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LC/MS/MS Analysis of 75 Endocrine Disrupting Compounds, Pharmaceuticals, and Personal Care Products in River Water
Loren Y Olson, Christopher D Borton, Hesham Ghobarah,Elliott B Jones, and Andre SchreiberLCMS Applications GroupApplied BiosystemsTM/MDS SciexTM
2 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Outline● Introduction
– What are endocrine distruptors (EDC)– Method development strategies and hardware
● 75 Analytes using QqQ scans on a 4000 Q Trap® System– Single injection with polarity switching– Simultaneous quantitation and confirmation
● 25 EDCs using a hybrid scan modes Q Trap® System– Multiple injection with dedicated ionization modes
(DuoSprayTM Source)– High throughput quantitation and spectral confirmation– Hybrid scan mode approach
3 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Introduction – Why Study Endocrine Disruptors?● Endocrine disruptors affect health of humans and aquatic ecosystems● Pesticides, industrial waste, pharmaceuticals, and personal care products
– Many act as hormone mimics● Endogenous hormones - potent effects on development
– Thus, ECDs can also have adverse effects, even at seemingly low levels● Criteria for testing not set or are outdated● Transgenerational effects / bioaccumulation● Population growth● New tools and techniques are needed
– Identify threats to the environment– Take action before adverse health effects are seen in organisms
4 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Strategies for LC/MS/MS Method Development
● Demonstrate feasibility of using different strategies LC/MS/MS for EDCs.– Screen, quantitate and confirm in environmental water samples.– ESI vs. APCI or DuoSprayTM Source– Polarity switching– Single vs. multiple injections– Performance and practicality
● Evaluate sample preparation– SPE– Direct detection - no derivatization
● Hardware and software automation– Increase efficiency data thoughput– Less manual review of data
● Data Reduction– Analyst® 1.4.1 and accessory software– Draw conclusions quickly and with confidence
5 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
HPLC Method
● Shimadzu LC system● Column: Phenomenex Ultracarb C18 250 x 4.6mm● Mobile Phase A: water + 0.01% formic acid● Mobile phase B: acetonitrile + 0.01% formic acid● Column oven temperature 28°C● Flow of 600µL/min● Injection of 50µL
6 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Mass Spec Method
● 4000 Q Trap® LC/MS/MS system● Turbo V™ source (ESI)● Heater temperature of 600°C● Polarity switching● 2 MRM transitions per compound● Total run time of 55min
7 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Turbo VTM Ionsource
• Flowrates: 10µL/min - 2 mL/min• Orthogonal spray• Excellent desolvation capacity• Entrainment of Taylor cone • Exhaust baffles to minimize tailing• Fast changeover from ESI to APCI
8 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
+1.5V Field Gradient
Collision Cell Entrance Collision Cell Exit
LINAC® Axial Field Gradient (LINAC=Linear Accelerator)
● Because of a field gradient, ions pass through the collision cell much faster than standard collision cells
● Q2 LINAC® collision cell eliminates cross-talk and allows faster MS/MS scanning without sensitivity losses
● Q2 rods are tilted and separate DC potentials are applied to each pair of rods to create an axial electric field
9 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Compound Type Compound TypeAcetaminophen Analgesic Estradiol 17a Estrogen
Ketoprofen Analgesic Estradiol 17b EstrogenCodeine Analgesic Ethynylestradiol Estrogen
Hydrocodone Analgesic Progesterone Estrogen Acetylsalicylic Acid Analgesic Equilin Estrogen replacement
Ibuprofen Analgesic Diethylstilbestrol Estrogen replacementAndorostenedione Androgen Simazine Herbicide
Testosterone Androgen Isoproturon HerbicideWarfarin Anti coagulant Chlorotoluron Herbicide
Diclofenac Anti-arthritic Atrazine HerbicideMeprobamate Anti-axiety Chloridazon HerbicideSulfadiazine Antibiotic Propazine Herbicide
Sulfamethoxazole Antibiotic Diuron HerbicideSulfathiazole Antibiotic Hexazinone Herbicide
Sulfamerazine Antibiotic Bromacil HerbicideSulfamethizole Antibiotic Metazachlor HerbicideSulfamethazine Antibiotic Metolachlor Herbicide
Sulfachloropyridazine Antibiotic 2,4-D HerbicideTrimethoprim Antibiotic DEET Insect Repellant
Sulfadimethoxine Antibiotic Bezafibrate Lipid RegulatorCiprofolxacin Antibiotic Clofibric Acid Metabolite of Lipid RegulatorPenicillin G Antibiotic Diazepam Muscle-relaxantAmoxicillin Antibiotic Norethisterone Ovulation InhibitorLincomycin Antibiotic Theophylline Stimulant
Oxytetracycline Antibiotic Theobromine StimulantChlortetracycline Antibiotic Caffeine Stimulant
Viginiamycin Antibiotic Oxybenzone SunscreenErythromycin Antibiotic Sildenafil Virility regulator
Roxithromycin Antibiotic Vardenafil Virility regulatorTylosone Tartrate Antibiotic Cotinine
Carbadox Antibiotic KetorolacTriclosan (Irgasan) Antibiotic Meclofenamic Acid
Chloamphenicol Antibiotic NifedipineFluoxetine Anti-depresant Indomethacin
Carbamazepine Anti-seizure DiatrizoatePentoxifylline Blood Viscosity Reducing Agent 2,4-Dichlorobenzoic Acid
Estrone Estrogen
Sample List
75
10 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
100 ppb Standard – ESI with Polarity Switching
ESI Positive65 analytes
ESI Negative10 analytes
11 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
2 MRMs Per Analyte Were Used for Confirmation
Diclofenac
ClofibricAcid
Warfarin
Ibuprofen
ChloramphenicolAcetylsalicylic Acid
12 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Linear Over 3-4 Orders of Magnitude (R2 > 0.995)
Theophylline 1Stimulant
Chlortoluron 1Pesticide
Sulfathiazole 1Antibiotic
Vardenafil 1Virility Regulator
Theophylline 2 Chlortoluron 2
Sulfathiazole 2 Vardenafil 2
181.4 / 124.1 213.1 / 72
181.4 / 96.1 213.1 / 140
256.3 / 156.1
256.3 / 92.1
490.3 / 72.1
490.3 / 114.1
13 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Solid Phase Extraction Results in a 100X Concentration of Analytes
1L RiverWater
1L Filtered
Filter
SPE
NEW
Condition SPE20 mL MeOH,40 mL Water.
SPE
Rea
dy
10mL
14 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Solid Phase Extraction Results in a 100X Concentration of Analytes
1L RiverWater
1L Filtered
Filter
SPE
NEW
Condition SPE20 mL MeOH,40 mL Water.
SPE
Rea
dy
10mL
50µL
15 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
SPE Recovery - Comparison of Pre and Post Extraction Analyte Spike
Pre vs Post Extraction Spike
020406080
100120
Linco
mycin
1Coti
nine 2
Cotinin
e 1Vard
enafi
l 1
Triclos
an (Irg
asan
) 1The
ophy
lline 1
Piroxic
am 1
2,4-D
1Diclo
fenac
1Ind
ometh
acin
1
Acetam
inoph
en 1
Carbad
ox 1
Clofibric
Acid 1
Hydroc
odon
e 1Virg
iniam
ycin
1Trim
ethopri
m 1Diaze
pam 1
Ketopro
fen 1
Bezafi
brate
1Ciprof
loxacin
1Fluo
xetin
e 1Atra
zine 1
Tyloso
ne Tart
rate 1
Metaza
chlor
1Tes
tosteron
e 1Sild
enafi
l 1Prop
azine
1Diuron
1
Sulfam
ethaz
ine 1
Perc
ent r
ecov
ery
Pre vs Post Extraction Spike
050
100150200250300350400
Estrad
iol 17
b 1
Roxith
romyc
in 1
Sulfam
ethizo
le 1
Sulfam
erazin
e 1
Sulfac
hlorop
yrida
zine 1
Chloram
phen
icol 1
Sulfad
iazine
1Ibu
profen
1Keto
rolac
1Warfa
rin 1
Chlorot
oluron
1Sim
azine
1Sulf
athiaz
ole 1
Norethi
steron
e 1
Sulfam
ethox
azole
1Meto
lachlo
r 1Hex
azino
ne 1
Andros
tened
ione 1
Estron
e 1Pen
toxify
lline 1
Oxybe
nzone
1
Meprob
ramate
1
Erythro
mycin
1Brom
acil 1
Sulfad
imeth
oxine
1
Carbam
azep
ine 1
Chlorid
azon
1DEET 1
Caffein
e 1
Perc
ent r
ecov
ery
100%
16 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Collection of River Water Samples
River 1 – Influent and effluent samples of a river in a rural area
River 2 – Influent and effluent samples of a river running through an urban area
17 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Chromatograms - River 1 – A Rural AreaInfluent Effluent
Pos
itive
ES
IN
egat
ive
ES
I 2,4 DHerbicide
CarbamazepineAnti-convulsive
18 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Chromatograms - River 1 – A Rural AreaInfluent Effluent
Pos
itive
ES
IN
egat
ive
ES
I 2,4 DHerbicide
SulfamerazineAntibiotic
19 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Influent [C] Effluent [C]Analyte ng/L (ppt) ng/L (ppt)
Erythromycin 3.08 53.5Carbamazepine 65.5 151.5
2,4-D ND 9.35DEET 1.49 1.67
Sulfamethoxazole 13.15 13.3Caffeine 40.95 23.5
Ciprofloxacin 3.805 NDCotinine 2.05 ND
Increases by >2XWithin 2XDecreases by >2X
Results Table - River 1 – A Rural Area
ND = not detected
20 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Influent Effluent
Pos
itive
ES
IN
egat
ive
ES
IChromatograms - River 2 – An Urban Area
21 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCPDetection Confirmation Using MRM Area Ratios
Detection and Confirmation Using MRM Area Ratios of Contaminants in River 2
22 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Results Table - River 2 – An Urban AreaInfluent [C] Effluent [C]
Analyte ng/L (ppt) ng/L (ppt)Oxybenzone ND 6.25 Increases by >2X
Bromacil ND 7.4 Within 2XDiazepam ND 0.3875 Decreases by >2XWarfarin ND 0.93
Triclosan (Irgasan) 5.9 31.4Codine 17.05 77.5Diuron 1.375 4.345
Trimethoprim 58.5 122.5Lincomycin 1.525 3.015
Carbamazepine 870 1305DEET 24 29.9
Ketorolac 2.49 3.06Meprobramate 85.5 97.5
Atrazine 1.075 0.875Sulfamethoxazole 95.5 74.5
Pentoxifylline 6.6 3.385Caffeine 57 13.5Cotinine 14.4 NDSimazine 1.01 ND
Norethisterone 1.15 NDErythromycin 134.5 ND
Tylosone Tartrate 4.275 ND2,4-D 3.24 ND
ND = not detected
23 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Example Reports from Ion Ratio Report Generator Software
24 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Example Reports from Ion Ratio Report Generator Software
25 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Example Reports
26 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
4000 Q Trap® Method for Optimized Quantitative Performance and Qualitative ID in Treated Tap Water● Not sample limited
– Multiple injections possible– Higher throughput chromatography
● Sensitivity, robustness, linearity, precision are a priority– DuoSprayTM Source– Dedicated polarity
● Smaller, more focused analyte list (25 total)● Spectral confirmation and the ability to do qualitative ID on
unknowns– MRM IDA (Information Dependant Acquisition)
27 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
DuoSprayTM Source - for Diverse Sets of Analytes
APCI Probe
Turbo Heater
ESI Probe
Corona DischargeNeedle
28 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
HPLC Method
● Shimadzu LC system● Column: Phenomenex Polar RP C18 50 x 2.0mm● Mobile Phase A: water + 0.1% formic acid● Mobile phase B: acetonitrile + 0.1% formic acid● Flow of 800µL/min● Injection of 10µL
29 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Mass Spec Method
● 4000 Q Trap® LC/MS/MS system● DuoSpray™ source (automated ESI and APCI switching)● Heater temperature of 600°C● Dedicated ionization – Positive or Negative only● 1 MRM transition per compound● Total run time of 4min
30 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
A Schematic of the 4000 Q Trap® Ion Pathlinear ion trap
3x10-5 Torr
LINAC
Q0 Q1 Q2 Q3
• Hybrid QqQ / Linear Ion Trap (LIT)• QqQ - selectivity and sensitivity• Fast and sensitive full scan performance• Greater Ion capacity and higher resolution• MS3 and other scan modes
31 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Radial Trapping
Radial Trapping
AxialTrapping
IQ3 ExitBarrier
LIT Trapping Ions
32 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
LIT Scanning
Radial Trapping
AxialTrapping
Exit Lens with Grid
33 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
The Structure of an IDA Experiment
MS2 1
DynamicBackgroundSubtraction
MS2 2
MS2 3
MS2 4
Survey 1
Survey 2
IDA Criteria
SurveyScans
DependentScans
34 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
LLOQ from a Higher Throughput Multiple Injection Approach.
LLOQ are based on a signal to noise of 10 and an injection volume of 10 µL.%RSD were 1 to 3%
Compound fg on column ppt Compound fg on column pptcarbofuran 14.9 1.5 Progesterone 340.1 34.0triazophos 17.9 1.8 Ethynyl estradiol 1000.0 100.0linuron 493.1 49.3 Chloramphenicol 13.3 1.3Acetominophen 200.0 20.0 2,4-D 87.7 8.8Erythromycin 7.5 0.8 Silvex 348.4 34.8Sulfamethoxazole 101.4 10.1 ibuprofen 362.3 36.2Tetracycline 1.9 0.2 Aldicarb - 116 frag 47.6 4.8Caffeine 314.0 31.4 Methomyl 21.7 2.2Oxybenzone 213.7 21.4 Aldicarb - NH4 18.9 1.9DEET 6.0 0.6 Hydrocodone 10.0 1.0Diazepam 153.8 15.4 Buspirone 2.2 0.2Atrazine 38.5 3.8 Androstenedione 135.1 13.5Estradiol 469.5 46.9 Testosterone 100.0 10.0
35 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
LLOQ from a Higher Throughput Multiple Injection Approach.Compound fg on column ppt Compound fg on column pptcarbofuran 14.9 1.5 Progesterone 340.1 34.0triazophos 17.9 1.8 Ethynyl estradiol 1000.0 100.0linuron 493.1 49.3 Chloramphenicol 13.3 1.3Acetominophen 200.0 20.0 2,4-D 87.7 8.8Erythromycin 7.5 0.8 Silvex 348.4 34.8Sulfamethoxazole 101.4 10.1 ibuprofen 362.3 36.2Tetracycline 1.9 0.2 Aldicarb - 116 frag 47.6 4.8Caffeine 314.0 31.4 Methomyl 21.7 2.2Oxybenzone 213.7 21.4 Aldicarb - NH4 18.9 1.9DEET 6.0 0.6 Hydrocodone 10.0 1.0Diazepam 153.8 15.4 Buspirone 2.2 0.2Atrazine 38.5 3.8 Androstenedione 135.1 13.5Estradiol 469.5 46.9 Testosterone 100.0 10.0
Positive ESINegative ESIPostive APCI
36 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Comparison - MRM vs. MRM IDA Experiments
Dedicated MRM runs
MRM IDA runs
37 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Automatically Generated EPI (MS/MS) Spectra for ID / Structural Elucidation
Triazophos
DEET
Atrazine
38 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Automatically Generated EPI (MS/MS) Spectra for ID / Structural Elucidation
Triazophos
DEET
Atrazine
39 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Library Search Confirms the Presence of Atrazine
40 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Conclusion● EDCs are growing in importance in environmental monitoring● Quantitative analysis of 75 EDCs by triple quadrupole
– Single injection with polarity switching– Simultaneous quantitation and confirmation
● Analysis of EDCs using a Q Trap® System– Multiple injection with dedicated ionization modes– High throughput quantitation and spectral confirmation
41 © 2006 Applera Corporation and MDS Inc.
LC/MS/MS Approach to Endocrine Disruptors and PPCP
Legal Acknowledgements● For Research Use Only. Not for use in diagnostic procedures.
● Applied Biosystems and TURBO V are trademarks and Applera corporation.
● API 4000 and DuoSpray are trademarks of Applied Biosystems/MDS Sciex, a joint venture between Applera Corporation and MDS, Inc.
● ANALYST, Q Trap, and LINAC are registered trademarks of Applied Biosystems/MDS Sciex, a joint venture between Applera Corporation and MDS, Inc.
● MDS and MDS Sciex are trademarks of MDS Inc.
● All other trademarks are the sole property of their respective owners.
Thank you for your time.Questions?
The authors would like to thank…
Renee Huang for logistical support and Tania Sasaki and Matt Champion editorial support, as well as the entire Foster City demo team and applied markets division of ABI.