Ten Second DART -MS: Ultra-Fast QC Screening of Foods and Juices with Next Generation DART systems

Ten Second DART -MS: Ultra-Fast QC Screening of Foods and Juices with Next Generation DART systems IonSense, Inc. 999 Broadway Suite 404 Saugus, MA 01906 USA

DART-SVP Ionization Source

DART -SVP Ion Source with automated sampler

Method Development Optimization of Desorption Conditions Rapid detection of Adulterants in Herbal Supplements Rapid Screening for Synthetic AntidiabeticDrug Adulteration in Herbal Dietary Supplements using Direct Ionization in Real Time Mass Spectrometry, Z Zhou, J. Zhang, W. Zhang, Y. Bai, and H. Liu Analyst, in press 2011

Optimization of DART Conditions Gas Temp Gas Flow

DART MS and MS/MS Triplicate Analysis

ID-CUBE : Built on DART Technology

ID-CUBE Ionization Source

1. Spot 5 µl aliquot or powder residue on the OpenSpot Card 2. Let sample dry for easy transport or analyze directly when wet 3. Insert sample cardinto the slot on ID-CUBE Source 4. Set the desired heating setting on ID-CUBE control box and press button for instant desorption

100 80 60 40 Nature s Bounty Omega-3 Fish Oil 144.13790 Positive Ion Mode 250 C NL: 5.37E6 DART-SVP 1-D Bounty Fish Oil 02_POS_20110 65 0.14-0.36 T: [100.00-1000.00 20 172.16920 287.26869 0 80 60 40 20 0 100 Relative Abundance 100 80 60 369.35073 144.13803 338.34105 187.12606 417.37163 584.52405 658.53942 842.72096 369.35081 [Cholesterol + H H 2 O]+ Elemental composition search on mass 369.35 m/z= 364.35-374.35 m/z Theo. Mass Delta (ppm) RDB equiv. 369.35073 369.35158-2.30 5.5 C 27 H 45 369.34756 8.60 1.5 C H O N [Cholesterol + H H 2 O]+ Diglycerides Composition 350 C 450 C Triglycerides 40 549.48683 851.71013 20 313.27308 615.49724 495.44004 771.64803 916.73767 231.17393 0 100 200 300 400 500 600 700 800 900 1000 m/z NL: 1.39E6 DART-SVP 1-D Bounty Fish Oil 02_POS_20110 SB: 15 2.30-2.3 [100.00-1000.00 NL: 2.48E5 DART-SVP 1-D Bounty Fish Oil 02_POS_20110 SB: 14 4.77-4.8 [100.00-1000.00

100 80 60 40 20 0 80 60 40 20 0 100 Relative Abundance 100 80 60 40 20 Nature s Bounty Omega-3 Fish Oil 123.04438 123.04434 165.05514 150 C 185.11148 255.23310 250 C 165.05508 255.23305 185.11136 350 C 227.20152 Negative Ion Mode 255.23303 281.24882 301.21771 301.21752 281.24881 301.21748 EPA 327.23324 165.97842 123.04434 227.20149 327.23326 Elemental composition search on mass 301.22 DHA 369.32514 369.32512 448.35675 448.35631 415.35826 0 100 150 200 250 300 350 400 450 m/z NL: 2.12E5 DART-SVP 1-D Bounty Fish Oil 01_NEG_20110 SB: 35 0.04-0.1 [100.00-1000.00 m/z= 296.22-306.22 m/z Theo. Mass Delta (ppm) RDB equiv. 301.21752 301.21730 0.72 6.5 C20H29O2 301.21596 5.18 7.0 C H O N Elemental composition search on mass 327.23 Composition NL: 6.33E5 DART-SVP 1-D Bounty Fish Oil 01_NEG_20110 Composition SB: 14 1.72-1.7 [100.00-1000.00 m/z= 322.23-332.23 m/z Theo. Mass Delta (ppm) RDB equiv. 327.23324 327.23295 0.88 7.5 C22H31O2 327.23480-4.76 0.0 C H O N NL: 4.91E5 DART-SVP 1-D Bounty Fish Oil 01_NEG_20110 SB: 15 3.63-3.6 [100.00-1000.00

100 80 60 40 20 0 80 60 40 20 0 100 Relative Abundance 100 80 Vitamin Shoppe Omega 3 Fish Oil 195.13765 285.22066 285.22076 331.26246 331.26237 331.26227 357.27781 430.37923 357.27790 430.37907 Positive Ion Mode Elemental composition search on mass 430.38 m/z= 425.38-435.38 m/z Theo. Mass Delta (ppm) RDB equiv. Composition 430.37923 430.38053-3.03 5.0 C29H50O2 430.36528 32.42 1.0 C H O Vitamin E as alpha-tocopherol 681.54365 No Diglycerides 250 C 350 C 450 C No Triglycerides 60 40 357.27769 379.31942 20 285.22053 0 100 200 300 400 500 600 700 800 900 1000 m/z NL: 4.73E6 DART-SVP 1-D Shoppe Fish Oil 01_POS_20110 SB: 49 0.15-0.3 [100.00-1000.00 NL: 6.11E6 DART-SVP 1-D Shoppe Fish Oil 01_POS_20110 SB: 13 2.13-2.1 [100.00-1000.00 NL: 7.27E6 DART-SVP 1-D Shoppe Fish Oil 01_POS_20110 SB: 15 4.51-4.5 [100.00-1000.00

100 80 60 40 20 Vitamin Shoppe Omega 3 Fish Oil 123.04438 150 C Negative Ion Mode 301.21773 EPA 189.12808 261.14988 333.20759 138.04009 235.13397 393.22902 426.24982 458.23926 NL: 1.25E5 DART-SVP 1-D Shoppe Fish Oil 01_NEG_20110 SB: 36 0.21-0.3 [100.00-1000.00 0 80 60 40 20 0 100 Relative Abundance 100 80 60 149.09650 250 C 281.24884 301.21748 327.23340 189.12814 253.21746 360.25224 429.37387 350 C 281.24883 301.21743 DHA Elemental composition search on mass 301.22 40 327.23333 20 429.37388 253.21747 360.25230 189.12813 408.25237 149.09652 462.37147 0 100 150 200 250 300 350 400 450 m/z NL: 2.03E6 DART-SVP 1-D Shoppe Fish Oil 01_NEG_20110 Composition SB: 13 1.91-1.9 [100.00-1000.00 m/z= 296.22-306.22 m/z Theo. Mass Delta (ppm) RDB equiv. 301.21748 301.21730 0.59 6.5 C20H29O2 301.20205 51.23 2.5 C H O Elemental composition search on mass 327.23 NL: 2.32E6 DART-SVP 1-D Shoppe Fish Oil Composition 01_NEG_20110 SB: 14 3.78-3.8 [100.00-1000.00 m/z= 322.23-332.23 m/z Theo. Mass Delta (ppm) RDB equiv. 327.23340 327.23295 1.36 7.5 C22H31O2 327.21770 47.99 3.5 C H O

Routine analysis of powders The tablet was sliced open and using a standard glass capillary tube a small amount of material was scarped from the inside of the tablet, transferred onto the mesh and directly screened for the FDA regulated API sildenafil.

Mexican sildenafil Tablet Scraping Relative Abundance 100 80 60 40 20 0 100 80 60 40 475.21220 475.21122 476.21497 477.20881 [M+H] + DART Spectrum [M+H] + Elemental Composition Simulation NL: 1.70E4 C 22 H 31 O 4 N 6 S: C 22 H 31 O 4 N 6 S 1 p (gss, s /p:40) Chrg 1 R: 50000 Res.Pwr. @FWHM 478.21363 479.21464 NL: 3.04E3 IDCUBE_Mexican Viagra_High_Tablet Elemental composition search Scrape_POS_1_20110822#106 on mass 475.21 RT: 0.99 AV: 1 SB: 16 0.75-0.83, m/z= 470.21-480.21 1.26-1.29 T: FTMS + p NSI Full ms m/z Theo. Mass Delta [100.00-1000.00] RDB Composition (ppm) equiv. 475.21122 475.21220-2.06 10.5 C22H31O4N6S 20 476.21513 477.21716 0 474 475 476 477 478 479 480 m/z

Pai You Guo Herbal Slim Capsules 100 80 60 40 20 0 80 60 40 20 0 100 Relative Abundance 100 80 60 40 20 0 Clearly contains Sibutramine 280.18265 280.18204 280.18191 [M+H] + Elemental Comp. Simulation 281.18593 281.18540 281.18525 282.17976 282.17913 282.17887 283.18313 283.18237 283.18213 284.18633 [M+H] + Capsule Powder Direct DART Spectrum [M+H] + Capsule Contents in ACN DART Spectrum 279 280 281 282 283 284 285 286 m/z NL: 7.35E4 C 17 H 27 N Cl: C 17 H 27 N 1 Cl 1 p (gss, s /p:8) Chrg 1 R: 50000 Res.Pwr. @FWHM NL: 1.68E5 ID-Cube_Pai You Elemental composition Guo_POWDER_POS_High_20110821#45 search on mass 280.18-51 RT: 0.46-0.53 AV: 7 SB: 5 0.01-0.05 m/z= 275.18-285.18 T: FTMS + p NSI Full ms m/z Theo. Mass Delta RDB Composition [100.00-1000.00] (ppm) equiv. 280.18204 280.18265-2.19 4.5 C 17H 27NCl 280.18401-7.04 1.5 C H O N NL: 1.72E6 ID-Cube_Pan You Guo_20 Elemental composition ug-ml_wet_pos_low_3_5 search on mass 280.18 Capsules_20110821#232-247 RT: m/z= 275.18-285.18 2.18-2.32 AV: 16 SB: 39 0.10-0.46 T: m/z Theo. Mass Delta RDB Composition FTMS + (ppm) p NSI Full equiv. ms [100.00-1000.00] 280.18191 280.18265-2.66 4.5 C 17H 27NCl 280.17997 6.91 0.0 C H O Cl

HCD Experiment -Pai You Guo Capsule Major Fragment Ion T: FTMS + p NSI Full ms2 1000.00@hcd20.00 [100.00-1000.00] 125.01518 100 HCD Gas on, 20 ev 90 80 Relative Abundance 70 60 50 40 30 20 111.11687 Minor Fragment Ion 139.03071 149.02315 Sibutramine [M+H] + 280.18225 10 179.06198 165.04634 199.16905 0 100 120 140 160 180 200 220 240 260 280 m/z

This image cannot currently be displayed. This image cannot currently be displayed. DART-SVP Ion Source Automated Sample Prep Simultaneous deposition of up to 96- liquid samples either from individual wells or rows of like samples

Mass Chromatograms for Standard Curve Development 96 samples in 20 minutes 10 X 200ppb to 50 ppm concentration

Post Run inspection of sample array

Mass Chromatograms 5ppm Standard Position 1 and 12 Blanks, Position 2-11 Sample Blanks in purple boxes

Standard Curve 200 o C, 2 mm/sec, 2 sec ionization (n = 10) 1.6E+06 Dimethoate Standard 1.4E+06 Ion Current 1.2E+06 1.0E+06 8.0E+05 6.0E+05 y = 142865x -14264 R² = 0.9943 4.0E+05 2.0E+05 0.0E+00 0 2 4 6 8 10 12 Parts per million

Standard Curve -Acephate 200 o C, 2 mm/sec, 2 sec ionization (n = 10)

Standard Curve -Malathion 200 o C, 2 mm/sec, 2 sec ionization (n = 10) Shows poor correlation potentially due to large dynamic range in ion trap instrument

Cranberry/Apple juice spiked with three pesticides at two concentrations Mass Chromatograms for Protonated Pesticides indicate detection of 500 ppb Acephateand Dimethoatedirect from juice without prep Malathionnot detected at that level All detected at 5ppm CranAppleJuice Spiked with 5ppm Acephate

CranAppleJuice Spiked with 5ppm Acephate

CranAppleJuice Spiked with 5ppm Acephate

CranAppleJuice Spiked with 5ppm Acephate

Analysis of Various Juices Replicate analysis of Juices for three pesticides96 samples in Four commercial samples of juice analyzed for three pesticides Left to right in clusters of 12 Cranberry/Apple Grape Lemon Tomato Orange Low ppb or no pesticides detected in commercial product Cran/App Grape Lemon Tomato Orange

Analysis of Juices Inspection of dimethoate detection Expanded view of small mass range shows many sample related ions * Peak at mass corresponding to protonated dimethoate labelled ( * ) However no peak detected at C13 isotope mass ( ** ) therefore dimethoatenot detected **

Summary DART is gaining as a tool for non-specific screening of solid and liquid foodstuffs Pre-screening allows investigators to sample a larger number of samples increasing the probability of detection of adulterants and pesticides The ability to complete quantitative as well as qualitative analyses is enabled by better automation including use of our high throughput X-Z Transmission Module New technologies are evolving as customers make request for integration of automation with off-line sampling technologies such as liquid sample handlers

Screening for Carbendazim DART-SVP API 4000 QTRAP IonSense, Inc. 999 Broadway Suite 404 Saugus, MA 01906 USA March 25, 2012 Prepared By: E. Crawford

Carbendazim Unapproved Fungicide for Citrus Fruits in US 1. Objectives: Generate standard curvedata for carbendazimin orange juice ranging from 1 ppb 5000 ppb. Screen for carbendazimin a variety of orange juicesfrom the EU (2 countries) and the USA. European maximum residue limit * (MRL) is set at 200 ppbfor oranges and carbendazimis notapproved for use on citrus fruits in the US. The US FDA has set an action limitof 10 ppb. 2. Analysis Overview: No sample preparation liquid from consumer juice beverages directly analyzedby transmission mode direct analysis in real time (DART) source coupled with triple quad. Positive ion mode; carbendazim transition m/z 192.2 160.2. * http://eur-lex.europa.eu/lexuriserv/lexuriserv.do?uri=oj:l:2011:152:0001:0021:en:pdf 34

Source parameters for DART-SVP DART-SVP Parameters 1. Heater: 250 C 2. He Flow: 2.5 L/min 3. Rail Speed: Constant 0.5 mm/s 35

DART-SVP Transmission Module Prep Workflow Stainless steel mesh screens sample surface allows liquid samples to be suspended on it 3 µl aliquot pipetted onto stainless steel mesh screen and allowed to completely dry before DART analysis 36

4.00E+05 3.50E+05 Carbendazim Standard Curve in Orange Juice 5.00E+04 4.00E+04 y = 65.454x R² = 0.9988 Average Peak Area (counts) 3.00E+05 2.50E+05 2.00E+05 1.50E+05 1.00E+05 3.00E+04 2.00E+04 1.00E+04 0.00E+00 0 100 200 300 400 500 5.00E+04 0.00E+00 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 Concentration (ppb) n= 3 per conc. 37

25 Estimated Carbendazim Levels: Screening Juices 20 Measured Level ppb 15 10 5 0 ND ND ND ND ND USA Apple USA Fruit Punch USA OJ 1 USA OJ 2 USA OJ 3 France OJ 1 Germany OJ 1 Fruit Juice Consumer Beverage Germany OJ 2 ND = Not Detected n= 8; except n = 16 for German OJs 38

Summary DART-SVP ion source coupled to the API 4000 QTRAP yielded LOD of 2 ppb and LOQ of 15 ppb. DART-SVP contact closure capability allows the user to acquire data directly through an Analyst sample queue. The standard curve generated for cardbendazim using the transmission DART method was very good with R 2 value 0.9988 across 10 5000 ppb. The reproducibility for an n = 3 across the concentration ranges was generally less than 18 % based on peak areas without internal standard correction. The two orange juices from Germany from the same manufacturer, but different lots yielded estimated carbendazim levels of 20 ppb, 10x below the Europe MRL, where the French orange juice contained only trace amounts. 39

Pesticide Screening US FDA utilizing Swabs Timothy Ng, Elizabeth Crawford, Brian Musselman IonSense, Inc. 999 Broadway Suite 404 Saugus, MA 01906 USA

Overview Thousands of samples imported daily Lessthan 0.2 percent inspected Official methods require extensive workup even with QuEChERs Hours of sample preparation 40 minute LC/MS Limit 8 samples per day per LC/MS New technology to protect Chinese customers and export industries

Method Development Thermo Exactive Settings DART- SVP Parameters 12-DIP-it Temp Profile to run 150, 250, 350 C Sample positioning in module: XX O XX O XX X = sample swab and O = blank swab wetted with solvent mixture Starting Temp: 150 C Temp increment: 100 C Sample speed: 0.5 mm/s Scan Parameters: Positive Ion Mode & Negative Ion Mode Resolution: Enhanced - 25,000 @ 4 Hz Fragmentation: None, HCD Gas Off Scan Settings: 1 µ-scan by 250 ms max inject time AGC Target: Ultimate Mass Accuracy(5e 5 ) Exactive Inlet Parameters: Capillary Temp: 200 C Capillary Voltage: 25 V Tube Lens Voltage: 120 V Skimmer Voltage: 26 V

Data

Temperature Profile of Swab - Major Ions Few Changes m/z

Temperature Profile of Swab Many Different Ions Detected at each temp

Mass Chromatogram for Strawberry Sample

At ultrahigh resolution hundreds of ions are detected. ToxIDprogram searches for accurate mass matches using a pesticide database. Many match mass values Mass Chromatograms used to determine relative abundance of different compound and their optimum temperature for desorption

Comparison of Theoretical isotope pattern (top) vs experimental result Lime Imazalil 350 C -1.615 ppm Relative Abundance 100 80 60 40 20 0 100 80 60 297.05560 297.05512 298.05869 299.05284 299.05212 300.05582 Theoretical Stable Isotope Pattern 301.05042 Experimental Result 302.05308 NL: 1.15E4 C 14 H 14 Cl 2 N 2 O +H: C 14 H 15 Cl 2 N 2 O 1 p (gss, s /p:40) Chrg 1 R: 25000 Res.Pwr. @FWHM EPA Tolerance level 10 ppm NL: 4.99E6 Lime_MiniPolyesterSwab_150_25 0_350C POS_25K_20120223#6 56 RT: 2.22 AV: 1 SB: 1 2.84 T: FTMS + p NSI Full ms [100.00-1000.00] 40 20 0 298.05871 300.05550 301.04898 297.25714 298.27343 299.28274 300.28913 302.05242 297 298 299 300 301 302 m/z

Comparison of Theoretical isotope pattern (top) vs experimental result Relative Abundance 100 80 60 40 20 0 100 80 60 192.07675 192.07655 Lime Carbendazim 350 C -1.093 ppm 193.07951 Theoretical Stable Isotope Pattern 194.08169 NL: 2.09E4 C 9 H 9 N 3 O 2 +H: C 9 H 10 N 3 O 2 p (gss, s /p:40) Chrg 1 R: 25000 Res.Pwr. @FWHM NL: 3.30E5 Lime_MiniPolyesterSwab_150_250_35 0C POS_25K_20120223#1280-1297 RT: 4.33-4.39 AV: 18 SB: 119 3.42-3.82 T: FTMS + p NSI Full ms [100.00-1000.00] 40 20 0 193.04942 194.05284 192.0 192.5 193.0 193.5 194.0 m/z

Relative Abundance Lime Dodemorph 350 C 100 80 60 40 20 282.27914-0.354 ppm First and second isotope mass values match however third isotope is off by 37ppm and therefore 283.28244 not correct m/z or abundance NL: 1.91E4 C 18 H 35 NO +H: C 18 H 36 N 1 O 1 p (gss, s /p:40) Chrg 1 R: 25000 Res.Pwr. @FWHM 0 100 80 60 40 282.27904 283.26365 284.28552 285.28843 Not detected NL: 3.84E4 Lime_MiniPolyesterSwab_150_25 0_350C POS_25K_20120223#6 56 RT: 2.22 AV: 1 SB: 1 2.76 T: FTMS + p NSI Full ms [100.00-1000.00] 20 284.29622 285.22237 0 282.5 283.0 283.5 284.0 284.5 285.0 285.5 m/z

Confirm by matching Stable Isotope Patterns Two legal fungicides detected while other compounds not confirmed Pesticide screen completed in minutes