1. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Multi-residues screening
by UPLC/QTOF
Experts: S.Berg, E.Lindfors, A.Niemi
L.Saari M.Rokka and M. Jestoi
Introduce: J.C.Yorke
2. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Screening process
Extraction process
Clean-up optional
Chromatography
Desolvatation Ionisation
Data acquisition
Data processing
Result
3. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Ultra performance liquid chromatography
• Use of liquid in very high pressure in order to separate
molecules in short and small i.d. column packed with
1,7µm
+ Elution of narrower peak
(increase of peak height decreases detection limit)
+ Increase of peak capacity by 2 or 3 times
(might be helpful to avoid co-elution)
+ Decrease run time of analysis (5-20 min)
- High pressure
- Track of the dead volume (can spoil the separation)
- Few columns (stationary phase) are available
4. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Quadrupole time of flight mass
spectrometer 1/6
Hybrid mass spectrometer coupling a quadrupole to an orthogonal
acceleration time of flight (Q-oa-TOF)
The continuous beam coming from the quad is accelerated by a pulse
applied every 45µs to a pusher plate initiates an orthogonal acceleration.
The ions into the packets have Kinetic energy in relationship with their
masses. The time of flight (TOF) is the time it takes an ion to travel the
length of the flight tube to reach the detector
Mass/charge= AxTOF2
If you inject a compound with a known mass (Lock mass) and
you measure its TOF you obtain A. So you are able to
determine M/z for every ions.
5. 19.05.15
kemian ja toksikologian tutkimusyksikkö
QTOF spectrometer 2/6
QTOF is a“No scanning” instrument allowing:
• MS
• MS/MS
• DDA
MS mode should be the starting point to obtain:
1. Full scan mass acquisition
2. Accurate mass measurement(<5ppm)
3. Enable to obtain spectrum of the molecule
6. 19.05.15
kemian ja toksikologian tutkimusyksikkö
QTOF spectrometer 3/6
Data processing:
Need a heavy data processing
The process can be done “manually”
• Process involves the reconstruction of ion
Chromatograms (narrow extraction window up to 0.01Da)
• Peaks detection
• Comparison of the spectrums obtained from these peaks
with reference spectrums
• Calculation of the mass difference of the unknown with
target component ( should be within 5 ppm)
7. 19.05.15
kemian ja toksikologian tutkimusyksikkö
QTOF spectrometer 4/6
Data processing
The process can be done automatically (need the
purchase of a software- Chromalynx)
We will obtain in that case:
• Raw TIC
• Accurate mass score
• Deconvulated spectrum
• Candidate list (identification of drugs in sample)
8. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Quadrupole time of flight mass
spectrometer 5/6
The use of narrow mass windows in reconstructed ion
chromatograms yields indeed highly selective chromatograms
10. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Definition of the project
Develop if possible, one extraction method followed
by UPLC/QTOF for screening veterinary drugs in
animal matrixes
• Should be ideally:
– Rapid
– Robust
– Generic
– Allow retrospective data processing
Might a little too much ideal!
11. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Definition of the project / matrixes
– Evira is currently analysing
the following matrixes
• Tissues ( muscle, liver,
kidney, fish)
• Plasma
• Urine
• Milk
• Eggs
• Honey
• All these analysis are made
with different animal
species
• These analysis are planned
annually in regards to the
national Finnish production
• All the samples are not
available at the same time
Retained matrix
12. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Definition of the project/ drugs
Two types of drugs are analysed with different philosophies
1. Banned substances, group A
Avoid false negative with low false positive at the lowest
detection limit
2. MRL substances, group B
Avoid false positive with precision for the quantification
around the MRL
Remark: The TOF accurate mass measurement is good
tool to identify substances and may be not the best tool
to quantify
13. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Definition of the project/ drugs
Group A
• Stibenes
• Thyreostasts
• Steroid
• Resorcylic acid
• Beta agonists
• Chloramphenicol
• Nitrofurans
• Nitroimidazols
Group B
• Antibacterials
• Tetracyclines
• Beta lactames
• Quinolones
• Sulfa drugs
• Macrolides?
• Anthelmintics
• Anticoccidials
• Carbamates
• Ionophores
• Sedatives
• NSAID
• Organochlorines
• Organophosphorous
• Mycotoxins
• Other pharmacologycally
active substances…
According to detection
capacity of the method
14. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Experimental1/2 work on standard
• We worked on chromatography on standards
– Goal to separated 70 veterinary drugs
– Achieved this separation in a short time
– To have reproducible runs
• Data detection
– Determine for each drug the capacity to be ionised in
electrospray positive or negative mode
– Evaluated the accuracy of the mass measure
– Got spectrums from each drugs
• Data processing
– Extracted ion chromatograms in order to detect peaks
(using Chrotool software)
15. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Experimental 2/2 work on extracts
• Screening process
2 liquid extraction protocols applied to:
• Pork musle tissues blank ( 5 different)
• Pork muscle tissue spiked
– 3 Spiked samples at the concentration of 1µg/g (solution
Nº1 of 45 vet drugs)
– 3 Spiked samples at the concentration of 0,1µg/g
(solution Nº2 of 7 vet drugs)
Evaluation of:
• Selectivity
• Sensivity
• Reproductibility
16. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Chromatography
• Aminoglycosides
• Anthelmintics: Avermectines
• Anthelmintics: Benzimidazoles
• Chloramphenicol
• Antibiotic nitroimidazol
• Antimicrobial,Fumagillin
• Betalactams
• Coccidiostats
• Gestagens
• Glucocorticoid
• Lincosamides
• Micotoxin
• Nitrofurans
• NSAID
• Quinolones
• Sedatives
• Sulfonamides
• Tetracyclines
• Thyreostat
• Triphenylmethan dye
70 Standards from stock solutions have been
evaporated to dry then reconstituted with ACN/Water
50/50 to give concentration from 0,5 to 10 µg/ml, then
injected on 2 columns
20. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Chromatography
We have not big choice because only few column are
available:
We had tested on standards two apolar columns 2,1mm
x100mm packed with a 1,7µm C18 and HSS T3
Solvent was a linear gradient H2O,HCO2,H, ACN
They were not big differences between the 2 columns
We got nice peak shape with reproducible retention time
No separation was achieved for aminoglycosides
Some problems with Coccidiostats (peak tailing)
21. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Detection
• Nitrofurans as well as Thyreostats were not detected (low
mass drugs)
– Might be due also by a bad tune of the cone voltage for
this drugs
• With the exception of Chloramphenicol and Fumagillin all
the drug were better detected in positive electrospray
ionisation
• With few exception mass measure accuracy was less than
10 ppm
• Quasi molecular ion was not always the biggest peak
• Adduct peaks (Na, K,NH4) as well dimere peak as been
seen
22. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Data acquisition
• Detection of the drug rely on accurate mass measurement
of the drug
Calculation of the molecular weight based on exact masses of the
most abundant natural isotope:
CxHyNzOt
12.011 x + 1.0079 y + 14.0067 z + 15.9994 t
Molecular ion ****
.
****
4 digit after the comma
used for the confirmation of positive
finding as isotopic ratio pattern
In order to be able to record the exact mass of the drug the QTOF
needs to acquire in parallel a lock mass ( we perfuse a compound that
we know the exact mass)
The tune of the instrument is done on this compound
Isotopic ions
23. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Desolvation ionisation
• Detection of the drug if it is ionised
– Rely on parameters:
• UPLC flow, solvents
• Temperatures: source and the gas
• Flow of the gas
• Voltage on electrodes
Mass/Charge
Fragments
Quasi Molecular ion
[Mw+H]+
or [Mw-H] +
Adduc
tions
Dimere
Solvent
Adduct
24. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Chromatography
• Aminoglycosides (No separation)
• Anthelmintics: Avermectines
• Anthelmintics: Benzimidazoles
• Chloramphenicol
• Antibiotic nitroimidazol
• Antimicrobial,Fumagillin
• Betalactams
• Coccidiostats
• Gestagens
• Glucocorticoid
• Lincosamides
• Micotoxin
• Nitrofurans (No detection)
• NSAID
• Quinolones
• Sedatives
• Sulfonamides
• Tetracyclines
• Thyreostat (No detection)
• Triphenylmethan dye
From the 74 drugs we decided to keep 52 drugs
(Not always detected
peak tailing)
25. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Chromatography
• We decided to shorten the retention time from 20
min to 8,5min
– We took a shorter C18 column 50mm instead
of 100 mm
– We change the gradient composition, 2 linear
gradient step
– we change the composition of gradient
26. 19.05.15
kemian ja toksikologian tutkimusyksikkö
RT min % B
0 0
4 30
6 100
7,5 100
UPLC Gradient
0
20
40
60
80
100
0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 5,5 6 6,5 7 7,5
RT min
%B(ACN)
Nitroimidazols
Quinolones
Lincosamides
Tetracyclines
Sedative
s
Sulfonamides
Benzimidazoles
Blactams
Triphenylmethandye
NSAID
Avemectines
Chromatography
27. Screening process/Liquid extraction protocols
PE1, Acid extraction
After an extraction by TCA the
samples are diluted 4,5 times
Weight 2g of minced meat in 15
ml polypropylene tube
Addition of the spiked solution*
(1ml ,90 %water),vortex and wait
15 min
Addition of 8 ml TCA 5%
Stir 10 min 100 rpm
Centrifugation 5min 4000G 10 C
Filtration 1ml 0,45µm
Injection 5µl
PE2, Organic extraction
After an extraction by ACN the samples
are concentrated 2 times
Weight 2g of minced meat in 15 ml
polypropylene tube
Addition of the spiked solution*(1ml ,
90%water),vortex and wait 15 min
Addition of 8 ml ACN
Stir 10 min 100 rpm
Centrifugation 5min 4000G 10 C
Evaporation of 5 ml of the supernatant to
dryness
Reconstituted the sample with 0,5ml
Formic acid 0,1%
Filtration 0,5ml 0,45µm
Injection 5µl
28. Screening process/results
PE1, Acid extraction
TCA samples are diluted 4,5
times
Clean extract
Increase of retention time due to
high acidity of the sample
Lack of sensitivity due to sample
dilution: 25% of the drugs are
detected
Selectivity not
evaluated
Reproductibility not
evaluated
Spectrums are useless because
of lack of sensitivity
The limit of detection of the Qtof
should be lowered by tuning the
instrument to promote the
ionisation of the drugs
PE2, Organic extraction
ACN samples are concentrated 2
times
Cloudy extract
might need to had a hexane
defating step prior the
evaporation of ACN
No shift of retention time
The narrow windows reconstructed
chromatograms are selective
For most of the drugs the method is
reproducible ( 2 to 17 %)
For most of the drugs the yield of
extraction is around 65 %
31. 19.05.15 kemian ja toksikologian tutkimusyksikkö
The avemectines are not detected in the standard solution at 2 µg/ml while they
are detected with a large relative standard deviation in the extract(1µg/g)
The benzimidazoles showed a large relative standard deviation in the extract
and a very high recovery due to may be a problem of ionisation of the extract.
Chloramphenicol and Fumagillin have been detected by ES- in an extract at the
concentration of 1µg/g. The variation of the response of the extracts has not been
evaluated(1 injection) nor the response of the standard (not been injected).
Detection limit should be expected around 10ppb.
Betalactams are detected as sodium adduct in the extracts at 1µg/g (with the
exception of amoxicillin: L.D.).We can hardly expect to see betalactams at100ng/g
because of a loss sensivity. Recovery and RSD are about 60% and 7-15% with
the exception of the cefalexin recovery 300%and 3%. The high recovery of
cefalexin can come by the fact they have less sodium adduct in the standard than
in the extract solution and the calculation is done with the sodium adduct (biggest
peak in the extract samples)
The Medroxyprogesterone acetate is detected in the extracts at 1µg/g. The high
recovery (500%) is not explained the RSD is about 17%.
Interpretation of the results from the extraction of Spiked
pork meat
32. 19.05.15 kemian ja toksikologian tutkimusyksikkö
The dexamethasone is detected in the extracts at 1µg/g. The recovery is 125%
and RSD is 5%. We can hardly expect to see dexamethasone at100ng/g because
of a loss sensivity.
Ochratoxin is detected in the extracts at 1µg/g. The recovery is 125% and RSD
is 15%.
NSAID are detected in the extracts at 1µg/g. The recovery is 75-102% and RSD
is 7-15%. We can expect to see 50 ng/g .
Quinolones are detected in the extracts at 1µg/g. The recovery is 320-240% and
RSD is 7-11,5%. We can expect to see 10ng/g because the signal is high.
Sulfonamides are detected in the extracts at 1µg/g. The recovery is 62--74% and
RSD is 2-30%. We can expect to see 10ng/g because the signal is high.
Tetracycline are detected in the extracts at 1µg/g. The recovery is 30-41% and
RSD is 7%. We can expect to see 50ng/g .
Triphenylmethan dye are detected in the extracts at 1µg/g. The recovery is
380% and RSD is 30% for Malachite green and 68% and RSD is 22% for
Leucomalachite green. We can expect to see 50ng/g
Interpretation of the results from the extraction of Spiked
pork meat
33. 19.05.15
kemian ja toksikologian tutkimusyksikkö
Improvement of the detection of the drug
• To be able to detect the drug the conditions of ionisation desolvation of
the drug should not be very different from the ones used for the lock
mass.( Hereafter the parameters for our lock Mass: LeucineEnkephalin)
– Capillary (kV) 3.0 -Extraction Cone 3.0
– Sampling Cone 30.0 - Ion Guide3.4
– Source Temperature (°C) 120- Desolvation Temperature (°C)400
– Cone Gas Flow (L/Hr) 20.0
– Desolvation Gas Flow (L/Hr) 1000.0
• The conditions of ionisation of our drugs seem to be much soft
– Sampling Cone 10-25 according to the drug (major parameter can
lead to detection limit 100 higher)
– Ion guide 1-2
Changing of compound for the lock mass
Changing the ion guide voltage
Programming the change of cone voltage in the method
34. 19.05.15
kemian ja toksikologian tutkimusyksikkö
MIX47+6 0.8 ppm I G1.6 SCone(14/0-4min, 30/3.8-8.5min) Scan0.1 IntScan0.02
Time
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10
%
-10
90
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10
%
-10
90
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10
%
-10
90
0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10
%
-10
90
031007005 Sm (Mn, 3x2) 1: TOF MS ES+
366.112 0.05Da
126
Area
1.48;214.0901;8.31
031007002 Sm (Mn, 3x2) 1: TOF MS ES+
366.112 0.05Da
88.8
Area
1.48;214.0888;5.44
031007005 Sm (Mn, 3x2) 1: TOF MS ES+
388.094 0.05Da
5.64
Area
1.47;214.0901;0.30
031007002 Sm (Mn, 3x2) 1: TOF MS ES+
388.094 0.05Da
6.70
Area
1.49;158.0269;0.41
Amoxocillin
Ion guide 3.4
Amoxocillin
Ion guide 1.6
Amoxocillin Na
Ion guide 1.6
Amoxocillin Na
Ion guide 3.4
+52.7%
-26.8%
Effect of the ion guide voltage on the analyte response
