A Generic LC-MS Method for the Analysis of Multiple of Drug of Abuse Classes with the Thermo Scientific Exactive TM System

Home , Norpropoxyphene

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

60 Ethinyl-Estradiol, 279.17434 50

Relative Abundance Relative 30

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/mL Oxymorphone 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

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

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 4.4 4.6 4.8 5.0 5.2 Time (min) Time (min)

Propoxyphene 162 ng/mL Propoxyphene-D5

Std 1000 ng_mlbld - m/z= 351.30-351.30 SM: 5 RT: 3.36 - 5.36 NL: 4.35E6 F: {0,0} + p ESI Full ms [ 320.00-1000.00] 174876bld - m/z= 340.23-340.23 SM: 5 RT: 3.37 - 5.37 NL: 7.59E 5 F: {0,0} + p ESI Full ms [ 320.00-1000.00] RT: 4.36 100 RT: 4.37 100 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 4.95 4.71 4.88 3.92 4.22 4.81 5.18 3.413.60 3.73 3.86 4.06 4.17 5.01 5.15 5.25 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 4.4 4.6 4.8 5.0 5.2 Time (min) Time (min) Forensic Toxicology Use Only 23 Blood samples XIC with mass accuracy of 2 ppm

Ritalinic Acid 293 ng/mL PCP-D5 used as Int Std

154147 - m/z= 220.13-220.13 SM: 5 RT: 1.81 - 4.81 NL: 2.30E6 F: FTMS {0,0} + p ESI Full ms [ 218.00-1000.00] 154147 - m/z= 249.24-249.24 SM: 5 RT: 4.30 - 7.02 NL: 1.30E6 RT: 3.31 F: FTMS {0,0} + p ESI Full ms [ 218.00-1000.00] 100 RT: 5.80 95 100 90 95 85 90 80 85 75 80 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 3.71 4.73 4.93 5.12 5.34 6.19 6.386.53 6.69 6.84 0 0 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2 6.4 6.6 6.8 7.0 Time (min) Time (min)

Forensic Toxicology Use Only 24 Conclusions

• Exactive ultra high resolution MS coupled to LC system • Well suited for quantitative analysis • Makes addition of new compound to the method easy • Sensitive and specific LC-MS platform • Robust for urine dilution protocol for many compounds • Method can be multiplexed for higher throughput

Forensic Toxicology Use Only 25 Acknowledgements

• Marta Kozak, Thermo Fisher Scientific

Forensic Toxicology Use Only 26