Ethanol Metabolites by Paper Spray Ionization: Method Development in Negative Ion Mode

Poster Note 64377

Maria C. Prieto Conaway,1 Chengsen Zhang,2 Nicholas E. Manicke,2 Kristine Van Natta,2 Marta Kozak1 1Thermo Fisher Scientific, San Jose CA; 2Indiana University-Purdue University, Indianapolis, IN Ethanol Metabolites by Paper Spray Ionization: Method Development in Negative Ion Mode Maria C. Prieto Conaway1, Chengsen Zhang2, Nicholas E. Manicke2, Kristine Van Natta1, Marta Kozak1 1Thermo Fisher Scientific, San Jose CA; 2Indiana University-Purdue University, Indianapolis, IN

FIGURE 1. Velox 360 paper spray system showing, clockwise from FIGURE 4. Normalized analyte to FIGURE 5. Normalized analyte to IS Overview top left: paper spray ion source, mechanism for dispensing solvent to IS areas vs. concentration plots areas vs. concentration plots for Purpose: Develop an ionization method in negative mode for screening of the sample, paper cassette indicating sample deposition, and spotted for spiked EtS in, top to bottom: spiked EtG in, top to bottom: neat, ethanol metabolites in urine using paper spray technology and mass paper cassette electrospraying into mass spectrometer inlet. neat, synthetic urine and urine synthetic urine and urine matrices. spectrometry for use in forensic toxicology. matrices. EtG does not ionize well under the substrate/solvent conditions used. Methods: Urine spiked with ethyl sulfate (EtS) and ethyl glucuronide (EtG) were analyzed as dried urine samples by paper spray ionization coupled to Thermo Scientific™ Orbitrap™ mass spectrometers (MS). Positive Y = 0.000803989+0.0019195*X R^2 = 0.9719 W: 1/X Y = 1.11275+0.00386917*X R^2 = 0.9159 W: 1/X

2.0 5

4 Neat control donor samples are analyzed after method development. 1.5 Neat 3 1.0 2 Results: Demonstrated the potential of paper spray ionization in negative 0.5

Area Ratio 1 Area Ratio mode MS for the screening of ethanol metabolites with minimum sample 0.0 0 0 200 400 600 800 1000 0 200 400 600 800 1000

Y = -0.00713621+0.0022996*X R^2 = 0.9750 W: 1/X Y = 1.14201+0.0103237*X R^2 = 0.7091 W: 1/X

preparation and no chromatography. Paper spray is easy to use and 2.5 15 provides answers in seconds. The potential of this technique for 2.0 10 1.5 Synthetic Synthetic quantitation of EtG and EtS is also explored. 1.0 urine 5 urine

20 Deuterium-labeled internal standards were added to all samples. 2.5 15 Urine Paper spray is a direct ionization technique that simplifies the mass 2.0 Urine 1.5 10

spectrometric analysis of compounds from biological fluids. Both 1.0 5 Area Ratio Area Ratio 0.5 Ethanol Metabolite m/z Fragment 1 Fragment 2 Molecular Structure 0 qualitative and quantitative analysis of small molecules from complex 0.0 0 200 400 600 800 1000 0 200 400 600 800 1000 matrices such as blood and urine are possible without time-consuming Concentration (ng/mL) Concentration (ng/mL) sample preparation and chromatography. Samples are collected and Ethyl sulfate 124.9914 79.9574 96.9601 stored in a simple paper cassette for easy shipment of samples to the forensic toxicology lab. Paper spray technology is therefore attractive for Ethyl sulfate–d5 130.0228 79.9574 97.9664 FIGURE 6. Positive donor urine samples, top to bottom, donor 1, donor 2, forensic toxicology compound screening. donor 3 and negative teetotaler urine donor blank. Mass accuracy was 6- EtG and EtS have been identified as specific and direct metabolites of 10 ppm. recent alcohol consumption. Small amounts of ethanol (0.1%) are Ethyl glucuronide 221.0666 75.0088 85.0296 124.99 → 79.9574

79.9566 converted into EtG and EtS by conjugation pathways in the liver, and their 100 NL: 6.73E4 50 Donor_1

0 presence in blood and urine are used as confirmation for ethanol 100 NL: 1.84E4 Ethyl glucuronide–d5 226.0981 75.0088 85.0296 50 Donor_2 consumption. EtG and EtS are detectable in serum for 10–14 h and in 0 100 NL: 7.32E4

50 Donor_3

urine for 25–44 h. Therefore, these metabolites close the gap between Abundance Relative 0 100 NL: 9.83E3 short-term markers (e.g., ethanol) and long-term markers (e.g., 50 Urine Blank with IS

abstinence in the United States are defined as less than 100 ng/mL for 0 NL: 1.49E4 100 Paper spray ionization has primarily been shown for the analysis of small Donor_2 EtG and less than 25 ng/mL for EtS. Small amounts of EtG and EtS are 50

molecules that ionize in positive mode. Conditions for negative ionization 0 endogenous in our bodies and can also be produced from regular use of 100 NL: 8.63E4 usually require a low surface tension solvent and a spray voltage about Donor_3 antiseptic gels, mouthwash and certain medications. Abundance Relative 50 2 0 NL: 6.95E3 500 V less than that used in positive mode . This is to avoid corona 100 Urine Blank In this work, we develop a negative ionization mode mass spectrometry discharge which would increase the spray current and decrease analyte 50 0 method that is compatible with paper spray ionization for the analysis of signal. All experiments shown were conducted with 100% methanol/ 75 76 77 78 79 80 81 82 83 84 85 m/z ethanol metabolites in urine and coupling to an Orbitrap mass 100 ppm acetic acid, which fulfills the low surface tension negative spectrometer. ionization requirement. Preliminary results for the screening of ethanol metabolites by paper spray Conclusion

Methods MS/MS are shown in Figures 2 and 3, with expected fragments for EtG . We have developed a negative ion mode method for paper spray Sample Preparation (m/z 221.07→75.0088, 85.0296) and EtS (m/z 124.99 →79.9574, 96.9601) technology coupled to Thermo Scientific Q Exactive mass and outstanding mass accuracies. spectrometers. EtS and EtG were spiked in urine with their corresponding deuterated internal standards (IS), EtS-d5 and EtG-d5 (all from Cerilliant). Standards Figures 4 and 5 show calibration curves for EtS and EtG, respectively. The . The Velox 360 system has been demonstrated as a forensic screening were spiked in three different matrices: urine, synthetic urine or solvent. ethyl sulfate anion electrosprays well from the Whatman (GE Healthcare technique for ethyl sulfate and ethyl glucuronide from urine samples in The concentration of IS was kept at 500 ng/mL in all samples. Three Life Sciences, Pittsburgh, PS) cellulose paper used in the VSC, which a single experiment and from a single urine sample spot, all in much positive donor samples were also analyzed. makes it amenable for quantitative analysis. %RSD (std. dev/mean x100) shorter timeframes than it is possible using LC MS techniques. was found to be between 5 to 20% for the concentration range of 10 to Ethanol Metabolites by Paper SprayEtS-d5 Ionization: and EtG-d5 were spiked intoMethod negative donor andDevelopment positive donor in Negative Ion Mode . As the electrospray signal from paper lasts ~40 s, it can accommodate 1000 ng/mL. EtG ionization showed poor reproducibility compared to EtS. urine samples and submitted to dilution/precipitation (1:3 urine to full scan MS at high resolving power and many MS/MS experiments for Optimizing solvent /substrate conditions for better ionization of EtG will be 1 2 methanol) and centrifugation for2 10 min at 3,000 g. Same protocol1 was 1 identification and confirmation in screening applications. Maria C. Prieto Conaway , Chengsen Zhang , Nicholas E. Manicke , Kristine Van Natta , Marta Kozakpursued in follow-up work. followed with synthetic urine and neat samples. The IS-containing . The ethyl sulfate anion electrosprays well with an extraction solvent 1 2 supernatant was then spiked with EtS and EtG to obtain calibration Positive donor samples were analyzed (Fig. 6) by the same methodology. Thermo Fisher Scientific, San Jose CA; Indiana University-Purdue University, Indianapolis, IN conducive to negative ionization and from cellulose paper which, in the curves: 190 uL supernatant plus 10 uL EtS and Etg at various Even though positive results were obtained, the variability obtained is too presence of an internal standard, makes it promising for quantitative concentrations to make a calibration curve in the range of 5 to 1000 large to consider the technique robust enough to evaluate the donor signal analysis. ng/mL. Eight microliters were loaded onto paper spray cartridges and quantitatively. Please, note some endogenous signal detected in the urine allowed to dry at room temperature for 10 minutes. The recommended from the negative donor. . Ethyl glucuronide, being more hydrophilic, does not electrospray well extraction solvent for negative ionization from paper was used, 100% from cellulose paper and would benefit from alternative substrates or

methanol with 100 ppm acetic acid. Spray stability in negative ion mode FIGURE 2. MS/MS spectra FIGURE 3. MS/MS spectra paper treatments to make the cellulose paper more suitable for the benefits from a low surface tension solvent such as methanol. confirming the presence of EtS in confirming the presence of EtG in ionization of EtG. negativeFIGURE ion mode 4. (TableNormalized 1) for analyte to negativeFIGURE ion mode 5. Normalized (Table 1) for analyte to IS MassFIGURE Spectrometry 1. Velox 360 paper spray system showing, clockwise from Overview top left: paper spray ion source, mechanism for dispensing solvent to spiked urineIS areas samples vs. concentration (200 ng/mL). plots spikedareas urine vs.samples concentration (200 ng/ mLplots). for References Data collected with a Thermo Data collected with a Thermo Purpose: Develop an ionization method in negative mode for screening of A commerciallythe sample, available paper cassetteVelox 360™ indicating paper spray sample system deposition, (Fig. 1, and spotted for spiked EtS in, top to bottom: spiked EtG in, top to bottom: neat, Scientific™ Q Exactive™ Plus MS. ™ ™ 1. Jalbermann et al, JCS 50 (2012) 51-56. ethanol metabolites in urine using paper spray technology and mass Prosoliapaper, Inc., cassette Indianapolis, electrospraying IN) that uses into Velox mass Sample spectrometer Cartridges inlet. (VSC) neat, synthetic urine and urine Scientificsynthetic Q Exactive urine and Plus urine MS. matrices. spectrometry for use in forensic toxicology. was coupled to a Thermo Scientific™ Q Exactive™ Focus MS. The Full matrices. EtG does not ionize well under the 2. Manicke et al, IJMS 300 (2011) 123-129. MS with Confirmation by Data Dependent MS/MS method was used. Data 124.99→79.9574, 96.9601 substrate/solvent conditions used. Methods: Urine spiked with ethyl sulfate (EtS) and ethyl glucuronide (EtG) NL: 1.44E4 Dependent spectra are triggered from an inclusion list containing the FTMS - p NSI Full ms2 [email protected] [50.00-145.00] 81.0346 100 95 were analyzed as dried urine samples by paper spray ionization coupled 90 precursor mass for each metabolite and internal standard. Resolving 85 For forensic toxicology use only. 80 79.0190

75 Y = 0.000803989+0.0019195*X R^2 = 0.9719 W: 1/X 70 Y = 1.11275+0.00386917*X R^2 = 0.9159 W: 1/X to Thermo Scientific™ Orbitrap™ mass spectrometers (MS). Positive 65 221.07→75.0088, 85.0296

power was set to 70,000 and 35,000 (FWHM at m/z 200) for full scan MS 60 2.0 5

55 Ethanol Metabolites by Paper Spray Ionization: Method Development in Negative50 Ion Mode 4 Neat Abundance 45 1.5

control donor samples are analyzed after method development. and MS/MS, respectively. Negative ions are generated directly from paper 40 Neat ©2015 Thermo Fisher Scientific. All Rights Reserved. 35 3 30 1.0 NL: 6.03E4 25 2

Relative The Velox 360™ system is the trade mark of Prosolia, Inc. Whatman is a registered brand of GE Healthcare Life Sciences. All other trademarks are the when an applied spray voltage (4.5 kV in negative ion mode) induces 20 79.9574 FTMS - p NSI Full ms2 [email protected] [50.00-245.00]

Results: Demonstrated the potential of paper spray ionization in negative 15 78.9591 0.5 85.0296 10 100 property of Thermo Fisher Scientific and its subsidiaries.

Area Ratio 1

1 2 2 1 1 Area Ratio 5 80.9982 95 80.7259 0 90 Maria C. Prieto Conaway , Chengsen Zhang , Nicholaselectrospray E. Manicke from the sharp, tipKristine of the paper Van (Figure Natta 1). The Q, ExactiveMarta Kozak 78.4 78.6 78.8 79.0 79.2 0.079.4 79.6 79.8 80.0 80.2 80.4 80.6 80.8 81.0 81.2 81.4 81.6 81.8 0 This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. modeEthanol MS for the screening Metabolites of ethanol metabolites by with minimum Paper sample Spray Ionization: Method Development in Negative Ion Mode 85 0 200 400 600 800 1000 0 200 400 600 800 1000 m/z 80 75

mass spectrometers use Higher Collision Dissociation (HCD) energy 70 Y = -0.00713621+0.0022996*X R^2 = 0.9750 W: 1/X 65 Y = 1.14201+0.0103237*X R^2 = 0.7091 W: 1/X 1 preparation and no chromatography. Paper spray is easy to use and2 60 NL: 3.77E4 15 Thermo Fisher Scientific, San Jose CA; Indiana University-Purdue University, Indianapolis, IN 2.5 55 1 2 2 1 1 50 experiments to fragment precursor ions. FTMS - p NSI Full ms2 [email protected] [50.00-145.00] provides answers in seconds. The potential of this technique for 2.0 45 Maria C. Prieto Conaway , Chengsen Zhang , Nicholas E. Manicke , Kristine Van Natta , Marta Kozak 40 97.0295 10 1.5 Synthetic 35 100 83.0139 Synthetic 95 30 75.0088 quantitation of EtG and EtS is also explored. 90 1.0 25 urine 5 urine Data Analysis 85 Abundance Relative 20 1 2 80 0.5 15 Area Ratio

75 Area Ratio 10 Thermo Fisher Scientific, San Jose CA; Indiana University-Purdue University, Indianapolis, IN 70 0 0.0 5 65 0 0 200 400 600 800 1000 60 0 200 400 600 800 1000 75 76 77 78 79 80 81 82 83 84 85 Data acquisition was with the Thermo Scientific™ TraceFinder™ software. 55 50 Abundance

m/z Table 1. Ethanol urine metabolites indicative of ethanol consumption. 45 Y = 0.671872+0.00205785*X R^2 = 0.8521 W: 1/X Introduction 40 Y = 1.1776+0.0123971*X R^2 = 0.6825 W: 1/X

35 All other analyses used Thermo Scientific™ Xcalibur™ platform tools. 20 30

25 Deuterium-labeled internal standards were added to all samples. Relative 96.96012.5 96.9931 20 15 15 2.0 Urine Paper spray is a direct ionization technique that simplifies the mass 10 5 Urine 97.0659 10 0 96.85 96.90 96.951.5 97.00 97.05 97.10 97.15 97.20 97.25

spectrometric analysis of compounds from biological fluids. Both 1.0 m/z 5 Area Ratio Area Ratio 0.5 Ethanol Metabolite m/z Fragment 1 Fragment 2 Molecular Structure 0 qualitative and quantitative analysis of small molecules from complex 0.0 0 200 400 600 800 1000 FIGURE 4. 0Normalized 200 400 600analyte 800 to1000 FIGURE 5. Normalized analyte to IS matrices such as blood and urine are possible without time-consuming FIGURE 1. Velox 360 paper spray system showing, clockwise from Overview Concentration (ng/mL) Concentration (ng/mL) top left:Ethyl paper sulfate spray ion124.9914 source, mechanism79.9574 for96.9601 dispensing solvent to IS areas vs. concentration plots areas vs. concentration plots for sample preparation and chromatography. Samples are collected and FIGURE 1. Velox 360 paper spray system showing, clockwise from FIGURE 4. Normalized analyte to FIGURE 5. Normalized analyte to IS Develop an ionization method in negative mode for screening of the sample, paper cassette indicating sample deposition, and spotted for spiked EtS in, top to bottom: spiked EtG in, top to bottom: neat, Purpose:storedOverview in a simple paper cassette for easy shipment of samples to the top left: paper spray ion source, mechanism for dispensing solvent to IS areas vs. concentration plots areas vs. concentration plots for ethanol metabolites in urine using paper spray technology and mass paper cassette electrospraying into mass spectrometer inlet. neat, synthetic urine and urine synthetic urine and urine matrices. forensic toxicologyDevelop lab. an Paper ionization spray method technology in negative is therefore mode for attractive screening for of the sample,Ethyl sulfate paper–d5 cassette130.0228 indicating 79.9574 sample deposition,97.9664 and spotted for spiked FIGUREEtS in, top 6. to Positive bottom: donor urinespiked samples, EtG in, top top to to bottom: bottom, neat, donor 1, donor 2, Purpose: matrices. EtG does not ionize well under the spectrometryforensic toxicologyfor use in forensiccompound toxicology. screening . paper cassette electrospraying into mass spectrometer inlet. neat, synthetic urine and urine synthetic urine and urine matrices. ethanol metabolites in urine using paper spray technology and mass donor 3 and negative teetotaler urinesubstrate/solvent donor blank. Mass conditions accuracy used. was 6- spectrometry for use in forensic toxicology. matrices. EtG does not ionize well under the Methods:EtG and Urine EtS spiked have beenwith ethylidentified sulfate as ( specificEtS) and and ethyl direct glucuronide metabolites (EtG of) 10 ppm. Ethyl glucuronide 221.0666 75.0088 85.0296 substrate/solvent conditions used. wererecent analyzedMethods: alcohol as dried Urineconsumption. urine spiked samples with Small ethyl by amounts sulfate paper ( EtSspray of )ethanol and ionization ethyl (0.1%) glucuronide coupled are (EtG) 124.99 → 79.9574 Y = 0.000803989+0.0019195*X R^2 = 0.9719 W: 1/X 79.9566 Y = 1.11275+0.00386917*X R^2 = 0.9159 W: 1/X to Thermowere Scientific™ analyzed Orbitrapas dried ™urine mass samples spectrometers by paper spray (MS). ionization Positive coupled 100 NL: 6.73E4

2.0 5 converted into EtG and EtS by conjugation pathways in the liver, and their 50 Donor_1

Y = 0.000803989+0.0019195*X R^2 = 0.9719 W: 1/X Y = 1.11275+0.00386917*X4 R^2 = 0.9159 W: 1/X Neat control donorto Thermo samples Scientific™ are analyzed Orbitrap after™ mass method spectrometers development. (MS). Positive 1.5 0

2.0 100 Neat 5 presence in blood and urine are used as confirmation for ethanol NL: 1.84E43 1.0 50 Donor_2 control donor samples are analyzed after method development. 1.5 4 Neat Ethyl glucuronide–d5 226.0981 75.0088 85.0296 Neat 2 consumption. EtG and EtS are detectable in serum for 10–14 h and in 0 3 Results: Demonstrated the potential of paper spray ionization in negative 0.5 100 NL: 7.32E4

1.0 Area Ratio 1 2 Area Ratio 50 Donor_3 0.0 0 urineResults for 25–:44 Demonstrated h. Therefore, the these potential metabolites of paper spray close ionization the gap betweenin negative 0.5 Abundance Relative

Area Ratio 0 1

mode MS for the screening of ethanol metabolites with minimum sample Area Ratio 0 200 400 600 800 1000 0 200 400 600 800 1000 100 NL: 9.83E3 0.0 0 mode MS for the screening of ethanol metabolites with minimum sample 50 Urine Blank with IS short-term markers (e.g., ethanol) and long-term markers (e.g., 0 200 400Y = -0.00713621+0.0022996*X600 800 R^2 =1000 0.9750 W: 1/X 0 200 400 600Y = 1.14201+0.0103237*X800 1000 R^2 = 0.7091 W: 1/X preparation and no chromatography. Paper spray is easy to use and 0 2.5 79.5 79.6 79.7 79.8 79.9 80.0 80.1 80.2 80.3 80.4 80.5 15 )1 Y = -0.00713621+0.0022996*X R^2 = 0.9750 W: 1/X Y = 1.14201+0.0103237*X R^2 = 0.7091 W: 1/X carbohydratepreparation-deficient and no chromatography.transferrin in serum Paper sprayThis permitsis easy to a usewide and range of . m/z provides answers in seconds. The potential of this technique for 2.5 2.0 15 10 provides answers in seconds. The potential of this technique for Results 2.0 1.5 Synthetic Synthetic applications, such as controlling patients in withdrawal treatment or 221.07 →10 85.0296 quantitation of EtG and EtS is also explored. 1.5 1.0 Synthetic urine Synthetic urine 5 85.0287 NL: 2.66E5 100 quantitation of EtG and EtS is also explored. 1.0 Area Ratio 0.5 urine 5 urine monitoring alcohol abstinence. Cutoff values for establishing alcohol Area Ratio Donor_1 Ethanol metabolites by paper spray ionization 0.5 0.0 Area Ratio 83.01300 Area Ratio 50 75.0079 0 200 400 600 800 1000 0.0 0 200 400 600 800 1000 0 81.0337 0 200 400 600 800 1000 0 200 400 600 800 1000 abstinence in the United States are defined as less than 100 ng/mL for 0 NL: 1.49E4 Table 1. Ethanol urine metabolites indicative of ethanol consumption. 100 Introduction Table 1. Ethanol urine metabolites indicative of ethanol consumption. Y = 0.671872+0.00205785*X R^2 = 0.8521 W: 1/X Y = 1.1776+0.0123971*XDonor_2 R^2 = 0.6825 W: 1/X Paper spray ionization has primarily been shown for the analysis of small Y = 0.671872+0.00205785*X R^2 = 0.8521 W: 1/X Y = 1.1776+0.0123971*X R^2 = 0.6825 W: 1/X Introduction 20 EtG and less than 25 ng/mL for EtS. Small amounts of EtG and EtS are

50 20 Deuterium-labeled internal standards were added to all samples. 2.5 Deuterium-labeled internal standards were added to all samples. 2.5 15 molecules that ionize in positive mode. Conditions for negative ionization 15 Urine 2.0 0 NL: 8.63E4 Urine Paperendogenous spray is a direct in our ionization bodies and technique can also that be producedsimplifies thefrom mass regular use of 2.0 100 Urine Paper spray is a direct ionization technique that simplifies the mass Urine 10 1.5 10 Donor_3 usually require a low surface tension solvent and a spray voltage about 1.5

Relative Abundance Relative 50 spectrometric analysis of compounds from biological fluids. Both 1.0 5 antisepticspectrometric gels, mouthwash analysis of andcompounds certain frommedications. biological fluids. Both 1.0 5 Area Ratio Area Ratio Area Ratio 2 Area Ratio 0.5 0.5 0 0 NL: 6.95E3 EthanolEthanol500 Metabolite Metabolite V less than m/zthatm/z usedFragment in positive 1 1 Fragment modeFragment. This 2 2Molecular is Molecularto avoid Structure corona Structure 100 0 qualitativequalitative and quantitative and quantitative analysis analysis of small of moleculessmall molecules from fromcomplex complex 0.0 0.0 0 200 4000 600200 Urine800 400Blank 1000 600 800 1000 0 200 0 400 200 600 400 800 600 1000 800 1000 In this work, we develop a negative ionization mode mass spectrometry discharge which would increase the spray current and decrease analyte 50 matrices suchmatrices as bloodsuch as and blood urine and are urine possible are possible without without time -timeconsuming-consuming Concentration (ng/mL) Concentration (ng/mL) Concentration (ng0 /mL) Concentration (ng/mL) methodsample that preparation is compatible and withchromatography. paper spray Samples ionization are for collected the analysis and of EthylEthylsignal. sulfate sulfate All experiments124.9914124.9914 shown79.957479.9574 were conducted96.960196.9601 with 100% methanol/ 75 76 77 78 79 80 81 82 83 84 85 sample preparation and chromatography. Samples are collected and m/z storedethanol in astored simple metabolites in apaper simple cassettein paper urine cassette and for easycoupling for shipment easy to shipment an ofOrbitrap samples of samples mass to the to the 100 ppm acetic acid, which fulfills the low surface tension negative spectrometer.forensic toxicology lab. Paper spray technology is therefore attractive for forensic toxicology lab. Paper spray technology is therefore attractive for EthylEthylionization sulfate sulfate–d5–d5 requirement. 130.0228130.0228 79.957479.9574 97.966497.9664 FIGUREFIGURE 6. Positive 6. Positive donor urinedonor samples, urine samples, top to bottom, top to donor bottom, 1, donor donor 2, 1, donor 2, forensic toxicology compound screening. forensic toxicology compound screening. donordonor 3 and Conclusion 3negative and negative teetotaler teetotaler urine donor urine blank. donor Mass blank. accuracy Mass wasaccuracy 6- was 6- Preliminary results for the screening of ethanol metabolites by paper spray 10 ppm. MethodsEtG and EtS have been identified as specific and direct metabolites of 10 ppm. EtG and EtS have been identified as specific and direct metabolites of EthylMS/MS glucuronide are shown221.0666 in Figures 75.0088 2 and 3, 85.0296with expected fragments for EtG . We have124.99 developed → 79.9574 a negative ion mode method for paper spray recent alcohol consumption. Small amounts of ethanol (0.1%) are Ethyl (glucuronidem/z 221.07→75.0088, 221.0666 85.0296)75.0088 and EtS85.0296 (m/z 124.99 →79.9574, 96.9601) 124.99 → 79.9574 recentSample alcohol Preparation consumption. Small amounts of ethanol (0.1%) are technology79.9566 coupled to Thermo Scientific Q Exactive mass 100 NL: 6.73E4 converted into EtG and EtS by conjugation pathways in the liver, and their Donor_1 50 79.9566 and outstanding mass accuracies. spectrometers. 100 NL: 6.73E4 converted into EtG and EtS by conjugation pathways in the liver, and their 0 Donor_1 presence in blood and urine are used as confirmation for ethanol 100 50 NL: 1.84E4 EtS and EtG were spiked in urine with their corresponding deuterated 50 0 Donor_2 presence in blood and urine are used as confirmation for ethanol Ethyl glucuronide–d5 226.0981 75.0088 85.0296 100 NL: 1.84E4 consumption. EtG and EtS are detectable in serum for 10–14 h and in 0 internal standards (IS), EtS-d5 and EtG-d5 (all from Cerilliant). Standards Figures 4 and 5 show calibration curves for EtS and EtG, respectively. The . The100 Velox 360 systemNL: 7.32E4 has been demonstrated as a forensic screening 50 Donor_2 Ethyl glucuronide–d5 226.0981 75.0088 85.0296 50 Donor_3 consumption. EtG and EtS are detectable in serum for 10–14 h and in 0 urine for 25–44 h. Therefore, these metabolites close the gap between Abundance Relative 100 ethyl sulfate anion electrosprays well from the Whatman (GE Healthcare 0 NL: 7.32E4 were spiked in three different matrices: urine, synthetic urine or solvent. technique100 for ethyl NL:sulfate 9.83E3 and ethyl glucuronide from urine samples in 50 Donor_3 50 Urine Blank with IS urine for 25short–44-term h. Therefore, markers (e.g. these, ethanol) metabolites and long close-term themarkers gap between(e.g., Abundance Relative 0 The concentration of IS was kept at 500 ng/mL in all samples. Three Life Sciences, Pittsburgh, PS) cellulose paper used in the VSC, which a single0 experiment100 and fromNL: 9.83E3 a single urine sample spot, all in much )1 79.5 79.6 79.7 79.8 79.9 80.0 80.1 80.2 80.3 80.4 80.5 short-termcarbohydrate markers (e.g.-deficient, ethanol) transferrin and long in serum-term markers. This permits (e.g. ,a wide range of 50 m/z Urine Blank with IS positive donor samples were also analyzed. makes it amenable for quantitative analysis. %RSD (std. dev/mean x100) shorter timeframes0 than it is possible using LC MS techniques. applications, such as controlling patients)1 in withdrawal treatment or Results 221.0779.5 79.6 →79.7 79.885.0296 79.9 80.0 80.1 80.2 80.3 80.4 80.5 carbohydrate-deficient transferrin in serum This permits a wide range of m/z . 85.0287 NL: 2.66E5 was found to be between 5 to 20% for the concentration range of 10 to 100 monitoring alcohol abstinence. Cutoff values for establishing alcohol Results . As the electrospray signal fromDonor_1 paper lasts ~40 s, it can accommodate applications,EtS-d5 and such EtG as- d5controlling were spiked patients into innegative withdrawal donor treatment and positive or donor Ethanol metabolites by paper spray ionization 50 75.0079 221.07 →83.0130 85.0296 81.0337 1000 ng/mL. EtG ionization showed poor reproducibility compared to EtS. 85.0287 NL: 2.66E5 100 abstinence in the United States are defined as less than 100 ng/mL for 0 monitoringurine samples alcohol abstinence.and submitted Cutoff to dilution/precipitation values for establishing (1:3 alcohol urine to 100full scan MS at high resolvingNL: power 1.49E4 andDonor_1 many MS/MS experiments for EthanolPaperOptimizing spraymetabolites ionization solvent by has /substrate paper primarily spray conditions been ionization shown for for better the analysis ionization of small of EtG will be 50 75.0079 D83.0130onor_2 EtG and less than 25 ng/mL for EtS. Small amounts of EtG and EtS are 81.0337 methanol) and centrifugation for 10 min at 3,000 g. Same protocol was 50identification and confirmation in screening applications. abstinence in the United States are defined as less than 100 ng/mL for molecules that ionize in positive mode. Conditions for negative ionization 0 NL: 1.49E4 pursued in follow-up work. 0 100 NL: 8.63E4 endogenous in our bodies and can also be produced from regular use of 100 Donor_2 Paper spray ionization has primarily been shown for the analysis of small Donor_3 EtG andfollowed less thanwith synthetic25 ng/mL urine for EtS and. Small neat amountssamples. ofThe EtG IS and-containing EtS are usually require a low surface tension solvent and a spray voltage about 50 antiseptic gels, mouthwash and certain medications. . Abundance Relative 50The ethyl sulfate anion electrosprays well with an extraction solvent molecules that ionize in positive mode. Conditions2 for negative ionization 0 supernatant was then spiked with EtS and EtG to obtain calibration Positive donor samples were analyzed (Fig. 6) by the same methodology. 0 NL: 6.95E3 NL: 8.63E4 endogenous in our bodies and can also be produced from regular use of 500 V less than that used in positive mode . This is to avoid corona 100 100 usually require a low surface tension solvent and a spray voltage about conducive to negative ionizationUrine Blank and fromDonor_3 cellulose paper which, in the antisepticcurves:In gels, this 190 work,mouthwash uL we supernatant develop and acertain negative plus 10medications. ionizationuL EtS and mode Etg mass at various spectrometry dischargeEven though which would positive increase results the were spray obtained, current and the decrease variability analyte obtained is too 50 Abundance Relative 50 2 presence of an internal standard, makes it promising for quantitative 500 V less than that used in positive mode . This is to avoid corona 0 0 NL: 6.95E3 concentrationsmethod that to is makecompatible a calibration with paper curve spray in ionization the range for of the 5 toanalysis 1000 of signal.large All to experiments consider the shown technique were conducted robust enough with 100% to evaluate methanol/ the donor signal 75 76100 77 78 79 80 81 82 83 84 85 m/z Urine Blank In this work, we develop a negative ionization mode mass spectrometry analysis.50 ng/mL.ethanol Eight metabolites microliters in were urine loaded and coupling onto paperto an Orbitrap spray cartridges mass and discharge100quantitatively. ppm whichacetic wouldacid, Please, which increase notefulfills thesome the spray low endogenous surface current tension and signal decrease negative detected analyte in the urine 0 method thatspectrometer. is compatible with paper spray ionization for the analysis of signal.ionization All experiments requirement. shown were conducted with 100% methanol/ 75 76 77 78 79 80 81 82 83 84 85 allowed to dry at room temperature for 10 minutes. The recommended from the negative donor. . Ethyl glucuronide, beingm/z more hydrophilic, does not electrospray well ethanol metabolites in urine and coupling to an Orbitrap mass 100 ppm acetic acid, which fulfills the low surface tension negative extraction solvent for negative ionization from paper was used, 100% Preliminary results for the screening of ethanol metabolites by paper spray Conclusionfrom cellulose paper and would benefit from alternative substrates or spectrometer. methanolMethods with 100 ppm acetic acid. Spray stability in negative ion mode ionizationFIGUREMS/MS arerequirement. 2. shownMS/MS in spectraFigures 2 and 3, with expectedFIGURE fragments 3. MS/MS for EtG spectra . We have developedpaper treatments a negative toion make mode the method cellulose for paper paper spray more suitable for the benefitsSample from Preparation a low surface tension solvent such as methanol. Preliminaryconfirming(m/z 221.07→75.0088, results the presence for the 85.0296) screening of EtS and inofEtS ethanol (m/z 124.99 metabolitesconfirming →79.9574, bythe paper96.9601) presence spray of EtG in technologyConclusion coupledionization to Thermoof EtG. Scientific Q Exactive mass Methods negativeand outstanding ion mode mass (Tableaccuracies. 1) for negative ion mode (Table 1) for spectrometers. EtS and EtG were spiked in urine with their corresponding deuterated MS/MS are shown in Figures 2 and 3, with expected fragments for EtG . We have developed a negative ion mode method for paper spray Mass Spectrometry spiked urine samples (200 ng/mL). spiked urine samples (200 ng/mL). Sample Preparationinternal standards (IS), EtS-d5 and EtG-d5 (all from Cerilliant). Standards (m/zFigures 221.07→75.0088, 4 and 5 show calibration 85.0296) curvesand EtS for (EtSm/z and124.99 EtG ,→79.9574, respectively. 96.9601) The . The VeloxtechnologyReferences 360 system coupled has beento Thermo demonstrated Scientific as Qa forensicExactive screening mass A commerciallywere spiked availablein three different Velox 360™matrices: paper urine spray, synthetic system urine (Fig. or solvent. 1, andDataethyl outstanding sulfatecollected anion mass with electrosprays accuracies.a Thermo well from the WhatmanData collected (GE Healthcare with a Thermo techniquespectrometers. for ethyl sulfate and ethyl glucuronide from urine samples in 1. Jalbermann et al, JCS 50 (2012) 51-56. EtS andProsolia EtGThe , were concentrationInc., spikedIndianapolis, in of urineIS was IN) with keptthat their atuses 500 corresponding Veloxng/mL Sample in all samples. deuterated Cartridges Three (VSC) ScientificLife Sciences,™ Q Pittsburgh, Exactive PS)™ Plus cellulose MS. paper usedScientific in the VSC,™ Qwhich Exactive ™ Plus MS. a single experiment and from a single urine sample spot, all in much internal standards (IS), EtS-d5 and EtG-d5 (all from Cerilliant). Standards Figures 4 and 5 show calibration curves for EtS and EtG, respectively. The . The Velox 360 system has been demonstrated as a forensic screening was coupledpositive donor to a Thermosamples Scientific™were also analyzed. Q Exactive™ Focus MS. The Full makes it amenable for quantitative analysis. %RSD (std. dev/mean x100) shorter timeframes2. Manicke than et it al,is possibleIJMS 300 using (2011) LC MS 123 techniques.-129. wereMS spiked with inConfirmation three different by matrices:Data Dependent urine, synthetic MS/MS methodurine or wassolvent. used. Data ethylwas sulfate found to124.99→anion be between electrosprays79.9574, 5 to 96.9601 20% well for fromthe concentration the Whatman range (GE of Healthcare 10 to technique for ethyl sulfate and ethyl glucuronide from urine samples in EtS-d5 and EtG-d5 were spiked into negative donor and positive donor . As the electrospray signal from paper lasts ~40 s, it can accommodate The concentration of IS was kept at 500 ng/mL in all samples. Three Life1000 Sciences, ngNL: 1.44E4/mL. EtGPittsburgh, ionization PS) showed cellulose poor paperreproducibility used in comparedthe VSC, towhich EtS. a single experiment and from a single urine sample spot, all in much Dependent spectra are triggered from an inclusion list containing the FTMS - p NSI Full ms2 [email protected] [50.00-145.00] 81.0346 urine samples and submitted to dilution/precipitation (1:3 urine to 100 full scan MS at high resolving power and many MS/MS experiments for 95 positive donor samples were also analyzed. makesOptimizing it90 amenable solvent for/substrate quantitative conditions analysis. for better %RSD ionization (std. dev/mean of EtG will x100)be shorter timeframes than it is possible using LC MS techniques. precursor mass for each metabolite and internal standard. Resolving 85 For forensic toxicology use only. 80 79.0190

methanol) and centrifugation for 10 min at 3,000 g. Same protocol was identification and confirmation in screening applications. 75 waspursued found70 into follow be between-up work. 5 to 20% for the concentration range of 10 to power was set to 70,000 and 35,000 (FWHM at m/z 200) for full scan MS 65 221.07→75.0088, 85.0296 EtS-d5 andfollowed EtG-d5 with were synthetic spiked urine into and negative neat samples. donor and The positive IS-containing donor 60 . As the electrospray signal from paper lasts ~40 s, it can accommodate 55 . 1000 ng/50 mL. EtG ionization showed poor reproducibility compared to EtS. The ethyl sulfate anion electrosprays well with an extraction solvent

Abundance 45

and MS/MS,supernatant respectively. was then spiked Negative with ionsEtS and are EtGgenerated to obtain directly calibration from paper Positive40 donor samples were analyzed (Fig. 6) by the same methodology. full©2015 scan Thermo MS Fisher at Scientific. high All Rightsresolving Reserved. power and many MS/MS experiments for urine samples and submitted to dilution/precipitation (1:3 urine to 35 30 conducive to negative ionization and from cellulose paper which, in the NL: 6.03E4 Optimizing25 solvent /substrate conditions for better ionization of EtG will be

Relative The Velox 360™ system is the trade mark of Prosolia, Inc. Whatman is a registered brand of GE Healthcare Life Sciences. All other trademarks are the 2whencurves: an applied 190 sprayuL supernatant voltage (4.5 plus kV10 inuL negativeEtS and Etg ion at mode) various induces Even though20 positive79.9574 results were obtained, the variabilityFTMS - p NSI obtained Full ms2 [email protected] [50.00 is-245.00] too Ethanol Metabolites by Paper Spray Ionization: Method Development in Negative Ion Mode methanol) and centrifugation for 10 min at 3,000 g. Same protocol was 15 78.9591 85.0296 identification and confirmation in screening applications. 10 100 presenceproperty of an of Thermo internal Fisher Scientific standard, and its subsidiaries. makes it promising for quantitative

pursued 5 in follow-up work. 80.9982 95 80.7259 large to0 consider the technique robust enough to evaluate the donor signal concentrations to make a calibration curve in the range of 5 to 1000 90 electrospray from the sharp tip of the paper (Figure 1). The Q Exactive 78.4 78.6 78.8 79.0 79.2 79.4 79.6 79.8 80.0 80.2 80.4 80.6 80.8 81.0 81.2 81.4 81.6 81.8 This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. followed with synthetic urine and neat samples. The IS-containing 85 analysis. m/z 80 . quantitatively. Please, note some endogenous signal detected75 in the urine The ethyl sulfate anion electrosprays well with an extraction solvent ng/mL. Eight microliters were loaded onto paper spray cartridges and mass spectrometers use Higher Collision Dissociation (HCD) energy Positive donor samples were analyzed (Fig. 6) by the70 same methodology. supernatant was then spiked with EtS and EtG to obtain calibration 65

60 from theNL: 3.77E4 negative donor. . Ethyl glucuronideconducive ,to being negative more ionizationhydrophilic, and does from not celluloseelectrospray paper well which, in the allowed to dry at room temperature for 10 minutes. The recommended 55 curves:experiments 190 uL supernatantto fragment precursorplus 10 uL ions. EtS and Etg at various Even thoughFTMS - p NSI Full ms2positive [email protected] [50.00- 145.00]results were obtained, the variability50 obtained is too 45

40 extraction solvent for negative ionization from paper was used, 100% 97.0295 from cellulosepresence paper of an and internal would standard,benefit from makes alternative it promising substrates for orquantitative 100 35 83.0139 concentrations to make a calibration curve in the range of 5 to 1000 large to consider95 the technique robust enough to evaluate30 75.0088 the donor signal FIGURE 290 . MS/MS spectra FIGURE 3. MS/MS25 spectra Datamethanol Analysis with 100 ppm acetic acid. Spray stability in negative ion mode 85 Abundance Relative 20 paper treatments to make the cellulose paper more suitable for the analysis. 80 15 75 quantitatively. Please, note some endogenous signal10 detected in the urine ng/mL. Eight microliters were loaded onto paper spray cartridges and 70 confirming the presence of EtS in confirming the5 presence of EtG in benefits from a low surface tension solvent such as methanol. 65 ionization of EtG. 0 60 75 76 77 78 79 80 81 82 83 84 85 Data acquisition was with the Thermo Scientific™ TraceFinder™ software. 55 . 50 allowed to dry at room temperature for 10 minutes. The recommended from theAbundance negative donor. Ethyl glucuronide, being more hydrophilic, does not electrospray well negative ion mode (Table 1) for negative ion mode (Tablem/z 1) for 45

40 All otherMass analyses Spectrometry used Thermo Scientific™ Xcalibur™ platform tools. 35 extraction solvent for negative ionization from paper was used, 100% spiked urine30 samples (200 ng/mL). spiked urine samples (200 ng/mL). from cellulose paper and would benefit from alternative substrates or 25 Relative 96.9601 96.9931 20 References 15 methanol Awith commercially 100 ppm availableacetic acid. Velox Spray 360™ stability paper sprayin negative system ion (Fig. mode 1, FIGUREData collected 2. 10MS/MS with spectra a Thermo DataFIGURE collected 3. MS/MS with a spectraThermo paper treatments to make the cellulose paper more suitable for the 5 97.0659 0 Scientific™96.85 Q96.90 Exactive96.95 97.00 97.05 ™97.10 Plus97.15 97.20 MS.97.25 ™ ™ 1. Jalbermann et al, JCS 50 (2012) 51-56. benefits fromProsolia a low, Inc., surface Indianapolis, tension IN) solvent that uses such Velox as methanol. Sample Cartridges (VSC) confirming the presencem/z of EtS in Scientificconfirming Q theExactive presence Plus of MS. EtG in ionization of EtG. was coupled to a Thermo Scientific™ Q Exactive™ Focus MS. The Full negative ion mode (Table 1) for negative ion mode (Table 1) for 2. Manicke et al, IJMS 300 (2011) 123-129. Mass SpectrometryMS with Confirmation by Data Dependent MS/MS method was used. Data spiked urine124.99→ samples79.9574, (200 96.9601 ng/mL ). spiked urine samples (200 ng/mL). References NL: 1.44E4 Dependent spectra are triggered from an inclusion list containing the FTMS - p NSI Full ms2 [email protected] [50.00-145.00] 81.0346 A commercially available Velox 360™ paper spray system (Fig. 1, Data collected100 with a Thermo Data collected with a Thermo 95

90 precursor mass for each metabolite and internal standard. Resolving 85 For forensic toxicology use only. 80 79.0190 1. Jalbermann et al, JCS 50 (2012) 51-56. Scientific ™ Q Exactive™ Plus MS. Scientific™ Q Exactive™ Plus MS. Prosolia, Inc., Indianapolis, IN) that uses Velox Sample Cartridges (VSC) 75 70 power was set to 70,000 and 35,000 (FWHM at m/z 200) for full scan MS 65 221.07→75.0088, 85.0296 60 55 was coupled to a Thermo Scientific™ Q Exactive™ Focus MS. The Full 50

Abundance 45 2. Manicke et al, IJMS 300 (2011) 123-129.

and MS/MS, respectively. Negative ions are generated directly from paper 40 ©2015 Thermo Fisher Scientific. All Rights Reserved. 35 124.99→79.9574, 96.9601 MS with Confirmation by Data Dependent MS/MS method was used. Data 30 NL: 6.03E4 25

NL: 1.44E415 78.9591 85.0296 10 100 property of Thermo Fisher Scientific and its subsidiaries.

Dependent spectra are triggered from an inclusion list containing the FTMS - p NSI5 Full ms2 [email protected] [50.00-145.00] 80.9982 95 80.725981.0346 100 0 90 electrospray from the sharp tip of the paper (Figure 1). The Q Exactive 78.4 78.6 78.8 79.0 79.2 79.4 79.6 79.8 80.0 80.2 80.4 80.6 80.8 81.0 81.2 81.4 81.6 81.8 95 This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 85 90 m/z 80 precursor mass for each metabolite and internal standard. Resolving 85 75 For forensic toxicology use only.

80 79.0190 70

mass spectrometers use Higher Collision Dissociation (HCD) energy 75 65 70 60 65 NL: 3.77E4 221.07→75.0088, 85.0296 power was set to 70,000 and 35,000 (FWHM at m/z 200) for full scan MS 55 60 experiments to fragment precursor ions. FTMS - p NSI Full ms2 [email protected] [50.00-145.00] 50 55 45 50 40 97.0295 Abundance 45 35 100 83.0139 and MS/MS, respectively. Negative ions are generated directly from paper 40 95 30 75.0088 ©2015 Thermo Fisher Scientific. All Rights Reserved. 35 90 25 30 Data Analysis 85 Abundance Relative 20 NL: 6.03E4 25 80 15

Relative The Velox 360™ system is the trade mark of Prosolia, Inc. Whatman is a registered brand of GE Healthcare Life Sciences. All other trademarks are the 20 75 FTMS - p NSI Full ms2 [email protected] [50.00-245.00] when an applied spray voltage (4.5 kV in negative ion mode) induces 79.9574 10

15 70 78.9591 5 85.0296 10 65 100 property of Thermo Fisher Scientific and its subsidiaries. 0 60 75 76 77 78 79 80 81 82 83 84 85 5 80.9982 95 55 80.7259 Data acquisition was with the Thermo Scientific™ TraceFinder™ software. 0 90 78.4 50 78.6 78.8 79.0 79.2 79.4 79.6 79.8 80.0 80.2 80.4 80.6 80.8 81.0 81.2 81.4 81.6 81.8 electrospray from the sharp tip of the paper (Figure 1). The Q Exactive Abundance m/z This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 45 85 40 m/z 80 35 75

All other analyses used Thermo Scientific™ Xcalibur™ platform tools. 30 mass spectrometers use Higher Collision Dissociation (HCD) energy 70 25 Relative 96.9931 96.9601 65 20

15 60 NL: 3.77E4 10 55

5 experiments to fragment precursor ions. FTMS - p NSI Full ms2 [email protected] [50.00-145.00]97.0659 50 0 96.85 96.90 96.95 97.00 97.05 97.10 97.15 97.20 97.25 45

40 97.0295m/z 100 35 83.0139 95 30 75.0088 90 25

Data Analysis 85 Abundance Relative 20

80 15 75 10 70 5 65 0 60 75 76 77 78 79 80 81 82 83 84 85 Data acquisition was with the Thermo Scientific™ TraceFinder™ software. 55 50 Abundance

m/z 45

40 All other analyses used Thermo Scientific™ Xcalibur™ platform tools. 35 30

25 Relative 96.9601 96.9931 20

5 97.0659 0 96.85 96.90 96.95 97.00 97.05 97.10 97.15 97.20 97.25

m/z EthanolEthanol Metabolites Metabolites by by Paper Paper Spray Spray Ionization: Ionization: Method Method Development Development in in Negative Negative Ion Ion Mode Mode 1 2 2 1 1 MariaMaria C. C. Prieto Prieto Conaway Conaway, 1Chengsen, Chengsen Zhang Zhang, 2Nicholas, Nicholas E. E. Manicke Manicke, 2Kristine, Kristine Van Van Natta Natta, 1Marta, Marta Kozak Kozak 1 1 2 Thermo1Thermo Fisher Fisher Scientific, Scientific, San San Jose Jose CA; CA; Indiana 2Indiana University University-Purdue-Purdue University, University, Indianapolis, Indianapolis, IN IN

Overview FIGUREFIGURE 1. Velox1. Velox 360 360 paper paper spray spray system system showing showing, clockwise, clockwise from from FIGUREFIGURE 4. Normalized4. Normalized analyte analyte to to FIGUREFIGURE 5. Normalized5. Normalized analyte analyte to ISto IS Overview toptop left: left: paper paper spray spray ion ion source, source, mechanism mechanism for fordispensing dispensing solvent solvent to to IS areasIS areas vs .vs concentration. concentration plots plots areasareas vs. vs. concentration concentration plots plots for for the sample, paper cassette indicating sample deposition, and spotted for spiked EtS in, top to bottom: spiked EtG in, top to bottom: neat, Purpose:Purpose: Develop Develop an anionization ionization method method in negative in negative mode mode for forscreening screening of of the sample, paper cassette indicating sample deposition, and spotted for spiked EtS in, top to bottom: spiked EtG in, top to bottom: neat, paper cassette electrospraying into mass spectrometer inlet. neat, synthetic urine and urine synthetic urine and urine matrices. ethanolethanol metabolites metabolites in urine in urine using using paper paper spray spray technology technology and and mass mass paper cassette electrospraying into mass spectrometer inlet. neat, synthetic urine and urine synthetic urine and urine matrices. matrices. EtG does not ionize well under the spectrometryspectrometry for foruse use in forensic in forensic toxicology. toxicology. matrices. EtG does not ionize well under the substrate/solventsubstrate/solvent conditions conditions used. used. Methods:Methods: Urine Urine spiked spiked with with ethyl ethyl sulfate sulfate (EtS (EtS) and) and ethyl ethyl glucuronide glucuronide (EtG (EtG) ) werewere analyzed analyzed as driedas dried urine urine samples samples by paperby paper spray spray ionization ionization coupled coupled Y = 0.000803989+0.0019195*X R^2 = 0.9719 W: 1/X Y = 1.11275+0.00386917*X R^2 = 0.9159 W: 1/X to Thermo Scientific™ Orbitrap™ mass spectrometers (MS). Positive Y = 0.000803989+0.0019195*X R^2 = 0.9719 W: 1/X Y = 1.11275+0.00386917*X R^2 = 0.9159 W: 1/X 2.0 5 to Thermo Scientific™ Orbitrap™ mass spectrometers (MS). Positive

2.0 5 control donor samples are analyzed after method development. 1.5 4 Neat Neat 4 control donor samples are analyzed after method development. 1.5 Neat Neat 3 1.0 3 1.0 2 Results: Demonstrated the potential of paper spray ionization in negative 0.5 2

Area Ratio 1 Results: Demonstrated the potential of paper spray ionization in negative 0.5 Area Ratio

Area Ratio 1 mode MS for the screening of ethanol metabolites with minimum sample 0.0 0 Area Ratio mode MS for the screening of ethanol metabolites with minimum sample 0 0.0 200 400 600 800 1000 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Y = -0.00713621+0.0022996*X R^2 = 0.9750 W: 1/X Y = 1.14201+0.0103237*X R^2 = 0.7091 W: 1/X preparation and no chromatography. Paper spray is easy to use and

Y = -0.00713621+0.0022996*X R^2 = 0.9750 W: 1/X Y = 1.14201+0.0103237*X R^2 = 0.7091 W: 1/X preparation and no chromatography. Paper spray is easy to use and 2.5 15 2.5 15 provides answers in seconds. The potential of this technique for 2.0 2.0 10 provides answers in seconds. The potential of this technique for 1.5 Synthetic Synthetic 10 1.5 Synthetic Synthetic quantitation of EtG and EtS is also explored. 1.0 urine 5 urine quantitation of EtG and EtS is also explored. 1.0 urine 0.5 Area Ratio 5 urine Area Ratio 0.5 0 Area Ratio 0.0 Area Ratio 0 200 400 600 800 1000 0 0.0 200 400 600 800 1000 0 Table 1. Ethanol urine metabolites indicative of ethanol consumption. 0 200 400 600 800 1000 0 200 400 600 800 1000 Introduction Table 1. Ethanol urine metabolites indicative of ethanol consumption. Y = 0.671872+0.00205785*X R^2 = 0.8521 W: 1/X Y = 1.1776+0.0123971*X R^2 = 0.6825 W: 1/X

Introduction Y = 0.671872+0.00205785*X R^2 = 0.8521 W: 1/X 20 Y = 1.1776+0.0123971*X R^2 = 0.6825 W: 1/X

Deuterium-labeled internal standards were added to all samples. 2.5 20 Deuterium-labeled internal standards were added to all samples. 2.5 15 Urine Paper spray is a direct ionization technique that simplifies the mass 2.0 15 2.0 Urine Urine Paper spray is a direct ionization technique that simplifies the mass 1.5 Urine 10 1.5 10 spectrometric analysis of compounds from biological fluids. Both 1.0 5 Area Ratio spectrometric analysis of compounds from biological fluids. Both Area Ratio 1.0 5

0.5 Area Ratio

Area Ratio 0 Ethanol Metabolite m/z Fragment 1 Fragment 2 Molecular Structure 0.5 qualitative and quantitative analysis of small molecules from complex Ethanol Metabolite m/z Fragment 1 Fragment 2 Molecular Structure 0.0 0 0 200 400 600 800 1000 qualitative and quantitative analysis of small molecules from complex 0 0.0 200 400 600 800 1000 0 200 400 600 800 1000 matrices such as blood and urine are possible without time-consuming 0 200 400 600 800 1000 matrices such as blood and urine are possible without time-consuming Concentration (ng/mL) Concentration (ng/mL) Ethyl sulfate 124.9914 79.9574 96.9601 Concentration (ng/mL) Concentration (ng/mL) samplesampleEthanol preparation preparation and Metabolitesand chromatography. chromatography. Samples Samples byare are collected Papercollected and and Spray Ionization:Ethyl sulfate 124.9914 Method 79.9574 Development96.9601 in Negative Ion Mode storedstored in a in simple a simple paper paper cassette cassette for foreasy easy shipment shipment of samplesof samples to theto the forensic toxicology lab. Paper spray technology1 is therefore attractive for 2 Ethyl sulfate–d5 130.02282 79.9574 97.9664 1 1 forensicMaria toxicology C. Prieto lab. Paper Conaway spray technology, Chengsen is therefore attractive Zhang for , NicholasEthyl E. sulfate Manicke–d5 130.0228, Kristine 79.9574 Van Natta97.9664 , Marta Kozak FIGUREFIGURE 6. Positive6. Positive donor donor urine urine samples, samples, top top to bottom,to bottom, donor donor 1, donor1, donor 2, 2, forensic toxicology compound screening. donor 3 and negative teetotaler urine donor blank. Mass accuracy was 6- forensic1 toxicology compound screening. 2 donor 3 and negative teetotaler urine donor blank. Mass accuracy was 6- EtG andThermo EtS have beenFisher identified Scientific, as specific and San direct metabolitesJose CA; of Indiana University-Purdue University, Indianapolis, IN 10 10ppm ppm. . EtG and EtS have been identified as specific and direct metabolites of Ethyl glucuronide 221.0666 75.0088 85.0296 124.99 → 79.9574 recentrecent alcohol alcohol consumption. consumption. Small Small amounts amounts of ethanolof ethanol (0.1%) (0.1%) are are Ethyl glucuronide 221.0666 75.0088 85.0296 124.99 → 79.9574 79.9566 100 NL: 6.73E4 converted into EtG and EtS by conjugation pathways in the liver, and their 79.9566 100 NL: 6.73E4 50 Donor_1 converted into EtG and EtS by conjugation pathways in the liver, and their 50 Donor_1

0 presence in blood and urine are used as confirmation for ethanol 100 0 NL: 1.84E4 presence in blood and urine are used as confirmation for ethanol 100 50 Donor_2NL: 1.84E4 Ethyl glucuronide–d5 226.0981 75.0088 85.0296 50 Donor_2 consumption. EtG and EtS are detectable in serum for 10–14 h and in 0 Ethyl glucuronide–d5 226.0981 75.0088 85.0296 100 NL: 7.32E4 consumption. EtG and EtS are detectable in serum for 10–14 h and in 0 100 50 Donor_3NL: 7.32E4

urine for 25–44 h. Therefore, these metabolites close the gap between Abundance Relative 50 Donor_3 0 100 urine for 25–44 h. Therefore, these metabolites close the gap between Abundance Relative 0 NL: 9.83E3 100 NL: 9.83E3 short-term markers (e.g., ethanol) and long-term markers (e.g., 50 Urine Blank with IS 50 Urine Blank with IS short-term markers (e.g., ethanol) and long-term markers (e.g., 0 )1 79.5 79.6 79.7 79.8 79.9 80.0 80.1 80.2 80.3 80.4 80.5 0 carbohydrate-deficient transferrin in serum This permits a wide range of 79.5 79.6 79.7 79.8 79.9 80.0 80.1 80.2 80.3 80.4 80.5 carbohydrate-deficient transferrin in serum. )1 This permits a wide range of FIGURE 1. Velox 360 paper spray system showing, clockwise from FIGURE 4. Normalized analyte tom/z FIGURE 5. Normalized analyte to IS . m/z applications,Overview such as controlling patients in withdrawal treatment or topResults Resultsleft: paper spray ion source, mechanism for dispensing solvent to IS areas vs. concentration plots221.07 → 85.0296areas vs. concentration plots for applications, such as controlling patients in withdrawal treatment or 221.07 → 85.0296 85.0287 NL: 2.66E5 100 85.0287 NL: 2.66E5 monitoring alcohol abstinence. Cutoff values for establishing alcohol the sample, paper cassette indicating sample deposition, and spotted for spiked EtS in, top to100 bottom: spiked EtG Donor_1in, top to bottom: neat, monitoringPurpose: Developalcohol abstinence.an ionization Cutoffmethod values in negative for establishing mode for screening alcohol of Ethanol metabolites by paper spray ionization 50 75.0079 83.0130 Donor_1 Ethanol metabolites by paper spray ionization 50 75.0079 81.0337 83.0130 81.0337 abstinence in the United States are defined as less than 100 ng/mL for paper cassette electrospraying into mass spectrometer inlet. neat, synthetic urine 0and urine synthetic urineNL: 1.49E4 and urine matrices. ethanol metabolites in urine using paper spray technology and mass 100 abstinence in the United States are defined as less than 100 ng/mL for 0 NL: 1.49E4 Paper spray ionization has primarily been shown for the analysis of small 100 Donor_2 EtG and less than 25 ng/mL for EtS. Small amounts of EtG and EtS are matrices. EtG does not ionizeDonor_2 well under the EtGspectrometry and less thanfor use 25 in ng forensic/mL for toxicology.EtS. Small amounts of EtG and EtS are Paper spray ionization has primarily been shown for the analysis of small 50 molecules that ionize in positive mode. Conditions for negative ionization 50 0 substrate/solventNL: 8.63E4 conditions used. endogenous in our bodies and can also be produced from regular use of molecules that ionize in positive mode. Conditions for negative ionization 100 0 endogenous in our bodies and can also be produced from regular use of 100 Donor_3NL: 8.63E4 Methods: Urine spiked with ethyl sulfate (EtS) and ethyl glucuronide (EtG) usually require a low surface tension solvent and a spray voltage about Donor_3 antiseptic gels, mouthwash and certain medications. usually require a low surface tension solvent and a spray voltage about Abundance Relative 50 antiseptic gels, mouthwash and certain medications. 2 Abundance Relative 50 were analyzed as dried urine samples by paper spray ionization coupled 0 NL: 6.95E3 500 V less than that used in positive mode . This2 is to avoid corona 100 0 NL: 6.95E3 500 V less than that used in positive mode . This is to avoid corona 100 Urine Blank Y = 0.000803989+0.0019195*X R^2 = 0.9719 W: 1/X Y = 1.11275+0.00386917*X R^2 = 0.9159 W: 1/X In thisto Thermowork, we Scientific™ develop a Orbitrap negative™ ionizationmass spectrometers mode mass (MS). spectrometry Positive 50 Urine Blank

discharge which would increase the spray current and decrease analyte 2.0 5 In this work, we develop a negative ionization mode mass spectrometry discharge which would increase the spray current and decrease analyte 50 0 control donor samples are analyzed after method development. 1.5 4 Neat method that is compatible with paper spray ionization for the analysis of signal. All experiments shown were conducted with 100% methanol/ 750 76 77Neat 78 79 80 81 82 83 84 85 method that is compatible with paper spray ionization for the analysis of 75 76 77 78 m/z79 80 81 3 82 83 84 85 signal. All experiments shown were conducted with 100% methanol/ 1.0 m/z ethanol metabolites in urine and coupling to an Orbitrap mass 100 ppm acetic acid, which fulfills the low surface tension negative 2 ethanolResults metabolites: Demonstrated in urine the potential and coupling of paper to anspray Orbitrap ionization mass in negative 0.5

Area Ratio 1

100 ppm acetic acid, which fulfills the low surface tension negative Area Ratio spectrometer. mode MS for the screening of ethanol metabolites with minimum sample ionization requirement. 0.0 0 spectrometer. ionization requirement. 0 200 400 600 800 1000 0 200 400 600 800 1000

Y = -0.00713621+0.0022996*X R^2 = 0.9750 W: 1/X Y = 1.14201+0.0103237*X R^2 = 0.7091 W: 1/X preparation and no chromatography. Paper spray is easy to use and Conclusion PreliminaryPreliminary results results for forthe the screening screening of ethanolof ethanol metabolites metabolites by paperby paper spray spray 2.5 Conclusion 15 Methodsprovides answers in seconds. The potential of this technique for 2.0 Synthetic 10 Methods MS/MSMS/MS are are shown shown in Figures in Figures 2 and 2 and 3, with3, with expected expected fragments fragments for forEtG EtG 1.5 . We. have developed a negative ion mode method for paper spraySynthetic quantitation of EtG and EtS is also explored. 1.0 We have developed a negative ion mode method for paper spray urine 5 urine

(m/z 221.07→75.0088, 85.0296) and EtS (m/z 124.99 →79.9574, 96.9601) 0.5 technology coupled to Thermo ScientificArea Ratio Q Exactive mass Sample Preparation Area Ratio (m/z 221.07→75.0088, 85.0296) and EtS (m/z 124.99 →79.9574, 96.9601) 0 Sample Preparation 0.0 technology coupled to Thermo Scientific Q Exactive mass and outstanding mass accuracies. 0 spectrometers.200 400 600 800 1000 0 200 400 600 800 1000 Tableand 1. outstanding Ethanol urine mass metabolites accuracies. indicative of ethanol consumption. spectrometers. EtS Introductionand EtG were spiked in urine with their corresponding deuterated Y = 0.671872+0.00205785*X R^2 = 0.8521 W: 1/X Y = 1.1776+0.0123971*X R^2 = 0.6825 W: 1/X

EtS and EtG were spiked in urine with their corresponding deuterated 20 internal standards (IS), EtS-d5 and EtG-d5 (all from Cerilliant). Standards FiguresDeuterium 4 and-labeled 5 show internal calibration standards curves were for EtS added and to EtG all, samples. respectively. The 2.5 . The Velox 360 system has been demonstrated as a forensic screening 15 internal standards (IS), EtS-d5 and EtG-d5 (all from Cerilliant). Standards Figures 4 and 5 show calibration curves for EtS and EtG, respectively. The 2.0 . The Velox 360 system has been demonstrated as a forensicUrine screening Paper spray is a direct ionization technique that simplifies the mass Urine ethyl sulfate anion electrosprays well from the Whatman (GE Healthcare 1.5 10 werewere spiked spiked in three in three different different matrices: matrices: urine urine, synthetic, synthetic urine urine or solvent.or solvent. ethyl sulfate anion electrosprays well from the Whatman (GE Healthcare techniquetechnique for forethyl ethyl sulfate sulfate and and ethyl ethyl glucuronide glucuronide from from urine urine samples samples in in spectrometric analysis of compounds from biological fluids. Both 1.0 5 Area Ratio The concentration of IS was kept at 500 ng/mL in all samples. Three Life Sciences, Pittsburgh, PS) cellulose paper used in the VSC, which Area Ratio 0.5 a single experiment and from a single urine sample spot, all in much EthanolLife Metabolite Sciences, Pittsburgh,m/z Fragment PS) cellulose 1 Fragment paper used 2 Molecular in the VSC, Structure which a single experiment and from a 0single urine sample spot, all in much Thequalitative concentration and quantitative of IS was analysis kept at of 500 small ng /mLmolecules in all samples. from complex Three 0.0 0 200 400 600 800 1000 makes it amenable for quantitative analysis. %RSD (std. dev/mean x100) 0 200 400 600 800 1000 positivepositivematrices donor donor such samples assamples blood were andwere also urine also analyzed. are analyzed. possible without time-consuming makes it amenable for quantitative analysis. %RSD (std. dev/mean x100) shortershorter timeframes timeframes than than it is it possible is possible using using LC LCMS MS techniques. techniques. was found to be between 5 to 20% for the concentration range of 10 to Concentration (ng/mL) Concentration (ng/mL) EtS-sampled5 and preparation EtG-d5 were and spiked chromatography. into negative Samples donor andare collectedpositive donorand Ethylwas sulfate found to be124.9914 between 579.9574 to 20% for the96.9601 concentration range of 10 to . As the electrospray signal from paper lasts ~40 s, it can accommodate EtS-d5 and EtG-d5 were spiked into negative donor and positive donor 1000 ng/mL. EtG ionization showed poor reproducibility compared to EtS. . As the electrospray signal from paper lasts ~40 s, it can accommodate urinestored samples in a simpleand submitted paper cassette to dilution/precipitation for easy shipment (1:3 of samples urine to to the 1000 ng/mL. EtG ionization showed poor reproducibility compared to EtS. full scan MS at high resolving power and many MS/MS experiments for urine samples and submitted to dilution/precipitation (1:3 urine to Optimizing solvent /substrate conditions for better ionization of EtG will be full scan MS at high resolving power and many MS/MS experiments for forensic toxicology lab. Paper spray technology is therefore attractive for EthylOptimizing sulfate–d5 solvent130.0228 /substrate 79.9574 conditions 97.9664 for better ionization of EtG will be FIGURE 6. Positive donor urine samples, top to bottom, donor 1, donor 2, methanol)methanol) and and centrifugation centrifugation for for10 10min min at 3,000at 3,000 g. Sameg. Same protocol protocol was was identificationidentification and and confirmation confirmation in screening in screening applications. applications. forensic toxicology compound screening. pursuedpursued in follow in follow-up- upwork. work. donor 3 and negative teetotaler urine donor blank. Mass accuracy was 6- followedfollowed with with synthetic synthetic urine urine and and neat neat samples. samples. The The IS- containingIS-containing . 10 ppm. The. The ethyl ethyl sulfate sulfate anion anion electrosprays electrosprays well well with with an anextraction extraction solvent solvent supernatantsupernatantEtG and EtSwas was have then then been spiked spiked identified with with EtS as EtS and specific and EtG EtG andto obtain todirect obtain calibrationmetabolites calibration of PositivePositive donor donor samples samples were were analyzed analyzed (Fig. (Fig. 6) by6) theby the same same methodology. methodology. Ethyl glucuronide 221.0666 75.0088 85.0296 conduciveconducive to 124.99 negativeto negative → 79.9574 ionization ionization and and from from cellulose cellulose paper paper which, which, in the in the curves:curves:recent 190alcohol 190 uL supernatantuLconsumption. supernatant plus Small plus 10 amounts10uL EtSuL EtS and of andethanol Etg Etg at (0.1%) variousat various are EvenEven though though positive positive results results were were obtained, obtained, the the variability variability obtained obtained is too is too presence of an internal79.9566 standard, makes it promising for quantitative concentrationsconverted into to makeEtG and a calibrationEtS by conjugation curve in pathwaysthe range in of the 5 toliver, 1000 and their large to consider the technique robust enough to evaluate the donor signal presence100 of an internalNL: standard, 6.73E4 makes it promising for quantitative 50 Donor_1 concentrations to make a calibration curve in the range of 5 to 1000 large to consider the technique robust enough to evaluate the donor signal analysis. 0 ng/mL.presence Eight inmicroliters blood and were urine loaded are used onto as paperconfirmation spray cartridgesfor ethanol and quantitatively. Please, note some endogenous signal detected in the urine analysis.100 NL: 1.84E4 ng/mL. Eight microliters were loaded onto paper spray cartridges and quantitatively. Please, note some endogenous signal detected in the urine 50 Donor_2 Ethyl glucuronide–d5 226.0981 75.0088 85.0296 consumption. EtG and EtS are detectable in serum for 10–14 h and in . Ethyl glucuronide0 , being more hydrophilic, does not electrospray well allowed to dry at room temperature for 10 minutes. The recommended from the negative donor. 100 allowed to dry at room temperature for 10 minutes. The recommended from the negative donor. . Ethyl glucuronide, beingNL: 7.32E4 more hydrophilic, does not electrospray well 50 Donor_3

urine for 25–44 h. Therefore, these metabolites close the gap between Abundance Relative extraction solvent for negative ionization from paper was used, 100% from cellulose0 paper and would benefit from alternative substrates or extraction solvent for negative ionization from paper was used, 100% from cellulose100 paper andNL: 9.83E3 would benefit from alternative substrates or methanolshort-term with markers100 ppm (e.g. acetic, ethanol) acid. Sprayand long stability-term markersin negative (e.g. ion, mode FIGURE 2. MS/MS spectra FIGURE 3. MS/MS spectra paper treatments50 to make Utherine Blank cellulose with IS paper more suitable for the FIGURE 2. MS/MS spectra 0 methanol with 100 ppm acetic acid. Spray)1 stability in negative ion mode FIGURE 3. MS/MS spectra paper treatments79.5 79.6 79.7 79.8 79.9 80.0 80.1to 80.2 make80.3 80.4 80.5 the cellulose paper more suitable for the carbohydrate-deficient transferrin in serum . This permits a wide range of confirming the presence of EtS in confirming the presence of EtG in m/z benefits from a low surface tension solvent such as methanol. confirming the presence of EtS in confirming the presence of EtG in ionizationionization of EtGof EtG. . benefitsapplications, from sucha low as surface controlling tension patients solvent in withdrawal such as methanol. treatment or Results 221.07 → 85.0296 negative ion mode (Table 1) for negative ion mode (Table 1) for 85.0287 negative ion mode (Table 1) for negative ion mode (Table 1) for 100 NL: 2.66E5 Massmonitoring Spectrometry alcohol abstinence. Cutoff values for establishing alcohol Donor_1 Mass Spectrometry spikedEthanol urine metabolites samples by(200 paper ng/mL spray). ionizationspiked urine samples (200 ng/mL). 50 75.0079 83.0130 spiked urine samples (200 ng/mL). spiked urine samples (200 ng/mL). References 81.0337 abstinence in the United States are defined as less than 100 ng/mL for References 0 A commercially available Velox 360™ paper spray system (Fig. 1, Data collected with a Thermo Data collected with a Thermo 100 NL: 1.49E4 AEtG commercially and less than available 25 ng/mL Velox for EtS 360™. Small paper amounts spray ofsystem EtG and (Fig. EtS 1, are PaperData collectedspray ionization with ahas Thermo primarily been shownData for the collected analysis withof small a Thermo Donor_2 Prosolia, Inc., Indianapolis, IN) that uses Velox Sample Cartridges (VSC) Scientific™ Q Exactive™ Plus MS. Scientific™ Q Exactive™ Plus MS. 1. Jalbermann1. Jalbermann50 et al,et JCSal, JCS 50 50(2012) (2012) 51 -5156.-56. moleculesScientific that™ Q ionize Exactive in positive™ Plus mode. MS. Conditions Scientific for negative™ Q ionization Exactive ™ Plus MS. 0 Prosoliaendogenous, Inc., in Indianapolis, our bodies and IN) can that also uses be Velox produced Sample from Cartridges regular use (VSC) of 100 NL: 8.63E4 was coupled to a Thermo Scientific™ Q Exactive™ Focus MS. The Full usually require a low surface tension solvent and a spray voltage about Donor_3 wasantiseptic coupled gels, to mouthwasha Thermo Scientific™ and certain Qmedications. Exactive™ Focus MS. The Full 2. Manicke Abundance Relative 50 et al, IJMS 300 (2011) 123-129. 2 2. Manicke et al, IJMS 300 (2011) 123-129. MS with Confirmation by Data Dependent MS/MS method was used. Data 124.99→79.9574, 96.9601 0 NL: 6.95E3 500 V less than124.99→ that79.9574, used in 96.9601positive mode . This is to avoid corona 100 MS with Confirmation by Data Dependent MS/MS method was used. Data Urine Blank NL: 1.44E4 In this work, we develop a negative ionization mode mass spectrometry 50 Dependent spectra are triggered from an inclusion list containing the dischargeFTMS - p NSINL: Full 1.44E4 ms2 [email protected] which [50.00-145.00] would 81.0346increase the spray current and decrease analyte Dependent spectra are triggered from an inclusion list containing the 100 FTMS - p NSI Full ms2 [email protected] [50.00-145.00] 95 81.0346 100 0 90 method that is compatible with paper spray ionization for the analysis of 95 75 76 77 78 79 80 81 82 83 84 85 signal.85 All experiments shown were conducted with 100% methanol/ precursor mass for each metabolite and internal standard. Resolving 90 For forensic toxicology use only. 80 79.0190

85 m/z precursor mass for each metabolite and internal standard. Resolving 75 For forensic toxicology use only. 80 70 79.0190 ethanol metabolites in urine and coupling to an Orbitrap mass 75 65 221.07→75.0088, 85.0296 power was set to 70,000 and 35,000 (FWHM at m/z 200) for full scan MS 100 ppm70 acetic acid, which fulfills the low surface tension negative 60 65 221.07→75.0088, 85.0296 power was set to 70,000 and 35,000 (FWHM at m/z 200) for full scan MS 55 60 50 spectrometer. 55

Abundance 45

ionization50 requirement. and MS/MS, respectively. Negative ions are generated directly from paper 40

Relative The Velox 360™ system is the trade mark of Prosolia, Inc. Whatman is a registered brand of GE Healthcare Life Sciences. All other trademarks are the 20 FTMS - p NSINL: Full 6.03E4 ms2 [email protected] [50.00-245.00] Conclusion when an applied spray voltage (4.5 kV in negative ion mode) induces 79.9574 25 15 78.9591

PreliminaryRelative 20 results for the screening of ethanol metabolitesFTMS - p NSI Full ms2 [email protected] paper [50.00-245.00] spray85.0296 The Velox 360™ system is the trade mark of Prosolia, Inc. Whatman is a registered brand of GE Healthcare Life Sciences. All other trademarks are the when an applied spray voltage (4.5 kV in negative ion mode) induces 10 79.9574 100 property of Thermo Fisher Scientific and its subsidiaries.

15 78.9591 5 80.9982 95 85.0296 10 80.7259 100 property of Thermo Fisher Scientific and its subsidiaries. Methods 0 90 electrospray from the sharp tip of the paper (Figure 1). The Q Exactive 78.4 78.65 78.8 79.0 79.2 79.4 79.6 79.8 80.0 80.2 80.4 80.6 80.8 81.0 81.2 81.4 81.6 81.8 95 80.9982 80.7259 85 . This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. MS/MS0 are shown in Figures 2 and 3, with expected fragments90 for EtG We have developed a negative ion mode method for paper spray electrospray from the sharp tip of the paper (Figure 1). The Q Exactive 78.4 78.6 78.8 79.0 79.2 79.4 79.6 79.8 80.0 80.2 80.4 80.6 80.8 81.0 81.2 81.4 81.6 81.8 m/z 80 This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 85 75 80 m/z mass spectrometers use Higher Collision Dissociation (HCD) energy 70 75

(m/z 221.07→75.0088, 85.0296) and EtS (m/z 124.9965 →79.9574, 96.9601) technology coupled to Thermo Scientific Q Exactive mass massSample spectrometers Preparation use Higher Collision Dissociation (HCD) energy 70 60 NL: 3.77E4 65 55 60 experiments to fragment precursor ions. FTMS - p NSINL: Full 3.77E4 ms2 [email protected] [50.00-145.00] 50 and outstanding mass accuracies. 55 spectrometers. 45 experiments to fragment precursor ions. FTMS - p NSI Full ms2 [email protected] [50.00-145.00] 50 40 EtS and EtG were spiked in urine with their corresponding deuterated 97.0295 45 100 35 40 83.0139 95 97.0295 30 100 75.008835 90 25 83.0139 95 30

85 Abundance Relative 75.0088 Data Analysis Figures 4 and 5 show calibration curves for EtS and20 EtG, respectively. The . internal standards (IS), EtS-d5 and EtG-d5 (all from Cerilliant). Standards 90 25 The Velox 360 system has been demonstrated as a forensic screening 80 15 Data Analysis 85 Abundance Relative 20 75 10 80 70 15 75 5 65 10 were spiked in three different matrices: urine, synthetic urine or solvent. ethyl sulfate70 anion electrosprays well from the Whatman0 (GE Healthcare technique for ethyl sulfate and ethyl glucuronide from urine samples in 60 75 5 76 77 78 79 80 81 82 83 84 85 65 55 0 Data acquisition was with the Thermo Scientific™ TraceFinder™ software. 60 75 76 77 78 79 80 81 82 83 84 85 50 Abundance

55 m/z Data acquisition was with the Thermo Scientific™ TraceFinder™ software. 45 50 The concentration of IS was kept at 500 ng/mL in all samples. Three Life Sciences,Abundance Pittsburgh, PS) cellulose paper used in the VSC, which a single experiment and from a single urine sample spot, all in much 40 m/z 45 35 All other analyses used Thermo Scientific™ Xcalibur™ platform tools. 40 30 35 25 All other analyses used Thermo Scientific™ Xcalibur™ platform tools. Relative 96.9931 makes it30 amenable96.9601 for quantitative analysis. %RSD (std. dev/mean x100) shorter timeframes than it is possible using LC MS techniques. positive donor samples were also analyzed. 20 25 Relative 96.9931 15 96.9601 20 10 15 5 97.0659 was found10 to be between 5 to 20% for the concentration range of 10 to 0 96.85 596.9 0 96.95 97.00 97.05 97.10 97.15 97.20 97.25 97.0659 0 . As the electrospray signal from paper lasts ~40 s, it can accommodate EtS-d5 and EtG-d5 were spiked into negative donor and positive donor 96.85 96.90 96.95 97.00 97.05 97.10 97.15 97.20 97.25 1000 ng/mL. EtGm/z ionization showed poor reproducibility compared to EtS. urine samples and submitted to dilution/precipitation (1:3 urine to m/z full scan MS at high resolving power and many MS/MS experiments for Optimizing solvent /substrate conditions for better ionization of EtG will be methanol) and centrifugation for 10 min at 3,000 g. Same protocol was identification and confirmation in screening applications. pursued in follow-up work. followed with synthetic urine and neat samples. The IS-containing . The ethyl sulfate anion electrosprays well with an extraction solvent supernatant was then spiked with EtS and EtG to obtain calibration Positive donor samples were analyzed (Fig. 6) by the same methodology. conducive to negative ionization and from cellulose paper which, in the curves: 190 uL supernatant plus 10 uL EtS and Etg at various Even though positive results were obtained, the variability obtained is too presence of an internal standard, makes it promising for quantitative concentrations to make a calibration curve in the range of 5 to 1000 large to consider the technique robust enough to evaluate the donor signal analysis. ng/mL. Eight microliters were loaded onto paper spray cartridges and quantitatively. Please, note some endogenous signal detected in the urine allowed to dry at room temperature for 10 minutes. The recommended from the negative donor. . Ethyl glucuronide, being more hydrophilic, does not electrospray well extraction solvent for negative ionization from paper was used, 100% from cellulose paper and would benefit from alternative substrates or methanol with 100 ppm acetic acid. Spray stability in negative ion mode FIGURE 2. MS/MS spectra FIGURE 3. MS/MS spectra paper treatments to make the cellulose paper more suitable for the benefits from a low surface tension solvent such as methanol. confirming the presence of EtS in confirming the presence of EtG in ionization of EtG. negative ion mode (Table 1) for negative ion mode (Table 1) for Mass Spectrometry spiked urine samples (200 ng/mL). spiked urine samples (200 ng/mL). References A commercially available Velox 360™ paper spray system (Fig. 1, Data collected with a Thermo Data collected with a Thermo Prosolia, Inc., Indianapolis, IN) that uses Velox Sample Cartridges (VSC) Scientific™ Q Exactive™ Plus MS. Scientific™ Q Exactive™ Plus MS. 1. Jalbermann et al, JCS 50 (2012) 51-56. was coupled to a Thermo Scientific™ Q Exactive™ Focus MS. The Full 2. Manicke et al, IJMS 300 (2011) 123-129. MS with Confirmation by Data Dependent MS/MS method was used. Data 124.99→79.9574, 96.9601

NL: 1.44E4 Dependent spectra are triggered from an inclusion list containing the FTMS - p NSI Full ms2 [email protected] [50.00-145.00] 81.0346 100 95

90 precursor mass for each metabolite and internal standard. Resolving 85 For forensic toxicology use only. 80 79.0190

70 power was set to 70,000 and 35,000 (FWHM at m/z 200) for full scan MS 65 221.07→75.0088, 85.0296 60 55 50

15 78.9591 85.0296 10 100 property of Thermo Fisher Scientific and its subsidiaries.

5 80.9982 95 80.7259 0 90 electrospray from the sharp tip of the paper (Figure 1). The Q Exactive 78.4 78.6 78.8 79.0 79.2 79.4 79.6 79.8 80.0 80.2 80.4 80.6 80.8 81.0 81.2 81.4 81.6 81.8 This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. 85 m/z 80 75 mass spectrometers use Higher Collision Dissociation (HCD) energy 70 65

60 NL: 3.77E4 55 experiments to fragment precursor ions. FTMS - p NSI Full ms2 [email protected] [50.00-145.00] 50 45

40 97.0295 100 35 83.0139 • • 95 30 75.0088 Thermo Scientific Poster Note MASCL PN64377-EN 0315S 3 90 25

Data Analysis 85 Abundance Relative 20

80 15 75 10 70 5 65 0 60 75 76 77 78 79 80 81 82 83 84 85 Data acquisition was with the Thermo Scientific™ TraceFinder™ software. 55 50 Abundance

m/z 45

40 All other analyses used Thermo Scientific™ Xcalibur™ platform tools. 35 30

25 Relative 96.9601 96.9931 20

5 97.0659 0 96.85 96.90 96.95 97.00 97.05 97.10 97.15 97.20 97.25

m/z Ethanol Metabolites by Paper Spray Ionization: Method Development in Negative Ion Mode Maria C. Prieto Conaway1, Chengsen Zhang2, Nicholas E. Manicke2, Kristine Van Natta1, Marta Kozak1 1Thermo Fisher Scientific, San Jose CA; 2Indiana University-Purdue University, Indianapolis, IN

4 Neat control donor samples are analyzed after method development. 1.5 Neat 3 1.0 2 Results: Demonstrated the potential of paper spray ionization in negative 0.5

Area Ratio 1 Area Ratio mode MS for the screening of ethanol metabolites with minimum sample 0.0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 preparation and no chromatography. Paper spray is easy to use and Y = -0.00713621+0.0022996*X R^2 = 0.9750 W: 1/X Y = 1.14201+0.0103237*X R^2 = 0.7091 W: 1/X 2.5 15 provides answers in seconds. The potential of this technique for 2.0 10 1.5 Synthetic Synthetic quantitation of EtG and EtS is also explored. 1.0 urine 5 urine

0.5 Area Ratio Area Ratio 0.0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 Table 1. Ethanol urine metabolites indicative of ethanol consumption. Introduction Y = 0.671872+0.00205785*X R^2 = 0.8521 W: 1/X Y = 1.1776+0.0123971*X R^2 = 0.6825 W: 1/X 20 Deuterium-labeled internal standards were added to all samples. 2.5 15 Urine Paper spray is a direct ionization technique that simplifies the mass 2.0 Urine 1.5 10 spectrometric analysis of compounds from biological fluids. Both 1.0 5 Area Ratio Area Ratio 0.5 Ethanol Metabolite m/z Fragment 1 Fragment 2 Molecular Structure 0 qualitative and quantitative analysis of small molecules from complex 0.0 0 200 400 600 800 1000 0 200 400 600 800 1000 matrices such as blood and urine are possible without time-consuming Concentration (ng/mL) Concentration (ng/mL) sample preparation and chromatography. Samples are collected and Ethyl sulfate 124.9914 79.9574 96.9601 stored in a simple paper cassette for easy shipment of samples to the forensic toxicology lab. Paper spray technology is therefore attractive for Ethyl sulfate–d5 130.0228 79.9574 97.9664 FIGURE 6. Positive donor urine samples, top to bottom, donor 1, donor 2, forensic toxicology compound screening. donor 3 and negative teetotaler urine donor blank. Mass accuracy was 6- EtG and EtS have been identified as specific and direct metabolites of 10 ppm. recent alcohol consumption. Small amounts of ethanol (0.1%) are Ethyl glucuronide 221.0666 75.0088 85.0296 124.99 → 79.9574

79.9566 converted into EtG and EtS by conjugation pathways in the liver, and their 100 NL: 6.73E4 50 Donor_1

50 Donor_3

urine for 25–44 h. Therefore, these metabolites close the gap between Abundance Relative 0 100 NL: 9.83E3 short-term markers (e.g., ethanol) and long-term markers (e.g., 50 Urine Blank with IS

0 )1 79.5 79.6 79.7 79.8 79.9 80.0 80.1 80.2 80.3 80.4 80.5 carbohydrate-deficient transferrin in serum . This permits a wide range of m/z applications, such as controlling patients in withdrawal treatment or Results 221.07 → 85.0296 85.0287 NL: 2.66E5 100 monitoring alcohol abstinence. Cutoff values for establishing alcohol Donor_1 Ethanol metabolites by paper spray ionization 50 75.0079 83.0130 81.0337 abstinence in the United States are defined as less than 100 ng/mL for 0 NL: 1.49E4 100 Paper spray ionization has primarily been shown for the analysis of small Donor_2 EtG and less than 25 ng/mL for EtS. Small amounts of EtG and EtS are 50 molecules that ionize in positive mode. Conditions for negative ionization 0 endogenous in our bodies and can also be produced from regular use of 100 NL: 8.63E4 usually require a low surface tension solvent and a spray voltage about Donor_3 antiseptic gels, mouthwash and certain medications. Abundance Relative 50 2 0 NL: 6.95E3 500 V less than that used in positive mode . This is to avoid corona 100 Urine Blank In this work, we develop a negative ionization mode mass spectrometry discharge which would increase the spray current and decrease analyte 50 0 method that is compatible with paper spray ionization for the analysis of signal. All experiments shown were conducted with 100% methanol/ 75 76 77 78 79 80 81 82 83 84 85 m/z ethanol metabolites in urine and coupling to an Orbitrap mass 100 ppm acetic acid, which fulfills the low surface tension negative spectrometer. ionization requirement. Preliminary results for the screening of ethanol metabolites by paper spray Conclusion Methods MS/MS are shown in Figures 2 and 3, with expected fragments for EtG . We have developed a negative ion mode method for paper spray Sample Preparation (m/z 221.07→75.0088, 85.0296) and EtS (m/z 124.99 →79.9574, 96.9601) technology coupled to Thermo Scientific Q Exactive mass and outstanding mass accuracies. spectrometers. EtS and EtG were spiked in urine with their corresponding deuterated internal standards (IS), EtS-d5 and EtG-d5 (all from Cerilliant). Standards Figures 4 and 5 show calibration curves for EtS and EtG, respectively. The . The Velox 360 system has been demonstrated as a forensic screening were spiked in three different matrices: urine, synthetic urine or solvent. ethyl sulfate anion electrosprays well from the Whatman (GE Healthcare technique for ethyl sulfate and ethyl glucuronide from urine samples in The concentration of IS was kept at 500 ng/mL in all samples. Three Life Sciences, Pittsburgh, PS) cellulose paper used in the VSC, which a single experiment and from a single urine sample spot, all in much positive donor samples were also analyzed. makes it amenable for quantitative analysis. %RSD (std. dev/mean x100) shorter timeframes than it is possible using LC MS techniques. was found to be between 5 to 20% for the concentration range of 10 to EtS-d5 and EtG-d5 were spiked into negative donor and positive donor . As the electrospray signal from paper lasts ~40 s, it can accommodate 1000 ng/mL. EtG ionization showed poor reproducibility compared to EtS. urine samples and submitted to dilution/precipitation (1:3 urine to full scan MS at high resolving power and many MS/MS experiments for Optimizing solvent /substrate conditions for better ionization of EtG will be methanol) and centrifugation for 10 min at 3,000 g. Same protocol was identification and confirmation in screening applications. pursued in follow-up work. followed with synthetic urine and neat samples. The IS-containing . The ethyl sulfate anion electrosprays well with an extraction solvent supernatant was then spiked with EtS and EtG to obtain calibration Positive donor samples were analyzed (Fig. 6) by the same methodology. conducive to negative ionization and from cellulose paper which, in the curves: 190 uL supernatant plus 10 uL EtS and Etg at various Even though positive results were obtained, the variability obtained is too presence of an internal standard, makes it promising for quantitative concentrations to make a calibration curve in the range of 5 to 1000 large to consider the technique robust enough to evaluate the donor signal analysis. ng/mL. Eight microliters were loaded onto paper spray cartridges and quantitatively. Please, note some endogenous signal detected in the urine allowed to dry at room temperature for 10 minutes. The recommended from the negative donor. . Ethyl glucuronide, being more hydrophilic, does not electrospray well extraction solvent for negative ionization from paper was used, 100% from cellulose paper and would benefit from alternative substrates or methanol with 100 ppm acetic acid. Spray stability in negative ion mode FIGURE 2. MS/MS spectra FIGURE 3. MS/MS spectra paper treatments to make the cellulose paper more suitable for the benefits from a low surface tension solvent such as methanol. confirming the presence of EtS in confirming the presence of EtG in ionization of EtG. negative ion mode (Table 1) for negative ion mode (Table 1) for Mass Spectrometry spiked urine samples (200 ng/mL). spiked urine samples (200 ng/mL). References A commercially available Velox 360™ paper spray system (Fig. 1, Data collected with a Thermo Data collected with a Thermo Prosolia, Inc., Indianapolis, IN) that uses Velox Sample Cartridges (VSC) Scientific™ Q Exactive™ Plus MS. Scientific™ Q Exactive™ Plus MS. 1. Jalbermann et al, JCS 50 (2012) 51-56. was coupled to a Thermo Scientific™ Q Exactive™ Focus MS. The Full 2. Manicke et al, IJMS 300 (2011) 123-129. MS with Confirmation by Data Dependent MS/MS method was used. Data 124.99→79.9574, 96.9601

NL: 1.44E4 Dependent spectra are triggered from an inclusion list containing the FTMS - p NSI Full ms2 [email protected] [50.00-145.00] 81.0346 100 95

90 precursor mass for each metabolite and internal standard. Resolving 85 For forensic toxicology use only. 80 79.0190 75

70 power was set to 70,000 and 35,000 (FWHM at m/z 200) for full scan MS 65 221.07→75.0088, 85.0296 60

Relative Abundance The Velox 360™ system is the trade mark of Prosolia, Inc. Whatman is a registered brand of GE Healthcare Life Sciences. All other trademarks are the when an applied spray voltage (4.5 kV in negative ion mode) induces 20 79.9574 FTMS - p NSI Full ms2 [email protected] [50.00-245.00] 15 78.9591 85.0296 10 100 property of Thermo Fisher Scientific and its subsidiaries.

60 NL: 3.77E4 55 experiments to fragment precursor ions. FTMS - p NSI Full ms2 [email protected] [50.00-145.00] 50 45

40 97.0295 100 35 83.0139 95 30 75.0088 90 25

Data Analysis 85 Abundance Relative 20 80 15 75 10 70 5 65 0 60 75 76 77 78 79 80 81 82 83 84 85 Data acquisition was with the Thermo Scientific™ TraceFinder™ software. 55 50 m/z 45

40 All other analyses used Thermo Scientific™ Xcalibur™ platform tools. 35 30

25 Relative Abundance 96.9601 96.9931 20

5 97.0659 0 96.85 96.90 96.95 97.00 97.05 97.10 97.15 97.20 97.25 m/z

For forensic toxicology use only.

www.thermofisher.com ©2016 Thermo Fisher Scientific Inc. All rights reserved. The Velox 360™ system is the trade mark of Prosolia, Inc. Whatman is a registered brand of GE Healthcare Life Sciences. All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries. This information is presented as an example of the capabilities of Thermo Fisher Scientific products. It is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.

Africa +43 1 333 50 34 0 Denmark +45 70 23 62 60 Japan +81 6 6885 1213 Russia/CIS +43 1 333 50 34 0 Australia +61 3 9757 4300 Europe-Other +43 1 333 50 34 0 Korea +82 2 3420 8600 Singapore +65 6289 1190 Austria +43 810 282 206 Finland +358 9 3291 0200 Latin America +1 561 688 8700 Sweden +46 8 556 468 00 Belgium +32 53 73 42 41 France +33 1 60 92 48 00 Middle East +43 1 333 50 34 0 Switzerland +41 61 716 77 00 Brazil +55 11 3731 5140 Germany +49 6103 408 1014 Netherlands +31 76 579 55 55 Taiwan +886 2 8751 6655 Canada +1 800 530 8447 India +91 22 6742 9494 New Zealand +64 9 980 6700 UK/Ireland +44 1442 233555 China 800 810 5118 (free call domestic) Italy +39 02 950 591 Norway +46 8 556 468 00 USA +1 800 532 4752 400 650 5118 PN64377-EN 0616S