Technology Transition Workshop DART Workshop – Ames, IA August 2008 Use of the AccuTOF-DART system for the forensic analysis of drugs of abuse Bob Steiner Virginia Department of Forensic Science Central Laboratory Richmond, VA 804-786-4707 x22347 robert.steiner@dfs.virginia.gov Technology Transition Workshop GLOSSARY OF TERMS: AccuTOF – Accurate mass Time Of Flight; mass spectrometer DART – Direct Analysis in Real Time; atmospheric pressure ion source Orifice 1 – inlet to the AccuTOF; raising voltage causes fragmentation of ions via collision induced dissociation Profile Spectrum – multiple data points to describe the mass peaks of a spectrum; have “chromatogram” appearance Centroid – to find the center of a mass peak collected in profile mode; gives resulting histogram spectrum as a mass vs. abundance pair PEG600 – polyethylene glycol polymer with an average MW of 600 Da; used for internal mass calibration Internal mass calibration – PEG600 spectrum run within a data file; allows to accurately set the mass axis for all spectra in that file Technology Transition Workshop GLOSSARY OF TERMS: Function switching – method that allows changing the orifice 1 voltage rapidly during data collection; ori 1 voltage changes every 0.25 sec Protonated or deprontonated molecule – the addition or subtraction of a hydrogen from the neutral molecule, depending on polarity of DART Millimass unit (mmu) – accuracy of TOF spectra measured to thousandths of a Dalton; acceptable spectra are < 5 mmu from calculated mass JEOL-DX file – text file of data points in a mass spectrum; can be easily exported to other software for analysis Technology Transition Workshop GLOSSARY OF TERMS: SearchFromList – library searching program; searches by empirical formula and matching user-library spectra Drugs_Neutral_Masses – library of empirical formulae of drugs Drug Prep Library_ori20 – library spectra of pharmaceuticals at orifice 1 voltage of 20 V; also libraries at ori30, 60 and 90 V Drug Std Library_ori20 – library spectra of primary standards at orifice 1 voltage of 20 V; also libraries at ori30, 60 and 90 V Mirror spectrum – display of unknown (positive) and known (negative) spectra in SearchFromList program Technology Transition Workshop DART METHODOLOGY How do I use the AccuTOF-DART for drug samples? Types of samples Best sampling methods Function Switching Calibration considerations Technology Transition Workshop HEADSPACE SAMPLING 1. Start acquisition 2. Open container near DART gas stream 3. Remove container Can’t get simpler than that!!!!!! Technology Transition Workshop SAMPLING OF SOLIDS Hold solid material in DART gas stream using tweezers Drawbacks: 1. Hard to hold 2. MESSY 3. Non-homogeneous samples Technology Transition Workshop MORE SOLID SAMPLING DRAWBACKS Coated tablets! GAS STREAM IS HOT!!! 4-chloro-2,5-dimethoxyamphetamine Technology Transition Workshop “DRIED ON TUBE” METHOD Deposit sample onto tubes with syringe Can measure how much sample is deposited Turned out not so good! Technology Transition Workshop “DRIED ON TUBE” METHOD Loading cap tubes with sample Even 1 uL “runs down” tube Inconsistent results obtained Technology Transition Workshop Injecting sample into gas stream with syringe Draw up samples from ALS vials Can measure amount injected Turned out not so good! Technology Transition Workshop LIQUID SAMPLING Dissolve sample into suitable solvent Dip capillary tube in liquid/hold “wand” in DART gas stream Advantages: 1. EASY!!! 2. Can control concentration of sample 3. Homogeneous sample Preferred method at DFS!!! Technology Transition Workshop DART sampling by hand is an ART! Consistency from one person to the next is difficult to obtain!!! Buy an AutoDART??? Technology Transition Workshop FUNCTION SWITCHING In general, ionization of molecules with DART produces hydride addition or subtraction products of the molecular ion. Methyl stearate spectrum showing [M+H]+ ion at 299.2931 Da. No ‘diagnostic’ ions Technology Transition Workshop Collision-Induced Dissociation Definition*: “An ion/neutral species interaction wherein the projectile ion is dissociated as a result of interaction with a target species. This is brought about by conversion of part of the translation energy of the ion to internal energy in the ion during collision.” High vacuum prevents this in EI Basis behind triple stage quadrupole systems Commonly done in LCMS systems to promote fragmentation Performed in the AccuTOF by raising orifice 1 voltage *http://mass-spec.lsu.edu/msterms/index.php/Collision-Induced_Dissociation Technology Transition Workshop EFFECT OF RAISING ORIFICE 1 VOLTAGE Orifice 1 = 15V Orifice 1 = 30V Technology Transition Workshop EFFECT OF RAISING ORIFICE 1 VOLTAGE Orifice 1 = 45V Orifice 1 = 60V Technology Transition Workshop Function Switching allows collection of data at SEVERAL orifice 1 voltages AT ONCE! Technology Transition Workshop Noscapine Orifice 1 = 20 Orifice 1 = 30 Orifice 1 = 60 Orifice 1 = 90 Function Switching results showing fragmentation at various orifice 1 voltages Technology Transition Workshop RUNNING A SAMPLE ON THE AccuTOF-DART TIC[2]; / ESI+ / cal7908 3 x10 Lock and chk stds Intensity (715231) PEG 0.258 1.007 More PEG 1.157 2.554 Sample 0.674 500 2.221 2.438 1.839 0.425 1.656 1.972 0 0 0.5 1.0 1.5 Time[min] 2.0 2.5 Technology Transition Workshop Mass calibrations Calibration at acquisition – PEG+H Internal mass calibration – temp_PEG Set at tune (after cleaning) Within your data file “Global” mass calibration “Fine tunes” your calibration Technology Transition Workshop “Run PEG before AND/OR after samples within your datafile! Intensity matters!!” Peggy the Dimetrodon TIC[2]; / ESI+ / jhp_310a_311a 3 x10 PEG Intensity (600571) PEG 2.486 3.085 500 0.705 0.971 0.389 1.920 1.3871.637 0 0 1.0 2.0 Time[min] 3.0 3.317 Technology Transition Workshop GOOD PEG vs BAD PEG Technology Transition Workshop TIC[2]; / ESI+ / cal1308 3 x10 Intensity (809700) Where to average your peak for best PEG intensity? TIC 0.474 500 0.291 0.674 0.524 0.558 0 0.30 0.40 0.50 Time[min] 0.60 0.70 MS[2];0.442..0.493;-1.0*MS[2];0.361..0.415; / ESI+ / cal1308 profile Intensity (6120) 371.22813 5000 30V data 459.27946 503.30537 327.19959 195.12221 239.14916 283.17463 547.33420 89.05961 133.08629 0 100 200 300 400 500 415.25460 459.27969 600 m/z MS[2];0.442..0.493;-1.0*MS[2];0.361..0.415; / ESI+ / cal1308 3 x10 centroid Area (23621) 20 503.30555 327.19988 10 195.12257 239.14967 283.17499 547.33415 87.04568 133.08689 0 100 200 300 400 m/z 500 600 Technology Transition Workshop TIC[2]; / ESI+ / cal1308 3 x10 Intensity (809700) TIC 0.474 Less intensity! 500 0.291 0.674 0.524 0.558 0 0.30 0.40 0.50 Time[min] 0.60 0.70 MS[2];0.429..0.571;-1.0*MS[2];0.356..0.412; / ESI+ / cal1308 profile Intensity (3373) 415.25622 503.30599 547.33324 327.20019 2000 195.12242 239.14952 283.17523 89.05965 133.08647 0 100 200 300 400 500 600 m/z MS[2];0.429..0.571;-1.0*MS[2];0.356..0.412; / ESI+ / cal1308 3 x10 centroid Area (14357) 415.25632 503.30621 547.33319 10 327.20052 195.12281 239.15015 283.17553 87.04657 133.08721 0 100 200 300 400 m/z 500 600 Technology Transition Workshop Centroiding peaks = difficult with low intensity ions MS[2];3.532..3.699;-1.0*MS[2];3.367..3.512; / ESI+ / jhp_272a_274a Intensity (659) 133.09083 500 Interfering peak or noise 0 133.00 133.10 m/z 133.20 MS[2];3.532..3.699;-1.0*MS[2];3.367..3.512; / ESI+ / jhp_272a_274a Intensity (3161) “tailing” due to kinetic energy spread in TOF 415.25405 2000 0 415.20 415.40 m/z 415.60 Technology Transition Workshop LOCK MASS AND CHECK STANDARDS Mix of cocaine, methamphetamine and nefazodone MWs span the mass range Cocaine is used as drift compensation “lock” at 304.1549 Da Methamphetamine (150.1283 Da) and Nefazodone (470.2323 Da) cover the low and high ends of mass range Adds another level of calibration applied to sample data Technology Transition Workshop LOCK MASS AND CHECK STANDARDS Cocaine 304.1549 304.1549 100 Rel. Abundance 80 60 40 Meth 150.1283 150.1296 Nefazodone 470.2323 20 470.2362 0 120 160 200 240 280 m/z 320 360 400 440 480 Technology Transition Workshop SAMPLE DATA TIC[2]; / ESI+ / af070701 3 x10 Sample signal is averaged, centroided and calibrations are applied for EACH orifice voltage Intensity (296800) 200 2.270 2.919 2.537 2.853 0 2.0 2.5 Time[min] 3.0 Spectra are saved in “JEOL-DX” format MS[2];2.225..2.317;-1.0*MS[2];2.042..2.192; / ESI+ / af070701 3 x10 Area (15434) 324.20773 192.08031 10 325.21241 192.17013 87.10397 192.24602 0 100 150 223.09892 200 250 m/z 324.48445 340.22848 300 350 400 Technology Transition Workshop SearchFromList Program – by Dr. Robert “Chip” Cody Technology Transition Workshop Match spectra search Load data file Load empirical formula library Adjust these to suit your search Empirical formula search Add or subtract from empirical formula Technology Transition Workshop SearchFromList empirical formula search result Spectrum file name From empirical formula library Lot number of std used to create lib spectrum must be < 5 mmu Technology Transition Workshop SearchFromList empirical formula search result Search result label Spectrum imported from Mass Center Technology Transition Workshop Match spectra search Choose library to match orifice 1 voltage for the spectrum you are searching DOUBLE CLICK on library entry to load all spectra! Search! Technology Transition Workshop Match spectra search results Blue = original spectrum Click on entries to view comparison spectra Red = comparison spectrum Technology Transition Workshop Suggested Data for casefiles • Print out: TIC[2] from Chromatogram view • Print out spectrum of cocaine lock mass and check stds • Save spectra as averaged, background subtracted, centroided, calibrated JEOL-DX files • Print out: SearchFromList search result for 20V spectrum • Print out: Other spectra or search results at other voltages – enough to characterize the spectrum Technology Transition Workshop FUNCTION SWITCHING - ADVANTAGES FOUR spectra collected every second Higher orifice 1 voltages result in more fragmentation Can lead to greater confidence for identification Except for mixtures, spectra are reproducible Technology Transition Workshop FUNCTION SWITCHING - DISADVANTAGES Mixture spectra – no prior chromatography (can be good OR bad!!) Less sensitivity – splitting ionization between four functions Finite database for searching – library databases need to be developed! Technology Transition Workshop Application of the DART to DX Currently being used as a screening tool for DX casework. Validation took over one year to complete. Approved by DFS Scientific Advisory Board and full VA Forensic Science Board, May 6-8, 2008. Technology Transition Workshop AccuTOF-DART Validation at DFS 1. DART screening method - LLOD determined for 7 drugs; daily calibration data to show stability of instrument 2. DART sampling methods – why we use methanol and MP tube ‘wands’ 3. Comparison of DART to GCMS – new technology vs. established technology; 553 samples run on each – ALL match in highest Scheduled drug found; DART typically showed more cmpds! 4. Selectivity study – can you tell the difference in DART spectra of drugs with the SAME empirical formula?? 5. “Rugustness” – same result on different days with different people?? Technology Transition Workshop LLOD DETERMINATION and INSTRUMENT STABILITY Conc. Drug Name 0.5 mg/mL 7/13/2007 0.1 mg/mL 7/13/2007 Exact Mass measured mass difference Alprazolam 309.0907 309.0906 0.0001 Butalbital 225.1239 225.1240 0.0001 Cocaine 304.1549 304.1526 0.0023 Heroin 370.1654 370.1636 0.0018 Methamphetamine 150.1283 150.1268 0.0033 Testosterone Propionate 345.2430 345.2409 0.0021 Trazodone 372.1591 372.1553 0.0038 Alprazolam 309.0907 309.0888 0.0019 Butalbital 225.1239 225.1246 0.0007 Cocaine 304.1549 304.1545 0.0004 Heroin 370.1654 370.1670 0.0016 Methamphetamine 150.1283 150.1295 0.0012 Testosterone Propionate 345.2430 345.2427 0.0003 Trazodone 372.1591 372.1626 0.0035 Technology Transition Workshop COMPARISON OF DART RESULTS WITH ANALYTICAL SCHEME – 553 SAMPLES! DART result GCMS confirmation 1161 heroin, papaverine, 6-MAM, quetiapine 1161 heroin 1665 cocaine, diltiazem, naproxen 1665 cocaine 1910 MDMA, caffeine, procaine 1910 MDMA, caffeine, procaine, dimethylsulfone 1976 cocaine, caffeine, benzocaine 1976 cocaine, caffeine Technology Transition Workshop SELECTIVITY STUDY 119.0872 Ori1 30V Easily distinguish methamphetamine from phentermine! Technology Transition Workshop SELECTIVITY STUDY Cocaine and scopolamine ori1 30V Technology Transition Workshop SELECTIVITY STUDY Cocaine and scopolamine ori1 90V Technology Transition Workshop SELECTIVITY STUDY LSD and LAMPA ori1 90V Technology Transition Workshop GHB SCREENING “Detection of GHB in various drink matrices via AccuTOF-DART” Bennett MJ, Steiner RR. J. Forensic Sci., in press* Spiked 50 beverages with GHB at “impairment levels” to determine if DART could detect GHB easily seen in all drink matrices Gave better results than using GHB color test! Done in NEGATIVE ion mode * slated for publication Jan 2009 Technology Transition Workshop GHB SCREENING blank [GHB-H]- Ocean Spray Cranberry Juice - blank Ocean Spray Cranberry Juice – 2 mg/mL spike with GHB Technology Transition Workshop DART Spectral Interpretation Technology Transition Workshop Molecular Weight Information Electron Impact Ionization DART Ionization • M+. [M+H]+ or [M-H]- •Unstable – leads to extensive fragmentation Very stable •Usually the peak @ highest mass, excluding isotope peaks •Look for “logical neutral losses” • Sometimes hard to find! Very little fragmentation (without help!) Proper terms: Protonated or deprotonated molecule Loss of stable neutrals Use SearchFromList to find Technology Transition Workshop Abundance # 3 7 2 : V E R A P A M I L --D I P 3 0 3 9500 9000 DART Spectral Characteristics 8500 8000 7500 EI - DIP 7000 6500 6000 5500 5000 verapamil – MW 454.61 Da 4500 4000 3500 3000 2500 2000 1500 1 5 1 5 8 1000 2 6 0 500 8 4 1 0 7 1 3 0 1 7 7 2 1 8 4 5 3 0 40 60 80 100120140160180200220240260280300320340360380400420440 m/z--> DART Technology Transition Workshop DART Spectral Characteristics Oxytetracycline MW 460.44 Da Stable neutral loss of H2O Technology Transition Workshop All the usual isotope clusters!! DART Spectral Characteristics Technology Transition Workshop Methamphetamine DART Spectral Characteristics CH2 CH3 CH H + N H CH3 - NH2CH3 Technology Transition Workshop DART Spectral Characteristics CH3 Phentermine CH2 CH3 C + NH2 H - NH3 Technology Transition Workshop DART Spectral Characteristics CH3 Heroin H N + CH3 CH2 CH _ CH2 OH + CH2 C C O O CH2 CH3 CH3 C O O _ CH3 O O OH C C O O NOT! Technology Transition Workshop DIMERS and Toto, too!! TRIMERS and ADDUCTS!!! OH MY!!!!!!!!! Technology Transition Workshop Dimers – hydrocodone/APAP (Lortab) Ori1 = 20V [M+H]+ Ori1 = 20V dimer Technology Transition Workshop C O CH 3 Acetaminophen dimer NH + O H H 19.1% O OH C NH CH3 Technology Transition Workshop Dimers – hydrocodone/APAP (Lortab) Ori1 = 30V Ori1 = 60V Dimer disappears! Technology Transition Workshop Elemental Composition Calculation 1/3/2008 10:08:11 AM Element Limits: C 0/40 H 0/50 N 0/10 O 0/10 Tolerance: 5.00 mmu Even or odd electron ion or both: BOTH Minimum unsaturation: -0.5 Maximum unsaturation: 100.0 Meas. mass u 451.227203 C18H21NO3 + C8 H9 N O2 Abund. Diff. % mmu 0.00 3.91 Hyd or Cod APAP [C26H30N2O5 + H] adduct!! Unsat. Compositions 12.5 C26 H31 N2 O5 WHAT’S THIS? Technology Transition Workshop Salsalate (disalicylic acid) – analgesic, anti-inflammatory OH 121 O MW = 258.0528 Da C O C O OH 137 In MEOH, ori1=20V Technology Transition Workshop Salsalate – ammonium adduct NH4OH on swab in DART sampling area [M+NH4–H2O]+ = 258.0766 Da [M+H]+ MW of Salsalate = 258.0528 Da Emp Form: C14H10O5 [salicylic acid+NH4–H2O]+ Charge carrier +NH4 in SFL Technology Transition Workshop Salsalate – ammonium adduct 276.086517 16.63 -2.52 2.01 2.00 0.66 -0.67 -0.67 1.0 9.5 4.0 9.0 14.0 8.5 C1 H12 N10 O7 C10 H10 N7 O3 C11 H16 N0 O8 C12 H12 N4 O4 C13 H8 N8 O0 C14 H14 N1 O5 [C14H10O5 + NH4]+ Technology Transition Workshop Salsalate – ammonium adduct Elemental composition Element Limits: C 0/40 H 0/50 N 0/10 O 0/10 Tolerance: 5.00 mmu Even or odd electron ion or both: BOTH Minimum unsaturation: -0.5 Maximum unsaturation: 100.0 Meas. mass u 138.053833 Abund. % 15.72 Diff. mmu 1.01 -0.33 -1.67 Unsat. Compositions 0.0 5.0 4.5 C4 H10 N0 O5 C5 H6 N4 O1 C7 H8 N1 O2 [C7H6O3 + NH4 – H2O]+ 258.072021 15.47 -1.93 -3.27 -4.61 3.94 5.0 10.0 9.5 14.0 C11 H14 N0 O7 C12 H10 N4 O3 NOT M+. but C14 H12 N1 O4 C18 H10 N0 O2 [C14H10O5+NH4–H2O]+ 259.063446 12.03 2.81 2.80 1.45 0.13 15.0 9.5 14.5 14.0 C13 H5 N7 O0 C14 H11 N0 O5 C15 H7 N4 O1 C17 H9 N1 O2 [C14H10O5+H]+ Technology Transition Workshop Salsalate – ammonium adduct OH [3(C7H4O2)+ NH4]+ O C O C O “Salicylate” dimer [2(C7H4O2) + H]+ “Salicylate” [C7H4O2 + H]+ OH “Salicylate” trimer [3(C7H4O2) + H]+ [3(C7H4O2) + H2O + NH4]+ Technology Transition Workshop Salsalate – ammonium adduct Elemental composition Element Limits: C 0/40 H 0/50 N 0/10 O 0/10 Tolerance: 5.00 mmu Even or odd electron ion or both: BOTH Minimum unsaturation: -0.5 Maximum unsaturation: 100.0 Meas. mass u 361.068268 Abund. % 7.36 Diff. mmu -2.94 -2.94 2.92 Unsat. Compositions 21.0 15.5 24.5 C20 H7 N7 O1 C21 H13 N0 O6 C28 H9 N0 O1 378.095795 15.06 -0.61 -1.96 -1.99 -4.64 3.89 15.0 20.0 14.5 19.0 23.5 C19 H14 N4 O5 C20 H10 N8 O1 C21 H16 N1 O6 [3(C7H4O2 + NH4]+ C24 H14 N2 O3 C28 H12 N1 O1 396.108185 7.08 -0.14 -0.14 -1.50 -2.82 -4.15 19.0 13.5 18.5 18.0 23.0 C20 H12 N8 O2 C21 H18 N1 O7 [3(C7H4O2)+H2O+NH4]+ C22 H14 N5 O3 C24 H16 N2 O4 C25 H12 N6 O0 “Salicylate” trimer [3(C7H4O2) + H]+ Technology Transition Workshop CH2 Cl N N CH2 O CH2 CH2 OH What is this??? CH Hydroxyzine (Vistaril) [M+H]+: 375.1839 Da In MEOH, ori1 20V Chlorinated! Technology Transition Workshop Elemental Composition Calculation 12/12/2007 11:25:13 AM Element Limits: C 0/40 H 0/50 N 0/10 O 0/10 Cl 1/1 Tolerance: 5.00 mmu Even or odd electron ion or both: BOTH Minimum unsaturation: -0.5 Maximum unsaturation: 100.0 Meas. mass u 537.233582 Abund. Diff. % mmu 5.76 3.53 2.19 0.84 -0.50 -0.50 -1.84 -3.15 -3.19 -4.50 4.05 2.70 -1.32 Elemental composition NOW WHAT????? Unsat. Compositions 6.5 6.0 5.5 10.5 5.0 10.0 15.0 9.5 14.5 19.0 18.5 22.5 C18 H34 N10 O7 Cl1 C20 H36 N7 O8 Cl1 C22 H38 N4 O9 Cl1 C23 H34 N8 O5 Cl1 C24 H40 N1 O10 Cl1 C25 H36 N5 O6 Cl1 C26 H32 N9 O2 Cl1 C27 H38 N2 O7 Cl1 C28 H34 N6 O3 Cl1 C32 H32 N5 O1 Cl1 C34 H34 N2 O2 Cl1 C39 H34 N0 O0 Cl1 Technology Transition Workshop CH2 Cl N N CH2 O CH2 CH2 OH CH Hydroxyzine empirical formula C21H27N2O2Cl Look at composition list and see if any are “terribly unreasonable” Technology Transition Workshop Elemental Composition Calculation 12/12/2007 11:25:13 AM Element Limits: C 0/40 H 0/50 N 0/10 O 0/10 Cl 1/1 Tolerance: 5.00 mmu Even or odd electron ion or both: BOTH Minimum unsaturation: -0.5 Maximum unsaturation: 100.0 Meas. mass u 537.233582 Hydroxyzine empirical formula C21H27N2O2Cl Apply Chemistry logic!! Abund. Diff. % mmu 5.76 3.53 2.19 0.84 -0.50 -0.50 -1.84 -3.15 -3.19 -4.50 4.05 2.70 -1.32 Unsat. Compositions 6.5 6.0 5.5 10.5 5.0 10.0 15.0 9.5 14.5 19.0 18.5 22.5 C18 H34 N10 O7 Cl1 C20 H36 N7 O8 Cl1 C22 H38 N4 O9 Cl1 C23 H34 N8 O5 Cl1 C24 H40 N1 O10 Cl1 C25 H36 N5 O6 Cl1 C26 H32 N9 O2 Cl1 C27 H38 N2 O7 Cl1 C28 H34 N6 O3 Cl1 C32 H32 N5 O1 Cl1 C34 H34 N2 O2 Cl1 C39 H34 N0 O0 Cl1 Not enough C! Too many N or O Not enough N or O or too many C Technology Transition Workshop Elemental Composition Calculation 12/12/2007 11:25:13 AM Element Limits: C 0/40 H 0/50 N 0/10 O 0/10 Cl 1/1 Tolerance: 5.00 mmu Even or odd electron ion or both: BOTH Minimum unsaturation: -0.5 Maximum unsaturation: 100.0 Meas. mass u 537.233582 From 2003 PDR: Hydroxyzine empirical formula C21H27N2O2Cl OK. NOW WHAT?? Abund. Diff. % mmu 5.76 -3.19 -4.50 4.05 Unsat. Compositions 9.5 14.5 19.0 C27 H38 N2 O7 Cl1 C28 H34 N6 O3 Cl1 Too many nitrogens! C32 H32 N5 O1 Cl1 Vistaril (hydroxyzine pamoate) Sucrose is C12H22O11 Inert ingredients: Magnesium stearate Sodium lauryl sulfate Starch Sucrose LOOK HERE!! Technology Transition Workshop Sucrose peaks! Sucrose – ori1=20V 289.0942 Technology Transition Workshop Hydroxyzine empirical formula: C21H27N2O2Cl Target compound: C27H38N2O7Cl BEST GUESS: C21H27N2O2Cl + C6H12O6 = C27H39N2O8Cl (not quite!) Hydrolyzed sucrose But, if subtract H2O: C27H37N2O7Cl (Still off by one!) Ionized to: [C27H37N2O7Cl + H]+ = 537.2367 Da (calculated) Technology Transition Workshop MIXTURES Magically disappearing ions! Codeine std ori 1 30V Codeine:APAP 20:1 Technology Transition Workshop MIXTURES Codeine:APAP 10:1 Codeine:APAP 5:1 Technology Transition Workshop MIXTURES Codeine:APAP 2:1 Codeine:APAP 1:1 Technology Transition Workshop “Today is done!”