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

Transcription

1 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

2 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 7-Aminoclonazepam 7-Aminoflunitrazepam 2-Hydroxy-ethyl-flurazepam Desalkylflurazepam Diazepam Hydroxy-alprazolam Hydroxy-triazolam Nordiazepam Lorazepam Oxazepam Temazepam Morphine Hydromorphone Oxymorphone Codeine Dihydrocodeine Hydrocodone Oxycodone Meperidine Normeperidine Ketamine Norketamine Butorphanol Fentanyl Norfentanyl Nalbuphine Alfentanil Sulfentanil Zolpidem Trazodone Venlafaxine Zopiclone Methylphenidate Ritalinic Acid Dextromethorphan Dextrophan Propoxyphene Norpropoxyphene 6-MAM 2

3 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 3

4 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 4

5 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 abuse group1 Other drugs of abuse group 2 ng/ml ng/ml 3 ng/ml 2- ng/ml ng/ml

6 Exactive uhram LC-MS system Resolution, at 1 scan per sec, at scans per sec Mass accuracy Sub ppm Scan speed Up to scans per second Mass range m/z - 4 Polarity switching 6

7 Power of mass resolution Ethinyl Estradiol Resolution: k, 3k, k, k Butyl-Phthalate, (ubiquitous background ion) Relative Abundance Ethinyl-Estradiol, m/z

8 Exactive LC/MS setup 8

9 Exactive LC/MS setup 9

10 Exactive quantitative analysis workflow Sample Preparation SPE or urine dilution LC-MS method PFP column, HESI source, Mass resolution, Data processing extract chromatograms with 2 ppm mass accuracy Custom reports

11 Urine sample preparation Benzodiazepines, opiates and Other drugs of abuse group 2 SPE Int. std addition Enzymatic Hydrolysis SPE Extraction Evaporation Reconstitution Other drugs of abuse group 1 Urine dilution Int. std addition X dilution with 2-% MeOH Inject Samples 11

12 Blood sample preparation Benzodiazepines, opiates other drugs of abuse group 2 SPE Int. std addition SPE Extraction Evaporation Reconstitution Other drugs of abuse group 1 Ritalinic Acid Precipitation/dilution Int. std addition ACN addition in ratio 1:1 Centrifugation 3 X dilution Inject Sample 12

13 LC method 1 x 2.1 mm, um PFP column Mobile phase A: mm NH4Ac,.1% FA in DIW B:.1% FA in ACN 7 min gradient 13

14 MS method HESI source Full scan data in compounds specific m/z range Resolution K Extract chromatograms using 2 ppm mass window 14

15 Opiates and Opioids in Urine Codeine Hydrocodone Oxymorphone D3 Morphine Hydromorphone Oxycodone D6 Normeperidine Dihydrocodeine D6 Oxycodone Oxymorphine Meperidine D6 dihydrocodeine Codeine D6 Hydrocodone D6 Meperidine Morphine D6 Hydromorphone D6 1

16 Opiates and Opioids in Urine - dynamic range Linearity range better than, ng/ml Hydrocodone Y = *X R^2 =.941 W: 1/X^2 11. Area Ratio Hydrocodone ng/ml Area Ratio Codeine Y = *X R^2 =.9461 W: 1/X^2 Codeine ng/ml Area Ratio Meperidine Y = *X R^2 =.94 W: 1/X^2 Meperidine ng/ml Area Ratio Morphine Y = *X R^2 =.9288 W: 1/X^2 Morphine ng/ml 16

17 Opiates and Opioids in Urine- dynamic range Area Ratio Linearity range, ng/mloxymorphone Y = *X R^2 =.9411 W: 1/X^2 Normeperidine Y = *X R^2 =.9434 W: 1/X^2. Normeperidine Area Ratio Oxymorphone ng/ml ng/ml Oxycodone Y = *X R^2 =.9 W: 1/X^2 Oxycodone Oxymorphone Y = *X R^2 =.9411 W: 1/X^2 Dihydrocodeine Area Ratio ng/ml Area Ratio ng/ml 17

18 GC-MS correlation No Analyte Exactive LC-MS GC-MS (ng/ml) (ng/ml) 1 Morphine Oxymorphone Oxycodone Oxymorphone Hydrocodone Hydromorphone Dihydrocodeine 7 74 Oxycodone Oxymorphone Opiates 7 Opiates 8 Morphine 22,62 37,94*** 8 Hydrocodone Hydromorphone Dihydrocodeine Only after dilution 18

19 6-MAM linearity and dynamic range 6AMSTD - m/z= SM: RT: NL: 3.16E F: {,} + p ESI Full ms [ ] RT: MAM 3-4 ng/ml 6-ACETYLMORPHINE Y = *X R^2 =.9984 W: 1/X Relative Intensity MAM ng/ml Time (min) 18 Area Ratio AMSTD - m/z= SM: RT: NL:.7E F: {,} + p ESI Full ms [ ] Relative Intensity RT: IS ng/ml NG/ML Time (min) 19

20 6-MAM NLCP compliance test Measured =.6 ng/ml STD WMOR - m/z= SM: RT: NL: 4.31E F: {,} + p ESI Full ms [ ] RT: Relative Intensity ng/ml of 6-MAM in the presence of, ng/ml Morphine Time (min)

21 6-MAM GC-MS Correlation - Samples No Exactive LC-MS GC-MS (ng/ml) (ng/ml)

22 Blood samples XIC with mass accuracy of 2 ppm Zolpidem 293 ng/ml Zolpidem-D bld - m/z= SM: RT: NL: 7.41E F: FTMS {,} + p ESI Full ms [ ] bld - m/z= SM: RT: NL: 1.97E6 F: FTMS {,} + p ESI Full ms [ ] RT: 4.68 RT: Relative Intensity Relative Intensity Time (min) Time (min) Dextrorphan 111 ng/ml m/z= SM: RT: NL: 1.28E6 F: FTMS {,} + p ESI Full ms [ ] RT: Relative Intensity Time (min) m/z= SM: RT: NL: 1.97E6 F: FTMS {,} + p ESI Full ms [ ] Relative Intensity Dextrorphan-D3 RT: Time (min)

23 Blood samples XIC with mass accuracy of 2 ppm Norpropoxyphene 168 ng/ml Norpropoxyphene-D bld - m/z= SM: RT: NL: 2.18E F: {,} + p ESI Full ms [ 3.-.] RT: Relative Intensity Time (min) bld - m/z= SM: RT: NL: 4.42E F: {,} + p ESI Full ms [ 3.-.] RT: Relative Intensity Time (min) Propoxyphene 162 ng/ml Propoxyphene-D bld - m/z= SM: RT: NL: 7.9E F: {,} + p E SI Full ms [ 3.-.] Relative Intensity RT: Time (min) Std ng_mlbld - m/z= SM: RT: NL: 4.3E6 F: {,} + p ESI Full ms [ 3.-.] Relative Intensity RT: Time (min)

24 Blood samples XIC with mass accuracy of 2 ppm Ritalinic Acid 293 ng/ml PCP-D used as Int Std m/z= SM: RT: NL: 2.3E6 F: FTMS {,} + p ESI Full ms [ ] RT: Relative Intensity Time (min) m/z= SM: RT: NL: 1.3E6 F: FTMS {,} + p ESI Full ms [ ] RT: Relative Intensity Time (min) 24

25 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 2

26 Acknowledgements Marta Kozak, Thermo Fisher Scientific 26