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

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 EEA and Norway Grants 2009-2014
Research and Scholarship Programme LV05
Research Scholarship

• 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
• 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

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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

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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

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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
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 Publications on Occurence of
Pharmaceuticals in the Environment

Journal of Hazardous Materials 175 (2010) 45–95 6

 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

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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

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Pharmaceuticals Studied

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Stages of the Research Project
Method development using
individual standard
compounds

Sample collection

Sample preparation

Method optimization

Method validation
 UV-HPLC
Caffeine standard RT 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 11
 UV-HPLC
Ketoprofen

Ketoprofen Analgesic / Anti-inflammatory

• 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
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 UV-HPLC
Coffeine

Fluoexetine

Diclofenac Analgesic / Anti-inflammatory

Ketoprofen

Metoprolol

Carbamazepine

Losartan Anti-hypertensive

Azythromycine
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 Conditions
Aim
to detect 10 pg/mL LC-MS-MS Results 0.0001 mg/mL
800 uL injection
5 uL/min flow rate

Acetaminophen
(Paracetamol) Analgesic / Antipyretic

Sulfamethoxazole

Antibiotics

Ketoprofen

Analgesic / Anti-inflammatory
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 Experimental Setup

To waste
From autosampler/Pump 1
Flow: 50 µL/min
0.1 % FA in H2O Load
Injection volume: 100 µL Filter
Particles
Pharmaceuticals
Salts/solvent

To column (ID 0.3 mm)

SPE

From pump 2
Flow: 5 µL/min
Credit Hanne Røberg-Larsen
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 Experimental Setup

To waste
From autosampler/Pump 1
Flow: 50 µL/min
0.1 % FA in H2O Inject
Injection volume: 800 µL Filter
Particles
Pharmaceuticals
Salts/solvent

To column (ID 0.3 mm)

SPE

From pump 2
Flow: 5 µL/min
Credit Hanne Røberg-Larsen
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 min
Retention time,

10
15
20
25
30
35

0
5
Atenolol 9.23
Acetaminophen (Paracetamol) 10.75
Trimethoprim 10.83
Ciprofloxacin 10.96
Caffeine 11.21
Xylazine hydrochloride 11.51
Azithromycin 11.64
Metoprolol tartrate salt 11.73

• Reverse phase chromatography

S-Propanolol hydrochloride 13.20
Erythromycin A dihydrate 13.57
Clarithromycin 14.63
Fluoxetine hydrochloride (Prozac) 14.96
Sulfamethoxazole 15.10
Carbamazepine
23 Compounds

16.70
Losartan potassium 17.34
Naproxen 19.35
Ketoprofen 19.94
Valsartan 20.31
Atorvastatin calcium 21.10
LC-MS-MS Results

Diclofenac 22.00
Ibuprofen 22.49
Simvastatin 26.20
Gemfibrosil 29.55
Conditions

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0.0001 mg/mL

30 min gradient
800 uL injection

• The Thermo Scientific™ TSQ Quantiva™ Triple Quadrupole Mass Spectrometer

5 uL/min flow rate
 Sample preparation
OSLO RIGA
• On-line SPE (solid phase • Off-line (extract purification on
extraction) phospholipid columns)
• Small volumes (1 mL) • Large volumes (5 L)
• Automatic • Manual
• No contamination • Possible lab contamination
• Time effective • Time consuming
Sample preperation

Results to be compared 18
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:

12 8.30
Concentration (ng/g)

10
8 Trout
6 Sturgeon
4
0.49 0.66 0.82
2
0
Enrofloxacin Trimethoprim
Antibiotic 19
 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

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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

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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

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Mobility: November/ December 2015

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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

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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

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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

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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

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