EPA Method 1694 with LC/MS/MS (QQQ)

EPA 1694: Pharmaceuticals and Personal Care Products in Water Soil, Sediment, and 

Biosolids by HPLC/MS/MS

Imma Ferrer1 E Michael Thurman1 and Jerry Zweigenbaum2Imma Ferrer1, E. Michael Thurman1, and Jerry Zweigenbaum2

1Center for Environmental Mass Spectrometry, University of Colorado,Boulder, CO and 2Agilent Technologies, Inc. Wilmington, DE

CEMS: Center for Environmental Mass Spectrometry

University ofColorado

Dept. CEAE

Center for EnvironmentalMass Spectrometry

Collaborators: Scientists,

MissionState-of-the-Art environmental analytical research to address

challenging environmental issues

USGS AgilentCollaborators: Scientists,

Instruments, National infrastructure

challenging environmental issuesProof-of-principle clearinghouse to test preliminary ideas

Experimental investigationsEducation and technical training

ServiceUniversity Wide

TeachingUniversity Wide

State-of-the ArtEnvironmental Analytical

ResearchResearch

Emerging contaminants• ECs are widely used, synthetic or organic 

compounds, measured in <μg/L concentrations, and not traditionally 

2009 <ppt

tested for in water quality assessments• Measuring emerging contaminants is an 

area of active environmental research. • Driven by improvements in analytical 

methods– At the sub pg/L levels!pgIn the area of PPCPs there are several methods addressing these analytes including EPA Method 1694.

Courtesy of USGS

gToday’s report describes the Agilent solution to Method 1694 (June 2008)

ECs in the NewsECs in the News

US: Phillie, DC, SouthernCalifornia

Europe: Spain, Germany,France, Italy

Japan Vietnam India ChinaJapan, Vietnam, India, China,Australia

Effects: Feminization of fish,Hormone targets, humaneffects??

EPA Response is Method 1694 andEPA Response is Method 1694 and 1698 for PPCPs and Hormones1694 Published in Dec 2007 about year agoLC/MS/MS method for PPCPS in soil  sediment  and LC/MS/MS method for PPCPS in soil, sediment, and water and biosolids 

EPA 1694 Consists of:75 Analytes20 internal standardsSPE for WaterLiquid extraction for solids that is combined into SPEqSingle transitions MS/MS by MRMRigid chromatographic RTs for identificationg g pAny deviations from method must be verifiedOur goal verify method with our Agilent instruments: LC and MS/MS by QQQ

Status of EPA Method 1694“Today’s Talk”

1. The EPA Method 1694 is demonstrated with the Agilent QQQmodel 6410 and will discuss 6460 at end.

Th A il t t l f EPA th d 6 i t f 6 l t2. The Agilent protocol for EPA method 1694 consists of 65 analytes(of 75 total analytes) and 17 labeled internal standards (of 20total), which are a mixture of pharmaceuticals and personal careproducts that are analyzed each by a single MRM transition.p y y g

3. The method also uses Agilent columns, C‐18 and HILIC.

Agilent App Note:EPA 1694

Ferrer, Thurman, and Zweigenbaum (2008): 5889-9665ENAvailable on Agilent web site

How does the EPA Method Work?How does the EPA Method Work?1000 mL sample:

Acid and Base1. Standard EPA protocol is used for

sample extraction, either solidsextraction (acetonitrile,filtration, and centrifugation) or

Acid and Base

Elute with Methanol to

Condition cartridge with filtration, and centrifugation) or

SPE for water samples (usingOasis HLB). See protocol PDFfrom EPA method for details atwww.epa.

Methanol to remove PPCPsMethanol and

Water

www.epa.

2. Extracts are then analyzeddirectly by the Agilent 6410LC/MS/MS i i l

Polymercartridge

LC/MS/MS using a singletransition for each compound asprescribed by EPA.

Effluent to waste3. Quantitation and QA/QC follow

EPA protocol.

Water and SolidsWater and Solids

SPESPEBase ExtractAcid Extract

EPA Method in pdf

l h d hAgilent Method with Additional ConfirmationAdditional Confirmation

To provide additional confirmation for the EPA method a second MRM transition was added for 60 of the 65 analytes analyzed.  This gives an even greater assurance of correct analysis than prescribed by EPAassurance of correct analysis than prescribed by EPA.

MRMsQuad Mass Filter (Q3)Quad Mass Filter (Q1)

C lli i C ll

Ions

Collision Cell

Spectrum with background

Q1 lets onlytarget ion 210

Collision cell breaks ion 210

Q3 monitors onlycharacteristic

210

gions (from ESI)

gpass through

210

apart fragments 158 and 191 from ion 210 for quant and qual222

268 280165 210158

191

qual.

158

190

191

170 210 250 290 190 210 150 170 190 210 160 190no chemical background

List of Group 1 CompoundsEPA 1694 46 A l tEPA 1694: 46 Analytes

Acetaminophen Codeine Lincomycin Sarafloxacin Sulfanilamide

Ampicillin Cotinine Lomefloxacin Sulfachloro‐pyridazine

Thiabendazole

Azithromycin Dehydronifedipine Miconazole Sulfadiazine Trimethoprim

Caffeine Digoxigenin Norfloxacin Sulfadi‐methoxine Tylosin

Carbadox Diltiazem Ofloxacin Sulfadiazine Virginiamycin

C b i Di h l O illi S lf di h i Di i *Carbamazepine 1,7‐Dimethyl‐xanthine

Oxacillin Sulfadi‐methoxine Digoxin*

Cefotaxime Diphenhydramine Oxolinic acid Sulfamerazine

Ci fl i E fl i P i illi  G S lf h iCiprofloxacin EnrofloxacinErythromycin

Penicillin G Sulfamethazine

Clarithromycin Flumequine Penicillin V Sulfamethizole

Cl illi Fl i R i h i S lf h l *C d f d Cloxacillin Fluoxetine Roxithromycin Sulfamethoxazole *Compound formed intractable Na adduct with current conditions.

List of Group 2, 3 and 4p ,Compounds: EPA 1694: 19 Analytes

Anhydrotetracycline Doxycycline Minocycline Triclocarban

Triclosan

Warfarin

Chlorotetracycline 4‐Epianhydrotetra‐cycline Tetracycline Albuteroly 4 p y y y

Meclocycline Cimetidine

Metformin

Demeclocycline 4‐Epitetracycline Gemfibrozil

Ibuprofen

Ranitidine

p

Naproxen

List of Labeled Internal Standards

13C‐15N‐Acetaminophen 13C2‐Erythromycin 13C6‐Sulfamethazine 13C3‐Trimethoprim

13C3‐Atrazine Fluoxetine‐d6 13C6‐Sulfamethoxazole Warfarin‐d5

13C3‐Caffeine Gemfibrozil‐d6 13C6‐2,4,5‐Tricloro‐phenoxyacetic acid

Carbamazepine‐d10(Extra compound, not EPA list)

C N Ci fl i C Ib f C6 T i l b13C3‐15N‐Cipro‐floxacin 13C3‐Ibuprofen 13C6‐Triclocarban

Cotinine‐d3 13C‐Naproxen‐d3 13C12‐TriclosanCotinine d3 13C Naproxen d3 13C12 Triclosan

Columns and GradientsColumns and GradientsThe column is a ZORBAX Eclipse Plus C18, narrow bore RR 2.1x100mm 3.5 micron column.  Part number 959793‐902.

Group 1: The gradient is from 10% acetonitrile, 90% 0.1 % formic acid in water,  held for 5 minutes at 0.2 mL/min, 5then to 0.3 mL/min in 1 minute.  In 24 min go to 60% ACN and finally to 100% ACN in 30 minutes.

Group 2: The gradient is from 10% acetonitrile, held for 10 minutes, and then to 100% ACN in 30 minutes at a flow rate of 0.2 mL/min.

Group 3: The gradient is from 40% acetonitrile/methanol and 60% water (0.1% ammonium acetate+0.1% Acetic Acid) 

fl fto 100% ACN/MeOH in 12.5 minutes at a flow rate of 0.2 mL/min.

C l d G diColumns and Gradients (Continued)(Continued)

The column for HILIC is a ZORBAX Rx‐SiL, narrow bore RR2.1x100mm 3.5 micron column.www.chem.Agilent.com/cag/lccustom_1page.aspGroup 4: The gradient is from 98% for 5 min acetonitrile to 70%p 4 g 9 5 7ACN in 12 minutes at a flow rate of 0.25 mins. The aqueous solventis 10 mM ammonium acetate, pH 6.7

Overview of LC/MS/MS Fractions

1. Group 1: Acidic Extraction by ESI +( l )(C18 column)

2  Group 2: Acidic Extraction by ESI +2. Group 2: Acidic Extraction by ESI +(C18 column)

3. Group 3: Acidic Extraction by ESI – (C18 column)

b ( l )4. Group 4: Basic Extraction by ESI + (HILIC column).

TIC: Group 1 CompoundsTIC Chromatogram for 1 transition

Extracted MRM’s

TIC: Group 1 CompoundsAgilent Improvement: 2 Transitions and 3 Time Segments

TIC: Group 2 Compounds4x10

1.3

1.4

1.5

+ TIC MRM (** -> **) Group 2_0.1ppm_EPA MethodNew grad.d

1 1

0 8

0.9

1

1.1

1.2

0.4

0.5

0.6

0.7

0.8

0

0.1

0.2

0.3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31Counts vs. Acquisition Time (min)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

TIC: Group 3 CompoundsTIC: Group 3 Compounds4x10

2

- TIC MRM (** -> **) 100 ppb WW Matrix_Negative JZ_Group3.d

1 1

1.5

1.6

1.7

1.8

1.9

1

1.1

1.2

1.3

1.4

0.5

0.6

0.7

0.8

0.9

0.1

0.2

0.3

0.4

0 5 1 1 5 2 2 5 3 3 5 4 4 5 5 5 5 6 6 5 7 7 5 8 8 5 9 9 5 10 10 5 11 11 5 12Counts vs. Acquisition Time (min)

0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12

TIC: Group 4 CompoundsTIC: Group 4 Compounds

MetforminAlbuterol

Ranitidine

Cimetidine

Calibration Curves in Wastewater Matrix

Acetaminophen

Agilent Improvement: use of Qualifier Ions

Carbamazepine with a qualifier transition

Sulfadimethoxinewith a qualifier transition

LC/TOF‐MS Analysis of Boulder, Colorado, y , ,Wastewater Outfall

LC/MS/MS Analysis of Boulder W t t O tf ll f EPA 1694Wastewater Outfall for EPA 1694

Group 1 with 2 transitionsp

C b i Di h h d iCarbamazepine

Carbamazepine

Diphenhydramine

Diphenhydramine256 > 167237=>194 256 => 167

Carbamazepine237=>179

Diphenhydramine256 => 152

Improvements Coming on 1694Shift Method to new Shift Method to new 6460 QQQUse of new OPT cartridges from AgilentRapid Chromatography

Subfemtogram, on‐column sensitivity  

“Dynamic Multiple Reaction      y pMonitoring”

Five Orders of Linearity

Fast Pos/Neg switching, 100 millisec

J t StJet Stream

Nebulizing gas

S h t d h th

Enhanced efficiency nebulizer

Nozzle voltage

Super-heated sheath gas

The super-heated sheath gas collimates the

Heated drying gas

p gnebulizer spray and creates a dramatically “brighter source” Resistive sampling

capillary

Patent PendingSpray without sheath gas

MassHunter “Dynamic yMRM”

Many applications require screening or quantitation of 100 – 1000 compounds 

in one MRM method !in one MRM method !

Food, Environmental and Forensic analysis

With out Dynamic MRM

Need to manually set up multiple time segments to maximize dwell timeTedious and fragile for chromatographic time shiftsTedious and fragile for chromatographic time shiftsWith Dynamic MRM

Automatic setup of overlapping time segments without user interventionEven less MRMs per time results in even longer dwell time / sensitivityUnaffected by chromatographic time shifts

P ti id Mi I j t dPesticide Mix Injected as 100 fg on column100 fg on‐column

Quantitation Methabenzthiazuron 9.874 712 0.1210 121.0Methiocarb sulfon 7.687 144 0.1485 148.5Q

Results

Compound RTRespons

eFinal Conc

Accuracy

3-OH Carbofuran 7.410 236 0.1286 128.6Acetamiprid 7.274 564 0.1113 111.3Aldicarb sulfone 4.665 144 0.1294 129.4Aldicarb sulfoxid 4.316 204 0.1911 191.1Aldi b 8 8 152 0 1233 123 3

Dioxacarb 7.300 526 0.1253 125.3Diuron 11.053 898 0.0878 87.8

et oca b su o 7.687 0 85 8 5Methiocarb sulfoxid 7.174 1003 0.0946 94.6Methomyl 5.294 407 0.1375 137.5Metosulam 10.085 59 0.1280 128.0Metoxuron 8.666 377 0.1088 108.8Metsulfuron methyl 8.924 31 0.1007 100.7Monalide 6.884 237 0.0666 66.6Monolinuron 10.314 74 0.0965 96.5Naptalam 9.503 15 0.1357 135.7Nicosulfuron 9 338 32 0.1486 148.6Aldicarb 8.585 152 0.1233 123.3

Amidosulfuron 8.858 13 0.0311 31.1Aminocarb 7.231 1087 0.1156 115.6Asulam 3.191 14 0.0883 88.3Azoxystrobin 12.101 223 0.1522 152.2Bendiocarb 7.300 526 0.1253 125.3Butocarboxym 8.463 152 0.1637 163.7Butocarboxym-sulfoxid 4.316 203 0.1239 123.9Buturon 11.086 63 0.0691 69.1C b d i 2565 0 1027 102 7

Ethiofencarb-sulfon 7.680 83 0.1325 132.5Fenobucarb 11.926 58 0.0840 84.0Fenuron 6.924 643 0.1074 107.4Flazasulfuron 11.167 20 0.0881 88.1Fluazifop 12.094 27 0.0607 60.7Flumequine 9.931 406 0.1438 143.8Flumetsulam 6.941 79 0.1058 105.8Fluometuron 11 053 898 0.0878 87.8

Nicosulfuron 9.338 32 0.1486 148.6Oxamyl 5.047 1120 0.1239 123.9Oxasulfuron 9.379 273 0.1353 135.3Propoxur 9.791 601 0.1491 149.1Prosulfurone 11.448 46 0.3549 354.9Pyracarbolid 9.911 913 0.1013 101.3Quinmerac 6.430 566 0.1278 127.8Rimsulfuron 10.231 12 0.0000 0.0Siduron 12.076 261 0.1457 145.7Sulcotrione 7 500 66 0 1240 124 0Carbendazim 7.212 2565 0.1027 102.7

Carbetamid 9.130 229 0.1395 139.5Carbofuran 9.886 713 0.1149 114.9Carboxine 10.111 82 0.1297 129.7Chlorotoluron 10.607 462 0.1473 147.3Chlorsulfuron 9.372 25 0.1586 158.6Cinosulfuron 9.324 115 0.1878 187.8Clothianidin 6.544 115 0.1391 139.1Cyazofamid 8.381 64 0.0817 81.7

Fluometuron 11.053 898 0.0878 87.8Fluroxypyr 7.409 328 0.1308 130.8Flutriafol 11.016 141 0.1222 122.2Foramsulfuron 10.328 20 0.1318 131.8Fuberidazole 8.535 1193 0.1590 159.0Imazamox 6.890 38 0.1031 103.1Imazapyr 5.558 101 0.1088 108.8Imazaquin 9.557 103 0.1222 122.2Imazethaphyr 8 163 0 1440 144 0

Sulcotrione 7.500 66 0.1240 124.0Tebuthiuron 10.041 1318 0.1203 120.3Thiabendazole 8.171 771 0.1602 160.2Thiacloprid 7.931 673 0.1237 123.7Thiametoxam 5.516 298 0.1076 107.6Thidiazuron 9.463 21 0.0770 77.0Thifensulfuron-methyl 9.009 45 0.1142 114.2Thiodicarb 10.600 85 0.1277 127.7Thiofanox sulfone 7.157 618 0.1114 111.4Thiofanox sulfoxide 6 881 188 0 1088 108 8Cycluron 11.375 400 0.1369 136.9

Demethon-S-methyl 9.902 278 0.1122 112.2Demethon-S-methyl-sulfon 5.657 291 0.1162 116.2Demeton-S-methyl sulfoxide 5.513 902 0.1223 122.3

Dichlorphen 5.520 191 0.1131 113.1Diethofencarb 12.110 311 0.1340 134.0Difenoxuron 11.188 476 0.1452 145.2

Imazethaphyr 8.390 163 0.1440 144.0Imidacloprid 6.608 59 0.1077 107.7Iodosulfuron methyl 11.314 14 0.0432 43.2Isocarbophos-Na 11.434 53 0.1337 133.7Isoproturon 11.112 666 0.1358 135.8Linuron 11.841 117 0.1945 194.5

Thiofanox sulfoxide 6.881 188 0.1088 108.8Thiofanox 10.675 202 0.1654 165.4Thiophanat-ethyl 11.402 201 0.1786 178.6Tralkoxydim 15.928 781 0.1267 126.7Triasulfuron 9.999 55 0.1511 151.1Tribenuron methyl 11.190 24 0.4215 421.5Tricyclazole 8.371 505 0.1441 144.1Triflusulfuron-methyl 12.286 34 0.1049 104.9

Atrazine LODs by Instrument

LC/TOF‐MS is 1.0 μg/L with 50 uL injectionLC/QQQ i     /L  i h    L i j iLC/QQQ is 0.1 μg/L with 50 uL injectionLC/QQQ “Jetstream” is <0.01 μg/L with 50 uL injection

Verified by our work and that of USGS in Lakewood (oral communication with Dr  Mark Burkhardt)(oral communication with Dr. Mark Burkhardt).

Thanks

Co‐authors: Drs. Imma Ferrer and Jerry ZweigenbaumD  Mi h l Z lt  f A il t f  h l i     t   Dr. Michael Zumwalt of Agilent for helping us set up HPLC/TOF‐MS in Boulder Lab and giving me this opportunity to speak today.pp y p yPhil Stremple of Agilent for Environmental Applications on EPA 1694.

Conclusions

Agilent 6410 LC/MS/MS has been set up to run the EPAmethod 1694 for pharmaceuticals and personal care productsusing Agilent LC supplies (C18 and HILIC column).using Agilent LC supplies (C18 and HILIC column).

Two transitions are available for definitive identification ofPPCPs in water.

The method has an Application Note describing the entire procedure 5889‐9665EN, Available on Agilent web site.