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EEA/Norway Grants 2009-2014 Programme LV05 NORWEGIAN-LATVIAN RESEARCH COOPERATION ACTIVITY Project Progress Project leader at UiO prof. Elsa LUNDANES Presented by Dr Silvija ABELE 15th of February, 2016 University of Oslo 1
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EEA and Norway Grants 2009-2014 Research and Scholarship Programme LV05 Research NFI/R/2014/010 Establishing of the scientific capacity for the management of pharmaceutical products residues in the environment of Latvia and Norway 218 432.00 EUR 01.06.2015 - 30.04.2017 Scholarship EEZ/NFI/S/2015/003 Educational collaboration between Norway and Latvia for establishment of an effective risk assessment and control of pharmaceutical residues 5971.00 EUR 01.08.2015 - 31.12.2015 2
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EEA/Norway Grants 2009-2014 Programme LV05 NORWEGIAN-LATVIAN RESEARCH COOPERATION ACTIVITY Establishing of the Scientific Capacity for the Management of Pharmaceutical Product Residues in the Environment of Latvia and Norway NFI/R/2014/010 3
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NO-LV RESEARCH COOPERATION ACTIVITY Research Project NFI/R/2014/010 Overall objective of the Project is 1) to enhance the growth of research-based knowledge 2) through the scientific cooperation between Norway and Latvia, 3) developing a scientific capacity for the administration of pharmaceutical residues in the Latvian and Norwegian environment 4
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Project Background and Justification Continuous increasing consumption of pharmaceutical products (PP) PP residues are introduced into the aquatic environment from: – municipal treatment plants – hospital effluents – livestock farming – households PP presence in the environment - a serious threat to environment and human health – antibiotics resistant bacteria strains – hormones endocrine disrupting effects 5
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Publications on Occurence of Pharmaceuticals in the Environment 6 Journal of Hazardous Materials 175 (2010) 45–95
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Project Concept The knowledge about PP, their short and long- term environmental impacts, possible degradation mechanisms and management strategies is essential Advanced high resolution liquid chromatography - mass spectrometric method will be developed to obtain information about the PP contamination status 7
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The Aim of the Study To develop and validate an ultrasensitive and selective analytical method for combined quantitative analysis of pharmaceuticals in the environmental water samples Ultra-High Performance Liquid Chromatography coupled to a hybrid quadrupole Orbitrap mass spectrometer (UHPLC- q-Orbitrap) will be used together with on-line sample preparation (SPE) Results obtained in Riga and Oslo will be compared 2-3 Peer-reviewed publications on analytical performance and application advantages of the elaborated method 8
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Pharmaceuticals Studied 9
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Stages of the Research Project Sample preparation Method validation Method optimization Sample collection Method development using individual standard compounds
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Caffeine standardRT 15 min Reverse phase chromatography Gradient 2% B to 95% B in 20 min Buff A = water with 0.1% FA Buff B = 95% ACN / 5 % water 0.1 % FA Buff A flow rate 50 µL/min Concentration 0.01 mg/mL Injection 50 µL UV-Detection 254 nm UV-HPLC 11
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Ketoprofen Reverse phase chromatography Gradient 2% B to 95% B in 20 min Buff A = water with 0.1% FA Buff B = 95% ACN / 5 % water 0.1 % FA Buff A flow rate 50 µL/min Concentration 0.01 mg/mL Injection 50 µL UV-Detection 280 nm Analgesic / Anti-inflammatory UV-HPLC 12
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Metoprolol Diclofenac Ketoprofen Fluoexetine Coffeine Carbamazepine Azythromycine Losartan UV-HPLC Anti-hypertensive Analgesic / Anti-inflammatory 13
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LC-MS-MS Results Ketoprofen Sulfamethoxazole Acetaminophen (Paracetamol) Analgesic / Antipyretic Antibiotics Analgesic / Anti-inflammatory Conditions 0.0001 mg/mL 800 uL injection 5 uL/min flow rate Aim to detect 10 pg/mL 14
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Experimental Setup Filter From autosampler/Pump 1 Flow: 50 µL/min 0.1 % FA in H 2 O Injection volume: 100 µL SPE From pump 2 Flow: 5 µL/min To column (ID 0.3 mm) To waste Load Particles Pharmaceuticals Salts/solvent Credit Hanne Røberg-Larsen 15
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Filter From autosampler/Pump 1 Flow: 50 µL/min 0.1 % FA in H 2 O Injection volume: 800 µL SPE From pump 2 Flow: 5 µL/min To column (ID 0.3 mm) Inject To waste Particles Pharmaceuticals Salts/solvent Experimental Setup Credit Hanne Røberg-Larsen 16
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LC-MS-MS Results Reverse phase chromatography The Thermo Scientific™ TSQ Quantiva™ Triple Quadrupole Mass Spectrometer Conditions 0.0001 mg/mL 800 uL injection 5 uL/min flow rate 30 min gradient Retention time, min 23 Compounds 17
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Sample preparation OSLO On-line SPE (solid phase extraction) Small volumes (1 mL) Automatic No contamination Time effective RIGA Off-line (extract purification on phospholipid columns) Large volumes (5 L) Manual Possible lab contamination Time consuming Results to be compared 18
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Occurence study of AB by UHPLC-Orbitrap-HRMS RIGA Occurence studies of 34 multi-class antibiotics were performed in fish samples obtained from 5 inland water farms in Latvia Presence of two compounds was determined: 19
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Outcome cooperation between the Latvian and Norwegian scientists on application of PPs analysis methods benefiting short and long-term management on the distribution of pharmaceuticals in the Latvian and Norwegian environment determination of their biodegradation processes and the methods reducing their hazardous impact on human health 20
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EEA/Norway Grants 2009-2014 Programme LV05 NORWEGIAN-LATVIAN RESEARCH COOPERATION ACTIVITY Educational collaboration between Norway and Latvia for establishment of an effective risk assessment and control of pharmaceutical residues EEZ/NFI/S/2015/003 21
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Synergy Aspect. Scholarship Activity Visits and/or the internship of several academic staff members and PhD students from the University of Latvia to the University of Oslo Visit of academic staff members of UiO to UL, and holding several lectures devoted to activities of the current Project to students of the Faculty of Chemistry 22
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Mobility: November/ December 2015 23
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Synergy Aspect. Research Activity Successful long-term scientific and educational cooperation between researchers of Latvian and Norwegian Universities Acquired knowledge of environmental object analysis Transfer of knowledge between research groups Involvement of PhD students, master and bachelor degree students Development of training and study materials Joint research publications 24
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People involved University of Latvia / BIOR Assoc. professor Vadims Bartkevics Professor Arturs Viksna Researcher Dr Silvija Abele Dr Dzintars Zacs Dr Ingars Reinholds Iveta Pugajeva Olga Muter 25
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People involved Oslo University Professor Elsa Lundanes Assoc. professor Steven Ray Wilson Researcher Dr Silvija Abele Master student Deniz Demir Bachelor student Sunniva F. Amundsen 26
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Future Tasks Sampling of inland water bodies and industrial waste waters in Latvia and Norway Application of the elaborated analytical methods (UHPLC-Orbitrap-HRMS) for the assessment of PPs in collected inland water and waste water samples Preparation of peer-reviewed publications (in progress) of analytical performance and application advantages of the elaborated sensitive high resolution MS methods for the analysis of pharmaceuticals 27
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