MSACL 2019 EU Abstract

Attempt to Develop a Fast LC-MS/MS Application for the Simultaneous Measurement of 12 Biogenic Monoamines

Damir Djulbic(1), Vera de Kleijne(1,2), Teun van Herwaarden(3), Maarten Broeren(2), and Marcel van Borren(1)
(1) Clinical Chemistry & Hematology Laboratory, Rijnstate Hospital Arnhem (2) Clinical Chemistry & Hematology Laboratory , Máxima Medisch Centrum, Veldhoven(3) Department of Laboratory Medicine, Radboudumc, Nijmegen. The Netherlands

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Marcel van Borren (Presenter) Rijnstate Hospital >> POSTER (PDF)

Presenter Bio: Clinical chemist with interest in endocrinology and LC-MS/MS

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION: Correct measurement of biogenic monoamines is essential in the clinical diagnosis and follow-up of pheochromocytoma, paraganglioma and carcinoid tumours. Currently we measure the catecholamines (adrenaline (A), noradrenaline (NA), dopamine(D)), their metabolites (vanillylmandelic acid (VMA) and homovanillic acid (HVA)) and the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) with two different fluorometric methods, while the metanephrines (metanephrine (MN), normetanephrine (NMN), 3-methoxytyramine (3MT) are measured with an electrochemical detection method. These three methods lack analytical specificity and sensitivity and are time consuming.
OBJECTIVES: To develop a fast, sensitive and specific LC-MS/MS assay for the simultaneous measurement of 12 biogenic monoamines.
METHODS: Optimal mass transitions (parent, quantifier and qualifier) and source settings for each of the 12 biogenic monoamines (tryptophan (TP), 5-HTP (5-hydroxytryptophan), 5-HT (5-hydroxytryptamin), 5-HIAA, A, NA, D, VMA, HVA, MN, NMN, 3-MT) were determined on a Shimadzu XR, LC-MS/MS 8060. For chromatographic separation a mixture of these 12 biogenic monoamines, an ACE C18-pentafluorophenyl column (2 µm, 150x3,0 mm), mobile phase A (2 mM ammonium formate in water with 0,05% formic acid) and mobile phase B (2 mM ammonium formate in acetonitril with 0,05% formic acid) were used. Percentages of mobile phase A and B, flow and temperature were varied to optimize separation. Weak anion exchange (WAX) and weak cation exchange (WCX) columns (Phenomenex and Waters) were used to study recovery of the compounds dissolved in water.
RESULTS: Mass transitions (parent>quantifier>qualifier) were optimized for TP (205.15>188.05>146.10), 5-HTP (221.10>204.15>162,10), 5-HT (176.90>160,20>115.10), 5-HIAA (192.00>146.20>91.30), A (184.00>166.15>107.00), NA (170.00>107.10>77.50), D (154.00>91.05>65.00), VMA (197.10>137.00>108.00), HVA (180.95>122.20>105,20), MN (198.20>148.30>165.30), NMN (166.20>79.30>77.30) and 3-MT (168.20>91.30>151.30). All 12 compounds were chromatographically separated with a minimal resolution of 1.2 when chromatography was started with 3% mobile phase B, increased to 11,5% during 4 minutes, and maintained for 4.5 minutes at 27,5%. The column was cleaned by 2,5 minutes 85% and 4 minutes 3% mobile phase B. Flow was 0,4 ml/min and column temperature was 30°C. Ionization proved optimal with 3 ml/min drying gas, 17 ml/min heating gas, 2 ml/min nebulizing gas, 300°C DL temperature, 400°C interface temperature, 500°C heating block and 2 kV interface voltage. Basic compounds showed acceptable recovery (30-98%) with WCX columns, whereas acidic compounds showed only recovery (10-118%) with WAX column.
CONCLUSION: The developed application is able to specifically measure 12 biogenic monoamines in less than 10 minutes. However basic and acidic compounds require separate sample preparation. Whether analytical sensitivity is sufficient remains to be determined.