P23a

Simultaneous Quantification of Plasma 3-Methoxytyramine, and by Ultraperformance LC-MSMS

Erdim Sertoglu (1), Namik Kemal Nazaroglu (2)

(1) University of Health Sciences, Gulhane School of Medicine, Department of Medical Biochemistry, Ankara, Turkey (2) Synlab Ankara Laboratory, Ankara, Turkey

SHORT ABSTRACT Methods and LC-MS/MS Conditions Metanephrine (MN), normetanephrine (NMN), and 3-methoxytyramine (MTY) are are produced Reversed-phase HPLC separation was performed using a Raptor HILIC-Si LC column (50 x 2.1 mm by O-methylation of the . In this study, we aimed to develop a rapid and sensitive (i.d.); 2.7 μm particle size) after extraction onto Oasis WCX (1 mL, 10 mg) 30 µm cartridges. mass spectrometry based method coupled to ultraperformance liquid chromatography (UPLC- Samples were injected at a flow of 0.6 mL/min using a gradient of mobile phases A (95:5 Water:ACN MS/MS) to measure these plasma metabolites for the diagnosis of + 30 mM ammonium formate) and B (15:85 Water:ACN + 30 mM ammonium formate). Details of neuroendocrine tumors. Reversed-phase HPLC separation was performed using a Raptor analysis conditions are presented in Table 2. HILIC-Si LC column (50 x 2.1 mm (i.d.); 2.7 μm particle size) after extraction onto Oasis WCX (1 mL, 10 mg) 30 µm solid-phase extraction cartridges. This method, with good precision, RESUTS sensitivity and linearity, can be used in clinical and research laboratories. Precision experiments to determine intra-day and inter-day precisions were performed using two replicates of level 2 control material (levels were 378 ng/L, 244 ng/L and 196 ng/L for MN, NMN and INTRODUCTION MTY, respectively) across three independent analytical runs. The intra-day and inter-day precisions were about 0.9-3.1 % for MN, 0.4-2.6 % for NMN and 3.9-6.44 % for MTY. Each run was controlled and paragangliomas are neuroendocrine tumors respectively derived from using a 4-point calibration curve. intra-adrenal and extra-adrenal chromaffin cells (1). Together, they are referred to as PPGL and produce catecholamines, such as , and (2). Traditional screening tests for PPGL have consisted of catecholamines in urine and plasma and catecholamine metabolites in urine, However, current literature have revealed that traditional blood or urine tests that measure only catecholamines are inadequate to diagnose catecholamine-producing neuroendocrine tumors due to heterogeneous pattern of catecholamine secretion and measurements of plasma-free or urinary fractionated metanephrines are being recommended as the initial biochemical test to diagnose these tumors (3-5). The metanephrine (MN) and normetanephrine (NMN) are produced by O-methylation of the catecholamines (epinephrine and norepinephrine) while dopamine is metabolized to methoxytyramine (MTY) (4, 6). Liquid chromatography with electrochemical detection (LC–ECD) was initially the method of choice for plasma metanephrine measurements. However, these methods are generally labor-intensive, require extensive sample preparation, and are time- consuming. The chromatographic run time is generally >20 min, which limits the number of assays that can be performed daily on a single instrument. Eventually, LC–ECD has since been largely superseded by LC with tandem mass spectrometry (LC–MS/MS) or immunoassay methods (7-9). In this study, we aimed to develop a rapid and sensitive MS based method coupled to ultraperformance LC (UPLC-MS/MS) to measure plasma NMN, MN and 3-methoxytyramine.

MATERIALS AND METHODS Chemicals and Reagents ClinCal® 4-Level lyophilised Serum Calibrator Set and 3-level ClinChek® Controls for

metanephrines were purchased from Recipe (Munich/Germany) while Metanephrine-D3 HCl and Normetanephrine-D3 HCl were purchased from Cerilliant (TX, USA). Calibrator concentrations are presented in Table 1. HPLC-grade acetonitrile and ammonium formate were obtained from Sigma. Oasis WCX (1 mL, 10 mg) 30 µm solid-phase extraction cartridges were purchased through Waters Corporation (Milford, MA, USA). And finally, Raptor HILIC-Si LC column (50 x 2.1 mm (i.d.); 2.7 μm particle size) was purchased from Restek (PA, USA)

Table 1. Calibrator Concentrations

Cal Level 1 Cal Level 2 Cal Level 3 Cal Level 4 Metanephrine (ng/L) 29,1 95,5 489 1437 Normetanephrine (ng/L) 38,5 191 580 1874 3-Methoxytyramine (ng/L) 19,3 94,8 480 1420

Table 2. Analysis conditions

Instrumentation LC : Thermo Dionex Ultimate 3000 MS/MS : Thermo Endura LC Conditions Solvent A : 95:5 WATER:ACN + 30 mM amonium formate pH :3 Solvent B : 15:85 WATER:ACN + 30 mM amonium formate pH :3 Figure 1. Calibration Curve (on the left side) and Chromatograms (on the right side) of Metanephrine, Normetanephrine and 3-methoxytyramine respectively. Gradient : Time (min) % B 0 100 % 1.0 100 % DISCUSSION 2.0 90 % Extraction using the Oasis WCX Elution columns resulted in low matrix effects and resulted in 2.1 90 % acceptable analytical accuracy and precision for all compounds. SPE extraction also avoided extra 2.5 70 % time or risk associated with evaporation and reconstitution. Raptor HILIC-Si LC column used for 2.6 100% separation resulted in rapid and efficient separation of all compounds. We highly recommend the use of this fast, reliable, and sensitive LC-MS/MS method for the monitoring plasma metanephrine 3.9 100% concentrations in clinical laboratories. 4.0 100% Column : Raptor HILIC-Si LC column (50 x 2.1 mm (i.d.); 2.7 μm Column Temperature : 30oC REFERENCES Injection volume : 15 µL 1. Lenders JW, Eisenhofer G, Mannelli M, Pacak K. Phaeochromocytoma. Lancet 2005. 366 665–675. Flow rate : 0.6 mL/min 2. Chen Y, Xiao H, Zhou X, Huang X, Li Y, Xiao H, Cao X. Accuracy Of Plasma Free Metanephrınes In The Dıagnosıs Of Autosampler syringe wash solvent Methanol And Paraganglıoma: A Systematıc Revıew And Meta-analysıs. Endocr Pract. 2017;23(10):1169- MS/MS Conditions Ion source : H-ESI (positive ion) 1177. Sheath gas flow (Arb) : 50 3. Young WF, Jr. Phaeochromocytoma: how to catch a moonbeam in your hand. Eur J Endocrinol. 1997;136:28-29 4. Rao D, Peitzsch M, Prejbisz A, Hanus K, Fassnacht M, Beuschlein F, et al. Plasma methoxytyramine: clinical utility with Aux Gas (Arb) : 15 L/min metanephrines for diagnosis of pheochromocytoma and paraganglioma. Eur J Endocrinol. 2017;177(2):103-113. Ion Transfer Tube Temp (°C) : 450°C 5. Eisenhofer G, Peitzsch M. Laboratory evaluation of pheochromocytoma and paraganglioma. Clin Chem. 2014;60(12):1486-99. Vaporizer Temp (°C) : 400°C 6. I.J. Kopin. Catecholamine metabolism: Basic aspects and clinical significance. Pharmacol Rev. 1985;37:333-364. Positive Ion (V) : 3000 V 7. Lagerstedt SA, O'Kane DJ, Singh RJ. Measurement of plasma free metanephrine and normetanephrine by liquid Detection : Multiple Reaction Monitoring chromatography-tandem mass spectrometry for diagnosis of pheochromocytoma. Clin Chem. 2004;50(3):603-11. Transitions : Metanephrine 180.185>165.175 180.185>148.15 8. Grebe SK & Singh RJ. LC–MS/MS in the clinical laboratory – where to from here? Clinical Biochemist. Reviews 2011;32:5–31. Normetanephrine 166.2>134.175 166.2>106.225 9. Manz B, Kuper M, Booltink E & Fischer-Brugge U. Development of enantioselective immunoassays for free plasma

Metanephrine -D3 183.195>151.2 183.195>168.175 metanephrines. Annals of the New York Academy of Sciences 2004;1018:582–7.

Normetanephrine-D3 169.195>109.2 169.195>137.175