Simultaneous extraction and determination of monoamine neurotransmitters in human urine for clinical routine testing based on a dual functional solid phase extraction assisted by phenylboronic acid coupled with liquid chromatography-tandem mass spectrometry

The major monoamine neurotransmitters, serotonin (5-HT) and catecholamines (i.e., norepinephrine (NE), epinephrine (E), and dopamine (DA)), are critical to the nervous system function, and imbalances of the neurotransmitters have been connected to a variety of diseases, making their measurement useful in a clinical setting. A simple, rapid, robust, sensitive, and specific LC-MS/MS method has been developed and validated for the simultaneous quantitation of urinary serotonin and catecholamines with low cost, which is ideal for routine clinical applications. A simple extraction from complex urine was accomplished using tailored solid phase extraction incorporating phenylboronic acid complexation on a 96-well HLB microplate for the sample extraction and resulted in significantly improved throughput, selectivity, and extraction recovery. Compared to 1-10 mL of urine typically used, this method required only 10 mu L. A rapid chromatographic elution with a total cycle time of 6 min per sample compared to reported run times of 19-75 min was achieved on a PFP column. The sensitivity of l and 2 ng mL(-1) for the detection of low abundant E and NE combined with the high coverage of 1024 ng mL(-1) for DA enabled the multi-analyte detection of these biogenic amines in a single run. Good linearity (2.0512, 1.0-512, 4.0-1024, and 4.0-1024 ng mL(-1) for NE, E, DA, and 5-HT, respectively), accuracy (87.6-104.0%), precision (<= 8.0%), extraction recovery (69.6-103.7%), and matrix effect (87.1-113.1% for catecholamines and 63.6-71.4% for 5-HT) were obtained. No autosampler carryover was observed. The analytes were stable for 5 days at 20 degrees C, 14 days at 4 degrees C, and 30 days at -20 degrees C and five freeze-thaw cycles. The easy sample preparation, rapid LC, and multi-analyte MS detection allow two 96-well plates of samples to be extracted within 2 h and analyzed on an LC-MS/ MS system within 24 h. The applicability and reliability of the assay were demonstrated by assessment of the reference interval for authentic urine specimens from 90 healthy individuals.