title: Simultaneous monitoring of monoamines, amino acids, nucleotides and neuropeptides by liquid chromatography-tandem mass spectrometry and its application to neurosecretion in bovine chromaffin cells.
abstract: The primary functions of adrenal medullary chromaffin cells are the synthesis and storage in their chromaffin vesicles of the catecholamines noradrenaline (NA) and adrenaline (AD), and their subsequent release into the bloodstream by Ca2+-dependent exocytosis under conditions of fear or stress (fight or flight response). Several monoamines, nucleotides and opiates, such as leucine-enkephalin (LENK) and methionine-enkephalin (MENK), are also co-stored and co-released with the catecholamines. However, other neurotransmitters have not been studied in depth. Here, we present a novel high-resolution liquid chromatography-tandem mass spectrometry approach for the simultaneous monitoring of 14 compounds stored and released in bovine chromaffin cells (BCCs). We validated the analytical method according to the recommendations of the EMA and FDA by testing matrix effect, selectivity, sensitivity, precision, accuracy, stability and carry-over. After testing on six batches of BCCs from different cultures, the method enabled simultaneous quantitative determination of monoamines (AD, NA, dopamine, serotonin, 5-hydroxyindoleacetic acid, histamine and metanephrine), amino acids (L-glutamic acid, γ-aminobutyric acid), nucleotides (adenosine 5'-diphosphate, adenosine 5'-monophosphate, cyclic adenosine 5'-monophosphate) and neuropeptides (LENK and MENK) in the intracellular content, basal secretion and acetylcholine induced secretion of BBCs. The high-resolution approach used here enabled us to determine the levels of 14 compounds in the same BCC batch in only 16 min. This novel approach will make it possible to study the regulatory mechanisms of Ca2+signaling, exocytosis and endocytosis using different neurotrophic factors and/or secretagogues as stimuli in primary BCC cultures. Our method is actually being applied to human plasma samples of different therapeutic areas where sympathoadrenal axis is involved in stress situations such as Alzheimer's disease, migraine or cirrhosis, to improve diagnosis and clinical practice. Copyright © 2016 John Wiley & Sons, Ltd.
authors.affiliation: Instituto-Fundación Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.|Servicio de Farmacología Clínica, Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Aneta
authors.initials: A
authors.lastname: Wojnicz
authors.affiliation: Instituto-Fundación Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Avendaño-Ortiz
authors.affiliation: Instituto-Fundación Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
authors.forename: Ricardo
authors.initials: R
authors.lastname: de Pascual
authors.affiliation: Instituto-Fundación Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.
authors.forename: Lucía
authors.initials: L
authors.lastname: Ruiz-Pascual
authors.affiliation: Instituto-Fundación Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.|Servicio de Farmacología Clínica, Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Antonio G
authors.initials: AG
authors.lastname: García
authors.affiliation: Instituto-Fundación Teófilo Hernando, Universidad Autónoma de Madrid, Madrid, Spain.|Departamento de Farmacología y Terapéutica, Facultad de Medicina, Universidad Autónoma de Madrid, Spain.|Servicio de Farmacología Clínica, Instituto de Investigación Sanitaria, Hospital Universitario de la Princesa, Universidad Autónoma de Madrid, Madrid, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Ruiz-Nuño
mesh_terms: D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D002138:Calibration; D002395:Catecholamines / Q000032:analysis* / Q000737:chemistry; D002417:Cattle; D019439:Chromaffin Cells / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D057230:Limit of Detection; D016014:Linear Models; D009479:Neuropeptides / Q000032:analysis* / Q000737:chemistry; D009489:Neurosecretion; D009711:Nucleotides / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D000596:Amino Acids; D002395:Catecholamines; D009479:Neuropeptides; D009711:Nucleotides
keywords: amino acids; bovine chromaffin cells; catecholamines; enkephalins; liquid chromatography-tandem mass spectrometry; nucleotides