title: 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.
abstract: 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 μL. 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-1for the detection of low abundant E and NE combined with the high coverage of 1024 ng mL-1for DA enabled the multi-analyte detection of these biogenic amines in a single run. Good linearity (2.0-512, 1.0-512, 4.0-1024, and 4.0-1024 ng mL-1for 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 °C, 14 days at 4 °C, and 30 days at -20 °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. Graphical abstract A simple, rapid, robust, sensitive and specific LC-MS/MS method combined with a dual functional solid phase extraction has been developed and validated for the simultaneous extraction and quantitation of monoamine neurotransmitters in human urine with low cost.
authors.affiliation: Pharmasan Labs, Inc., 373 280th Street, Osceola, WI, 54020, USA. xiaoguang.li@pharmasan.com.
authors.forename: Xiaoguang Sunny
authors.initials: XS
authors.lastname: Li
authors.affiliation: Pharmasan Labs, Inc., 373 280th Street, Osceola, WI, 54020, USA.
authors.forename: Shu
authors.initials: S
authors.lastname: Li
authors.affiliation: Pharmasan Labs, Inc., 373 280th Street, Osceola, WI, 54020, USA.|NeuroScience Inc., 373 280th Street, Osceola, WI, 54020, USA.
authors.forename: Gottfried
authors.initials: G
authors.lastname: Kellermann
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D015306:Biogenic Monoamines / Q000737:chemistry* / Q000652:urine*; D001897:Boronic Acids / Q000737:chemistry* / Q000652:urine; D002853:Chromatography, Liquid; D045424:Complex Mixtures / Q000652:urine; D005260:Female; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D018377:Neurotransmitter Agents / Q000652:urine*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D016482:Urinalysis / Q000379:methods*; D055815:Young Adult
chemical_list: D015306:Biogenic Monoamines; D001897:Boronic Acids; D045424:Complex Mixtures; D018377:Neurotransmitter Agents; C010686:benzeneboronic acid
keywords: Catecholamine; Liquid chromatography-tandem mass spectrometry; Monoamine neurotransmitter; Phenylboronic acid-based solid phase extraction; Serotonin; Urine