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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

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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−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 (2.0–512, 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 °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.

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

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Acknowledgements

The authors acknowledge Pheng Yang for providing technical assistance and Melissa Ahrens for critical reading of the manuscript.

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Authors and Affiliations

  1. Pharmasan Labs, Inc., 373 280th Street, Osceola, WI, 54020, USA

    Xiaoguang (Sunny) Li, Shu Li & Gottfried Kellermann

  2. NeuroScience Inc., 373 280th Street, Osceola, WI, 54020, USA

    Gottfried Kellermann

Authors
  1. Xiaoguang (Sunny) Li
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  2. Shu Li
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  3. Gottfried Kellermann
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Correspondence to Xiaoguang (Sunny) Li.

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All clinical studies involving human urine samples were conducted in adherence to the procedure approved by Pharmasan/Neuroscience Institutional Review Board (IRB).

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The authors declare that they have no conflict of interest.

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Li, X.(., Li, S. & Kellermann, G. 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. Anal Bioanal Chem 409, 2859–2871 (2017). https://doi.org/10.1007/s00216-017-0231-z

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  • DOI: https://doi.org/10.1007/s00216-017-0231-z

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