A Generic LC-MS Method for the Analysis of Multiple of Drug of Abuse Classes with the Thermo Scientific Exactive TM System Kent Johnson Fortes lab, Wilsonville Oregon Forensic Toxicology Use Only List of drug of abuse candidates for LC-MS analysis Sample matrix: urine and blood Benzodiazepines Opiates Other drugs group 1 Other drugs group 2 7-Aminonitrazepam Morphine Ketamine Methylphenidate 7-Aminoclonazepam Hydromorphone Norketamine Ritalinic Acid 7-Aminoflunitrazepam Oxymorphone Butorphanol Dextromethorphan 2-Hydroxy-ethyl-flurazepam Codeine Fentanyl Dextrophan Desalkylflurazepam Dihydrocodeine Norfentanyl Propoxyphene Diazepam Hydrocodone Nalbuphine Norpropoxyphene Hydroxy-alprazolam Oxycodone Alfentanil 6-MAM Hydroxy-triazolam Meperidine Sulfentanil Nordiazepam Normeperidine Zolpidem Lorazepam Trazodone Oxazepam Venlafaxine Temazepam Zopiclone Forensic Toxicology Use Only 2 Methods Employed Prior to LC-MS • Benzodiazepines GC-MS • Opiates GC-MS • Other drugs of abuse group 1 ELISA • Other drugs of abuse group 2 not analyzed before Forensic Toxicology Use Only 3 Why switch to LC-MS method? Benefits of replacing GC-MS • Faster less need for chromatographic separation • Less sample prep no derivatization • No thermal instability benzodiazepines analysis • No volatility limitations Benefits of replacing immunoassay • Lower consumables cost • More specific More cost efficient and analytically more universal Forensic Toxicology Use Only 4 Goal • Develop fast, easy to use, generic LC-MS method to analyze multiple classes of drugs of abuse in urine • Method has to meet industry standards for • Precision • Accuracy • Limit of quantitation • Robustness • With simplest sample prep procedure Opiates Benzodiazepines 6-MAM Other drugs of Other drugs of abuse abuse group1 group 2 10 ng/mL 10 ng/mL 3 ng/mL 2-50 ng/mL 10 ng/mL Forensic Toxicology Use Only 5 Exactive uHRAM LC-MS system • Resolution •100,000 at 1 scan per sec •10,000 at 10 scans per sec •Mass accuracy •Sub ppm • Scan speed •Up to 10 scans per second • •Mass range •m/z 50 - 4000 • Polarity switching Forensic Toxicology Use Only 6 Power of mass resolution – Ethinyl Estradiol Resolution: 10k , 30k , 50k , 100k Butyl-Phthalate, 279.15909 (ubiquitous background ion) 100 90 80 70 60 Ethinyl-Estradiol, 279.17434 50 40 Relative Abundance Relative 30 20 10 0 279.12 279.14 m/z 279.16 279.18 279.20 Forensic Toxicology Use Only 7 Exactive LC/MS setup Forensic Toxicology Use Only 8 Exactive LC/MS setup Forensic Toxicology Use Only 9 Exactive quantitative analysis workflow Sample Preparation SPE or urine dilution LC-MS method PFP column, HESI source, Mass resolution 100,000 Data processing extract chromatograms with 2 ppm mass accuracy Custom reports Forensic Toxicology Use Only 10 Urine sample preparation Benzodiazepines, opiates and Other drugs of abuse Other drugs of abuse group 2 group 1 Urine dilution SPE Int. std addition 20 X dilution with 25-50% MeOH Int. std addition Enzymatic Hydrolysis SPE Extraction Evaporation Reconstitution Inject Samples Forensic Toxicology Use Only 11 Blood sample preparation Other drugs of abuse Benzodiazepines, opiates group 1 other drugs of abuse group 2 Ritalinic Acid SPE Precipitation/dilution Int. std addition Int. std addition SPE Extraction ACN addition in ratio 1:1 Evaporation Centrifugation Reconstitution 30 X dilution Inject Sample Forensic Toxicology Use Only 12 LC method • 150 x 2.1 mm, 5 um PFP column • Mobile phase • A: 20 mm NH4Ac, 0.1% FA in DIW • B: 0.1% FA in ACN • 7 min gradient Forensic Toxicology Use Only 13 MS method • HESI source • Full scan data in compounds specific m/z range • Resolution 100K • Extract chromatograms using 2 ppm mass window Forensic Toxicology Use Only 14 Opiates and Opioids in Urine Hydrocodone Codeine Oxymorphone D3 Morphine Hydromorphone Oxycodone D6 Normeperidine Dihydrocodeine D6 Oxycodone Meperidine D6 Oxymorphine Hydrocodone D6 Codeine D6 dihydrocodeine Meperidine Morphine D6 Hydromorphone D6 Forensic Toxicology Use Only 15 Opiates and Opioids in Urine - dynamic range Linearity range better than 10 – 10,000 ng/mL Hydrocodone Y = 0.0744648+0.000852152*X R^2 = 0.9401 W: 1/X^2 11.0 10.5 Codeine Y = 0.0702431+0.000851922*X R^2 = 0.9461 W: 1/X^2 10.0 9.5 10.5 9.0 10.0 9.5 8.5 Hydrocodone 9.0 Codeine 8.0 8.5 7.5 8.0 7.0 7.5 6.5 7.0 6.0 6.5 5.5 6.0 AreaRatio 5.0 5.5 4.5 Area Ratio 5.0 4.0 4.5 3.5 4.0 3.0 3.5 2.5 3.0 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 2000 4000 6000 8000 10000 12000 0 2000 4000 6000 8000 10000 ng/mL ng/mL Meperidine Morphine Y = 0.075201+0.000849812*X R^2 = 0.9540 W: 1/X^2 Y = 0.054877+0.000912426*X R^2 = 0.9288 W: 1/X^2 10.5 11.5 10.0 11.0 9.5 10.5 Morphine 10.0 9.0 Meperidine 9.5 8.5 9.0 8.0 8.5 7.5 8.0 7.0 7.5 6.5 7.0 6.0 6.5 5.5 6.0 5.5 5.0 Area Ratio AreaRatio 5.0 4.5 4.5 4.0 4.0 3.5 3.5 3.0 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 2000 4000 6000 8000 10000 0 2000 4000 6000 8000 10000 ng/mL ng/mL Forensic Toxicology Use Only 16 Opiates and Opioids in Urine- dynamic range Linearity range 10 – 10,000 ng/mLOxymorphone Y = 0.0572347+0.000794734*X R^2 = 0.9411 W: 1/X^2 Normeperidine Y = 0.0229096+0.00023572*X R^2 = 0.9434 W: 1/X^2 10.0 3.0 9.5 9.0 2.8 Normeperidine 8.5 Oxymorphone 2.6 8.0 2.4 7.5 2.2 7.0 6.5 2.0 6.0 1.8 5.5 1.6 5.0 Area Ratio Area 4.5 1.4 Area Ratio Area 4.0 1.2 3.5 1.0 3.0 0.8 2.5 2.0 0.6 1.5 0.4 1.0 0.2 0.5 0.0 0.0 0 2000 4000 6000 8000 10000 0 2000 4000 6000 8000 10000 ng/mL ng/mL Oxymorphone Y = 0.0572347+0.000794734*X R^2 = 0.9411 W: 1/X^2 Oxycodone Y = 0.0712176+0.000849364*X R^2 = 0.9510 W: 1/X^2 10.0 11.0 9.5 10.5 Dihydrocodeine Oxycodone 9.0 10.0 8.5 9.5 8.0 9.0 7.5 8.5 8.0 7.0 7.5 6.5 7.0 6.0 6.5 5.5 6.0 5.0 5.5 Area Ratio Area 4.5 Area Ratio 5.0 4.0 4.5 3.5 4.0 3.0 3.5 3.0 2.5 2.5 2.0 2.0 1.5 1.5 1.0 1.0 0.5 0.5 0.0 0.0 0 2000 4000 6000 8000 10000 0 2000 4000 6000 8000 10000 ng/mL ng/mL Forensic Toxicology Use Only 17 GC-MS correlation Exactive LC-MS GC-MS No Analyte (ng/mL) (ng/mL) 1 Morphine 289 343 2 Oxymorphone 117 219 3 Oxycodone 167 166 3 Oxymorphone 121 174 4 Hydrocodone 651 650 4 Hydromorphone 503 552 4 Dihydrocodeine 57 74 5 Oxycodone 1833 1822 5 Oxymorphone 593 574 Only 6 Opiates 0 0 after dilution 7 Opiates 0 0 8 Morphine 22,562 37,954*** 8 Hydrocodone 6061 7336 8 Hydromorphone 3492 2706 8 Dihydrocodeine 1084 1104 Forensic Toxicology Use Only 18 6-MAM linearity and dynamic range 6AMSTD10 - m/z= 328.15-328.15 SM: 5 RT: 2.10 - 4.10 NL: 3.16E5 F: {0,0} + p ESI Full ms [ 327.00-335.00] RT: 3.10 100 95 90 85 80 75 6-MAM 10 ng/mL 70 65 6- MAM 3 - 400 ng/mL 60 55 50 6-ACETYLMORPHINE 45 RelativeIntensity 40 Y = -0.0899199+0.0613337*X R^2 = 0.9984 W: 1/X 35 26 30 25 20 24 15 10 5 3.40 3.75 22 0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min) 20 18 6AMSTD10 - m/z= 334.19-334.19 SM: 5 RT: 2.10 - 4.10 NL: 5.70E5 16 F: {0,0} + p ESI Full ms [ 327.00-335.00] RT: 3.10 100 14 95 90 12 Ratio Area 85 80 IS 20 ng/mL 10 75 70 65 8 60 55 6 50 45 4 RelativeIntensity 40 35 30 2 25 20 0 15 0 50 100 150 200 250 300 350 400 10 5 NG/ML 3.91 3.41 3.77 4.04 0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min) Forensic Toxicology Use Only 19 6-MAM NLCP compliance test Measured =10.6 ng/mL STD10 W50000MOR - m/z= 328.15-328.15 SM: 5 RT: 2.11 - 4.11 NL: 4.31E5 F: {0,0} + p ESI Full ms [ 327.00-335.00] RT: 3.11 100 95 90 85 10 ng/mL of 6-MAM 80 in the presence of 50,000 ng/mL 75 Morphine 70 65 60 55 50 45 RelativeIntensity 40 35 30 25 3.32 3.48 20 2.862.96 3.57 3.40 3.69 2.25 2.81 15 2.35 2.54 3.76 3.87 3.98 10 5 0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 Time (min) Forensic Toxicology Use Only 20 6-MAM GC-MS Correlation - Samples Exactive LC-MS GC-MS No (ng/mL) (ng/mL) 1 16.6 15.4 2 59.9 56.8 3 21.4 20.6 3 18.4 14.6 4 13 10.8 4 7.8 6.6 Forensic Toxicology Use Only 21 Blood samples XIC with mass accuracy of 2 ppm Zolpidem 293 ng/mL Zolpidem-D6 174876bld - m/z= 308.18-308.18 SM: 5 RT: 2.18 - 7.18 NL: 7.41E5 174876bld - m/z= 314.21-314.21 SM: 5 RT: 2.18 - 7.18 NL: 1.97E6 F: FTMS {0,0} + p ESI Full ms [ 223.00-1000.00] F: FTMS {0,0} + p ESI Full ms [ 223.00-1000.00] RT: 4.68 RT: 4.68 100 100 95 95 90 90 85 85 80 80 75 75 70 70 65 65 60 60 55 55 50 50 45 45 RelativeIntensity 40 RelativeIntensity 40 35 35 30 30 25 25 20 20 15 15 10 10 5 5 5.50 5.84 6.63 5.41 5.68 6.22 0 0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time (min) Time (min) Dextrorphan 111 ng/mL Dextrorphan-D3 176372 - m/z= 261.20-261.20 SM: 5 RT: 1.88 - 6.88 NL: 1.97E6 F: FTMS {0,0} + p ESI Full ms [ 232.00-1000.00] 176372 - m/z= 258.19-258.19 SM: 5 RT: 1.88 - 6.88 NL: 1.28E 6 F: FTMS {0,0} + p ESI Full ms [ 232.00-1000.00] RT: 4.38 100 RT: 4.38 100 95 95 90 90 85 85 80 80 75 75 70 70 65 65 60 60 55 55 50 50 45 45 RelativeIntensity 40 Relative Intensity 40 35 35 5.53 30 30 25 25 20 20 15 15 5.06 10 10 5 5 6.03 5.54 4.90 5.22 6.27 6.58 4.77 5.80 6.19 6.52 0 0 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 Time (min) Time (min) Forensic Toxicology Use Only 22 Blood samples XIC with mass accuracy of 2 ppm Norpropoxyphene 168 ng/mL Norpropoxyphene-D5 174876bld - m/z= 331.24-331.24 SM: 5 RT: 3.34 - 5.34 NL: 4.42E5 F: {0,0} + p ESI Full ms [ 320.00-1000.00] 174876bld - m/z= 326.21-326.21 SM: 5 RT: 3.34 - 5.34 NL: 2.18E5 F: {0,0} + p ESI Full ms [ 320.00-1000.00] RT: 4.34 100 RT: 4.34 100 95 95 90 90 85 85 80 80 75 75 70 70 65 65 60 60 55 55 50 50 45 45 RelativeIntensity 40 RelativeIntensity 40 35 35 30 30 25 25 4.51 20 20 15 15 10 10 5 5 4.45 4.64 4.79 4.58 4.70 5.07 0 0 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 3.4 3.6 3.8 4.0 4.2