Printing: One platform for determination of urinary and plasma metabolites This poster is 48” wide by relevant for diagnostic assessement of neuroendocrine tumors 36” high. It’s designed to be Rajska M. | Prochazkova P. | Bartonikova D. | Loucka P | Minar J. | Radina M. | Garcic L. printed on a large-format printer. Polok Rajska M. | Loucka P. | Minar J. | Barvikova M. SPADIA Lab, a.s. Customizing the Dr. Slabihoudka Ostrava Poruba, Czech Republic [email protected] Content: The placeholders in this Introduction Metabolic pathway COMT poster are formatted for Neuroendocrine tumors (NETs), represent diverse group of disorders, which exhibit secretion of many hormonally Dopamine 3-MT active substances and thus cause various endocrine syndroms. Diagnostic alghorithm is based on the clinical you. Type in the MAO MAO symptomathology, morphological methods, laboratory diagnostics and RTG methods. For the detection of NETs in COMT Dihydroxyphenylacetic DBH HVA early stage the estimation of hormonally active substances and their metabolites is very important. acid placeholders to add text, or The analysis of catecholamine metabolites, both in plasma and in 24 hour urine collection, is of importance for the diagnosis of tumors of the sympathoadrenal system, such as pheochromocytomas or paragangliomas. In case of COMT click an icon to add a table, carcinoid tumors the diagnostic assessment is based on determination of 5-hydroxy-indolacetic acid in 24 hour urine Noradrenaline NMN collection and supported by additional determination of serotonin in serum. MAO MAO chart, SmartArt graphic, COMT Dihydroxymandelic PNMT VMA The aim of this work was to develop methods in such a way as to maintain the same configuration acid picture or multimedia file. MAO of the LC-MS/MS system (INSTRUMENT, ANALYTICAL COLUMNS, MOBILE PHASES). Methods for wider group of MAO COMT analytes relevant for diagnostic asessement of NETs were developed as follows: Adrenaline MN To add or remove bullet I. Metanphrine (MN) and Normetanephrine (NMN) in plasma II. Metanphrine (MN) and Normetanephrine (NMN) in 24 hour urine collection points from text, click the III. Serotonine (SER) in serum MAO, Ald-DH IV. Vanilmandelic (VMA), homovanillic (HVA) and 5-hydroxyindolacetic acid (5-HIAA) in 24 hour urine collection Serotonin 5-HIAA Bullets button on the Home LC-MS/MS method based on HILIC chromatography and ESI ionization Mass spectrometry conditions tab. Instrumentation: Materials used for determination of methods parameters: I. II. III. IV. If you need more Ionization Positive positive positive negative ºC • Agilent 6490 Triple Quadrupole • Purchased from Sigma-Aldrich: Gas temperature 135 135 135 135 ºC placeholders for titles, LC/MS System - Standards: MN, NMN, HVA, VMA, 5-HIAA, SER Gas flow 12 12 12 12 l/min ® ® • SeQuant ZIC HILIC HPLC 3.5mm, 100Å, - Deuterated IS: MN-d3, NMN-d3, HVA-d5, VMA-d3, 5-HIAA-d5 Nebulizer 45 45 45 45 Psi content or body text, make a 100 x 2.1 mm PEEK coated HPLC column • Purchased from Recipe: SGT 400 400 400 400 ºC SGF 10 10 10 10 l/min copy of what you need and Chromatographic separation - ClinCal®Serum Calibrator Set for Metanephrines Capillary 4000 4000 4000 3000 V • Binary pump: Gradient elution - ClinCheck®Serum Control for Metanephrines Nozzle voltage 0 0 0 1500 V drag it into place. MPA: 50mM NH COOH in H O - ClinCheck®Urine Control for Biogenic Amines 4 2 PowerPoint’s Smart Guides MPB: ACN - IS for serotonine (5-Hydroxy-N-methyltryptamine) Mass transition conditions ◦ • Purchased from Chromsystems • Column oven: 45 C Dwell Frag CAV will help you align it with Analyte Prec Prod CE [V] - Plasma Control Endocrine Hormones/Metabolites [ms] [V] [V] t [min] %A %B 0.0 20 80 • Patient samples – plasma (K3EDTA); 24h urine collection-acidified MN 180 148 70 380 11 4 everything else. 0.5 20 80 MN* 180 165 70 380 11 4 1.5 40 60 Sample preparation: MN-d3 183 151 70 380 11 4 Want to use your own 2.8 40 60 MN-d3* 183 168 70 380 11 4 2.9 20 80 Metanephrines in plasma 6.0 20 80 NMN 166 134 70 380 14 4 pictures instead of ours? No • 200ml of sample (calibrator, control, plasma) mix shortly with 50ml of IS (5mg/l of MN-d3 and NMN-d3 in ACN) NMN* 166 106 70 380 18 4 NMN-d3 169 137 70 380 10 4 problem! Just click a picture, Fig.1. Chromatogram for metanephrines in plasma • Precipitate with 1ml of isopropanol - mix vigorously for 30sec and centrifugate – 13000g/10min NMN-d3* 169 109 70 380 18 4 t [min] %A %B press the Delete key, then 0.0 20 80 • Transfer 1ml of supernatant to a glass tube and evaporate under SER 177 160 100 380 10 4 1.0 20 50 a slight stream of nitrogen; Reconstitution in 100ml of 80% ACN SER* 177 115 100 380 20 4 click the icon to add your 2.0 50 50 Serotonine in serum SER-IS 192 160 100 380 15 4 2.1 20 80 picture. 4.0 20 80 • 20ml of sample (calibrator, control, serum) mix for 30 sec with 180ml SER-IS* 192 115 100 380 25 4 of precipitant (80% ACN with IS for serotonine) and centrifuge – 10000g/5min HVA 181 137 75 380 4 4 HVA* 181 122 75 380 16 4 Metanephrines in urine HVA-d5 186 142 75 380 4 4 Fig.2. Chromatogram for serotonine in serum • 40ml of sample (calibrator, control, urine) mix shortly with 15ml of IS HVA-d5* 186 172 75 380 16 4 t [min] %A %B (1mg/l of MN-d3 and NMN-d3 in ACN) VMA 197 138 75 380 10 4 0.0 20 80 • Adjust samples pH and hydrolyze for 30min at 95ºC 0.5 20 80 VMA* 197 137 75 380 24 4 1.5 40 60 • Transfer 40ml of hydrolyzed sample, add 360ml of 80% ACN and mix VMA-d3 200 141 75 380 10 4 3.0 40 60 shorty and centrifuge – 10000g/5min VMA-d3* 200 140 75 380 24 4 3.2 20 80 VMA, HVA, 5-HIAA in urine 15.0 20 80 5-HIAA 190 144 75 380 6 4 • 10ml of sample (calibrator, control, urine) mix shortly with 10ml of IS (10mg/l of HVA-d5, VMA-d3, 5-HIAA-d5 in ACN), 5-HIAA* 190 146 75 380 24 4 5-HIAA-d5 195 151 75 380 6 4 Fig.3. Chromatogram for HVA, VMA, 5-HIAA in urine • Add 80ml of 80% ACN, mix for 30 sec and centrifuge – 10000g/5min 5-HIAA-d5* 195 149 75 380 24 4 Analytical parameters Conclusions Parameter P_MN P_NMN U_MN U_NMN S_SER U_HVA U_VMA U_-5HIAA Simple and fast approaches Calib. Range 0.140 – 7.175 0.196 – 9.956 0.120 – 10.140 0.021 – 10.890 4.7 – 2362.7 0.9 – 274.0 0.8 – 252.3 0.8 – 261.0 • Methods are simple, fast and ensures grater throughput of nmol/l nmol/l mmol/l mmol/l nmol/l mmol/l mmol/l mmol/l samples than HPLC with ECD detection 0,057 0,103 0,020 0,021 4.7 0.9 0.8 2.9 • Sample prep. requires precipitation with isopropanol or ACN LLQ (CV=8.7%) (CV=9.6%) (CV=2.6%) (CV=1.5%) (CV=11.4%) (CV=10.5%) (CV=11.3%) (CV=6.5%) but there is no need for the step of derivatization Recovery (%) 95 – 99 89 – 98 95 – 101 85 – 92 86 – 90 80 – 89 92 – 101 94 – 105 Satisfactory analytical parameters Intra-assay MEAN±SD 0.118 ± 0.006 0.778 ± 0.019 0.887 ± 0.036 1.734 ± 0.102 552.3 ± 21.6 15.2 ± 0.8 26.2 ± 1.9 14.1 ± 1.2 • LLQ, Recoveries precition (CV=5.4%) 2.4 4.1 5.9 3.7 5.3 7.3 8.3 • Methods are reliable in terms of precision Inter-assay MEAN±SD 0.295 ± 0.012 0.630 ± 0.051 0.905 ± 0.034 1.657 ± 0.075 553.7 ± 57.3 27.3 ± 2.8 27.1 ± 2.6 23.0 ± 2.8 precision (CV=4.2%) 8.1 3.8 4.5 10.7 10.4 9.6 12.1 • The calibration curves provides linearity in the relevant ranges One system configuration for neuroendocrine tumor markers. Developed methods slightly differ in sample prep and gradient elution. They are suitable for routine practice in diagnostic lab .