliomas. Catecholamines are characterized by a monoamine-linked benzene ring with two vicinyl hydroxyl groups (catechol). Epinephrine is a secondary amine, while norepinephrine and dopamine are primary amines. Under neutral and alkaline conditions, the catechol group makes the catecholamines vulnerable to oxidation to the quinone species. Metanephrines lack a catechol group, having a methoxy group adjacent to the hydroxyl group, and are thus more stable. These compounds are highly polar and hydrophilic, with negative log D and log P values. These structural properties make sample preparation and analysis difficult.
DPX developed a method that uses a diphenylborinic acid (DPBA) complexation with styrene divinyl benzene prior to Dispersive Pipette XTRaction in order to minimize oxidation and maximize analyte recoveries from urine. During elution, the complexation is reversed with acid and the solution is ready for LC-MS/MS analysis. Extractions were performed using a Hamilton Nimbus96 liquid handler in less than 15 minutes. This workflow can be adapted on other Hamilton systems (STAR, STARlet or VANTAGE). The method described is highly reproducible and provides the necessary sensitivity for clinical applications.
|1 COMPLEX ANALYTES||Add DPBA to Urine|
|2 CONDITION||Aspirate and dispense|
100 % MeOH
0.2 M NH4Cl ph 8.5
|3 BIND ANALYTES||Aspirate and dispense Sample Solution x4|
|4 WASH||0.2 M NH4Cl pH 8.5|
|5 ELUTE ANALYTES||10% MeOH in 1 M Formic Acid|
Pre-scripted Application for Hamilton Robotics
|DPX Product||Sample Volume||Offline Processing||Online Processing|
|XTR tips with 5 mg RP in 1 mL Hamilton||300 µL||Solution Prep||< 15 minutes|