Screening of Pharmaceuticals and Personal Care Products in ...

Screening of Pharmaceuticals and Personal Care
Products in Effluents and Surface Waters with
LCMS Q-TOF
Anthony Gravell
National Laboratory Service
Environment Agency

Agenda
• Pharmaceuticals in the environment - Why look for them?
• Analytical Challenges for LCMS Screening
• Compounds found in effluents and surface waters
• Confirmation using targeted MS/MS
• Summary

Pharmaceuticals – Why look for them?
• Widespread detection in environmental samples
• Hormones have significant EDC effects
• Anti-biotic resistance
• “Pseudo-persistent”
• European Union’s Water Framework Directive – New proposal to
add pharmaceuticals to Annex X

Pharmaceuticals – Why look for them?
• Exposure of pharmaceuticals to
aquatic life most likely from STW
discharges
• Environmental risks due are an
important issue for the EA regulator
• Chemical Investigation programme
recently undertaken by UK water
companies

http://www.epa.gov/ppcp/pdf/drawing.pdf

National Geographic, April 2010.
Pharmaceuticals in Fish

Analytical Challenges for LCMS Screening
Detect and identify isobaric
compounds
Isolate compounds from matrix
components
Must Haves
• High Resolution
Searching for unknowns
Large number of compounds
• High mass accuracy
• Wide dynamic range

Resolution FWHM Resolution
100%
Apex
R
m
m
50%
FWHM
400
• Quadrupole MS R 571
0.7
400
• Time of Flight MS R 25000
0.0160
500.10 500.11
500.13 500.14 500.15 500.16 500.17 500.18 500.19 500. m/z
Mass
Assignment

Resolution FWHM Resolution
Desisopropyl-Atrazine
C 5 H 8 ClN 5
Theoretical m/z: 174.0541
Experimental m/z:
174.0540
Mass Error: -0.57 ppm
Resolution: 25,207
Allidochlor
C 8 H 12 ClNO
Theoretical m/z: 174.0680
Experimental m/z:
174.0678
Mass Error: -1.15 ppm
Resolution: 25,556
Sample
0.3pg A:0.1pg B
Blank
No Chemical
Interference

Measuring mass accuracy
Mass error = (exact mass) – (accurate mass)
Mass error in parts per million(ppm) = mass error x 10 6
exact mass

Importance of resolution and mass accuracy
Labetalol
C19H24N2O3
Extracted Ion Chromatogram 400ppm

Importance of resolution and mass accuracy
Significant improvement in selectivity
Extracted Ion Chromatogram 10ppm
Extracted Ion Chromatogram 400ppm

Sample collection & extraction
Primary and final effluent
POCIS
SPE disks
Disk extraction equipment

LC parameters
1290 Infinity LC system
Column: Waters Atlantis T3, 2.1 x 150mm, 3.5 µm @ 40°C
Flow: 0.4 ml/min
Injection Volume: 5µL
Solvent A: 2mM Ammonium Acetate, Water, 0.05% formic acid
Solvent B: 2mM Ammonium Acetate, Methanol, 0.05% formic acid
Gradient: Time (min) %B
0 5
25 100
30 100
Equilibration time 10mins

MS parameters
Source parameters :
ESI Jetsream source
Drying Gas Temp: 300°C
Drying Gas Flow: 10l/min
Nebuliser: 50 p.s.i.
Sheath Gas Temp: 350°C
Sheath Gas Flow: 12 l/min
VCcap:
3000V
Fragmentor: 175V
Nozzle voltage: 1000V
Aquisition parameters:
ESI Positive:
2 & 4GHz modes
Scan Range:
80-950 m/z
Scan Rate:
3Hz
Internal Mass Calibration
Refererence ions: 121.05087 m/z, 922.009798 m/z

Identification and scoring
MS Score based on
Monoisotopic mass
(Mass Match)
Isotope distribution
(Abundance Match)
Theoretical isotope distribution and
spacing generated from the molecular
formulae contained in the database.
Score is assigned for each of the three
criteria and combined into a final score
for each target compound in sample
Isotope spacing
(Spacing Match)

What did we find?
Compounds found in effluents and surface waters

Personal Care Product
Cetirizine – Antihistamine
Extracted Ion
Chromatogram
Match factor > 98

Prescribed Pharmaceutical
Lamotrigine Anti-convulsant
Extracted Ion
Chromatogram
Match factor > 98

Personal Care Product
Orlistat – Weight loss aid sold
under trade name ‘alli’
Extracted Ion
Chromatogram
Log Kow: 8.19 Probably associated with suspended
particulates in effluent
Match factor > 98

PPCPs found in final effluent
Tgt Name
Cpd 1: Sotalol
Cpd 2: Atenolol
Cpd 3: Ranitidine
Cpd 4: Trimethoprim
Cpd 5: Amisulpride
Cpd 6: Lidocaine
Cpd 7: Metoclopramide
Cpd 9: Tramadol
Cpd 8: Tramadol
Cpd 10: Lamotrigine
Cpd 11: Mirtazapine
Cpd 12: Celiprolol
Cpd 13: Quinine
Cpd 14: Labetalol
Cpd 15: Venlaxafine
Cpd 16: Bisoprolol
Cpd 17: Propranolol
Cpd 18: Citalopram
Cpd 19: Flecainide
Cpd 20: Mebeverine
Tgt Score
98.19
98.16
92.48
99.59
97.4
99.71
75.91
99.87
99.9
98.86
91.14
98.63
98.76
78.41
98.75
95.33
89.72
99.89
99.39
97.31
RT Diff
-0.015
0.031
0.068
-0.003
0.019
0.188
0.046
-0.494
0.044
-0.03
0.207
-0.081
0.069
-0.17
0.035
0.056
0.046
-0.01
-0.013
0.01
Mass
Error
(ppm)
-0.78
2.33
0.32
0.17
1.08
-0.58
0.51
0.36
0.32
-0.21
0.96
-0.99
0.39
-1.6
0.08
-2.64
0.19
0.1
-0.42
0.25
Tgt Formula
C12 H20 N2 O3 S
C14 H22 N2 O3
C13 H22 N4 O3 S
C14 H18 N4 O3
C17 H27 N3 O4 S
C14 H22 N2 O
C14 H22 Cl N3 O2
C16 H25 N O2
C16 H25 N O2
C9 H7 Cl2 N5
C17 H19 N3
C20 H33 N3 O4
C20 H24 N2 O2
C19 H24 N2 O3
C17 H27 N O2
C18 H31 N O4
C16 H21 N O2
C20 H21 F N2 O
C17 H20 F6 N2 O3
C25 H35 N O5
Beta blocker
Anti-depressant

PPCPs found in final effluent (cont.)
Tgt Name
Cpd 21: Diltiazem-Cis
Cpd 22: Carbamazepine
Cpd 23: Fexofenadine
Cpd 24: Amitriptyline
Cpd 25: Erythromycin
Cpd 26: Losartan
Cpd 27: Cetirizine
Cpd 28: Naproxen
Cpd 29: Bezafibrate
Cpd 30: Clarithromycin
Cpd 32: Valsartan
Cpd 31: Dipyridamote
Cpd 33: Irbesartan
Cpd 34: Ketoconazole
Cpd 35: Atorvastatin
Cpd 36: Telmisartan
Cpd 37: Clopidogrel
Cpd 38: Dioctyl sulfosuccinate
Cpd 39: Orlistat
Tgt Score
98.99
99.75
98.91
97.31
99.73
88.33
97.46
99.42
95.39
97.82
95.74
99.15
98.93
78.76
85
97.87
96.52
98.88
98.45
RT Diff
0.024
0.003
-0.039
0.104
0.103
-0.037
0.011
-0.091
-0.113
0.077
-0.064
0.432
0.019
0.419
0.013
0.09
0.089
0.023
0.055
Mass
Error
(ppm)
-0.19
0.12
0.61
0.65
-0.1
0.79
0.15
-0.2
0.66
-0.45
0.05
-0.33
-0.61
0.29
-1.63
-0.56
0.4
0.24
0.1
Tgt Formula
C22 H26 N2 O4 S
C15 H12 N2 O
C32 H39 N O4
C20 H23 N
C37 H67 N O13
C22 H23 Cl N6 O
C21 H25 Cl N2 O3
C14 H14 O3
C19 H20 Cl N O4
C38 H69 N O13
C24 H29 N5 O3
C24 H40 N8 O4
C25 H28 N6 O
C26 H28 Cl2 N4 O4
C33 H35 F N2 O5
C33 H30 N4 O2
C16 H16 Cl N O2 S
C20 H38 O7 S
C29 H53 N O5
Calcium channel
blocker
angiotensin-II
receptor
antagonist

PPCPs found in final effluent (cont.)
Tgt Name
Cpd 21: Diltiazem-Cis
Cpd 22: Carbamazepine
Cpd 23: Fexofenadine
Cpd 24: Amitriptyline
Cpd 25: Erythromycin
Cpd 26: Losartan
Cpd 27: Cetirizine
Cpd 28: Naproxen
Cpd 29: Bezafibrate
Cpd 30: Clarithromycin
Cpd 32: Valsartan
Cpd 31: Dipyridamote
Cpd 33: Irbesartan
Cpd 34: Ketoconazole
Cpd 35: Atorvastatin
Cpd 36: Telmisartan
Cpd 37: Clopidogrel
Cpd 38: Dioctyl sulfosuccinate
Cpd 39: Orlistat
Tgt Score
98.99
99.75
98.91
97.31
99.73
88.33
97.46
99.42
95.39
97.82
95.74
99.15
98.93
78.76
85
97.87
96.52
98.88
98.45
RT Diff
0.024
0.003
-0.039
0.104
0.103
-0.037
0.011
-0.091
-0.113
0.077
-0.064
0.432
0.019
0.419
0.013
0.09
0.089
0.023
0.055
Mass
Error
(ppm)
-0.19
0.12
0.61
0.65
-0.1
0.79
0.15
-0.2
0.66
-0.45
0.05
-0.33
-0.61
0.29
-1.63
-0.56
0.4
0.24
0.1
Tgt Formula
C22 H26 N2 O4 S
C15 H12 N2 O
C32 H39 N O4
C20 H23 N
C37 H67 N O13
C22 H23 Cl N6 O
C21 H25 Cl N2 O3
C14 H14 O3
C19 H20 Cl N O4
C38 H69 N O13
C24 H29 N5 O3
C24 H40 N8 O4
C25 H28 N6 O
C26 H28 Cl2 N4 O4
C33 H35 F N2 O5
C33 H30 N4 O2
C16 H16 Cl N O2 S
C20 H38 O7 S
C29 H53 N O5
Antibiotic
Treatment of
hyperlipidaemia &
cholesterol reducing

PPCPs found in surface water
plus a lot more!

Confirmation by MS/MS - Standard
Ofloxacin
Propanolol
Fluoxetine
Oxytetracycline
Erythromycin

Confirmation by MS/MS - Sample
TIC
Propranolol
EICs
Ofloxacin
Oxytetracycline
Erythromycin
(not present)
Fluoxetine

Confirmation of Fluoxetine by MS/MS
Precursor ion
A
Mass error = 0.8ppm
A

Confirmation of Propranolol by MS/MS
Average mass error = -2.67ppm

Summary
• Significant number of pharmaceuticals identified
• Various therapeutic classes found in all matrices
• Mass accuracy is a valuable tool for target identification
• High resolution ensures good mass accuracy with real samples
• How much resolution is required depends on matrix

Summary (cont.)
• Use of MS/MS with accurate mass provides unequivocal
compound identification
• Screening method shows potential for determining efficiency
of various WWTW treatment options
• POCIS provides higher sampling rates and lower LODs

Further work
• Use passive samplers to detect low concentrations of EDC’s
• Look at metabolites of pharmaceuticals
• Chirality and different effects / toxicity
• Link to High Resolution GC/MS

Acknowledgements
Prof. Graham Mills
University of Portsmouth
Agilent Technologies
Dwr Cymru / Welsh Water

Any questions?