LC/MS/MS Analysis of 75 Endocrine Disrupting Compounds ... WCTOC PPCP ED.pdf · LC/MS/MS Analysis...

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



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– Thus, ECDs can also have adverse effects, even at seemingly low levels● Criteria for testing not set or are outdated● Transgenerational effects / bioaccumulation● Population growth● New tools and techniques are needed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



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



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



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



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



+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



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



100 ppb Standard – ESI with Polarity Switching

ESI Positive65 analytes

ESI Negative10 analytes



2 MRMs Per Analyte Were Used for Confirmation

Diclofenac

ClofibricAcid

Warfarin

Ibuprofen

ChloramphenicolAcetylsalicylic Acid



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



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



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



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%



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



Chromatograms - River 1 – A Rural AreaInfluent Effluent

Pos

itive

ES

IN

egat

ive

ES

I 2,4 DHerbicide

CarbamazepineAnti-convulsive



Chromatograms - River 1 – A Rural AreaInfluent Effluent

Pos

itive

ES

IN

egat

ive

ES

I 2,4 DHerbicide

SulfamerazineAntibiotic



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



Influent Effluent

Pos

itive

ES

IN

egat

ive

ES

IChromatograms - River 2 – An Urban Area


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



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



Example Reports from Ion Ratio Report Generator Software



Example Reports from Ion Ratio Report Generator Software



Example Reports



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)



DuoSprayTM Source - for Diverse Sets of Analytes

APCI Probe

Turbo Heater

ESI Probe

Corona DischargeNeedle



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



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



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



Radial Trapping

Radial Trapping

AxialTrapping

IQ3 ExitBarrier

LIT Trapping Ions



LIT Scanning

Radial Trapping

AxialTrapping

Exit Lens with Grid



The Structure of an IDA Experiment

MS2 1

DynamicBackgroundSubtraction

MS2 2

MS2 3

MS2 4

Survey 1

Survey 2

IDA Criteria

SurveyScans

DependentScans



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



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



Comparison - MRM vs. MRM IDA Experiments

Dedicated MRM runs

MRM IDA runs



Automatically Generated EPI (MS/MS) Spectra for ID / Structural Elucidation

Triazophos

DEET

Atrazine



Automatically Generated EPI (MS/MS) Spectra for ID / Structural Elucidation

Triazophos

DEET

Atrazine



Library Search Confirms the Presence of Atrazine



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



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.

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