Analysis of Drugs in Hair using GC/GC/MS CAT Meeting 3/2/07

Transcription

1 Analysis of Drugs in Hair using GC/GC/MS CAT Meeting 3/2/07 Objective Develop procedures for the analysis of drugs in hair using two dimensional GC systems Christine Moore, Cynthia Coulter, Sumandeep Rana Immunalysis Corporation, CA Reasons for use Limited specimen volume degraded sample, oral fluid, hair Need for improved sensitivity over standard EI/GC/MS Need to remove matrix/background effect Intuitive improvements Fast oven Sharper peak shapes, improved resolution Injection volume High volume injectors (PTV, ProSep) Detection mode CI more sensitive than EI

2 Advanced improvements Two dimensional gas chromatography Micro-fluidic switch (Deans switch) Cryofocusing cold-trapping of analyte on second analytical column Gas Chromatography Two-dimensional GC (GC:GC) Serial columns As different in polarity as possible Deans (micro-fluidic) switch allows flow to be diverted from primary column to analytical column Switch off: DB-35MS effluent goes to FID (no cut) Switch on: DB-35MS effluent is cut to DB-1MS FID FID restrictor solenoid valve (off) restrictor solenoid valve (on) Inlet PCM 32.2 psi 19.1 psi DB-35MS Inlet PCM 32.2 psi 19.1 psi DB-35MS MSD DB-1MS --- clean helium --- He + effluent MSD DB-1MS --- clean helium --- He + effluent

3 Critical parameters Column phases and dimensions * Injection and switching pressures Deans switch calculator Restrictor length Length of time heart cut window is open affects background, therefore sensitivity * Zhu et al. J Chromatogr A 2006;1105: * Harynuk J et al. J Chromatogr A 2005; 1070: * Ong et al. J Chromatogr A 2002; 962: Method development 1. With the switch off, inject high concentration of analyte of interest This ensures all specimen goes to FID 2. Determine time of elution from first column 3. Turn switch on before and after that time and capture analyte onto the analytical column Gas chromatography (continued) Cryogenic focusing Used to cold trap the analyte as it passes through analytical column Operated through Back Inlet software of system Cooled using compressed air

4 Inlet MSD FID JAS Cryo-trap re-focuses drug on analytical column restrictor DB-35MS solenoid valve (on) PCM 32.2 psi 19.1 psi Cryo Cryogenic focusing Focuser cooled from oven temp (280 o C) to lower temperature (e.g.100 o C) as fast as possible: 777 o C/min Held at low temperature to allow analyte to cold trap Re-heat focuser at 777 o C/min to 280 o C releases analyte to MSD DB-1MS Application: THCA in hair THC is active ingredient Major urinary metabolite is THCA Parent THC is detected in higher concentrations in hair than THCA However, to avoid environmental contamination concerns, detection of the metabolite THCA is required Literature Marijuana positivity rates in hair are much lower than in urine Level of THCA detected in hair (users): pg/mg (1) pg/mg (2) Daily users ; Workplace, x=1.5 pg/mg (3) (1) Sachs H. For Sci Int 2000;107(1-3):239 (2) Uhl M. For Sci Int 1997;84(1-3):281 (3) Moore C et al. J Anal Tox; (7):555

5 Extraction Procedure Calibrators, controls, specimens: 20 mg hair Add internal standard:1 pg/mg D3-THCA Calibration curve: Negative, 0.05, 0.1, 0.5, 1.0, 5.0 pg/mg Add DI water (0.5 ml); 2N NaOH (0.5 ml) Heat 75 o C, 15 min Cool; centrifuge (2500 rpm; 15 min) Extraction Procedure Pour supernatant into glass tubes containing: acetic acid (1 ml) 1 M acetic acid (3 ml) 0.1 M sodium acetate buffer (ph 4; 2 ml) Condition SPE columns (hydrophobic/cation exchange): Hexane:EtAc (75:25 v,v 2 ml) Methanol (3 ml); DI water ( 3 ml) 0.2M hydrochloric acid ( 1 ml) Extraction Procedure Load samples Wash DI water ( 2 x 3 ml) 0.1 M HCl:acetonitrile (70:30 v,v, 3 ml) Elute THCA: Hexane:EtAc (75:25, v,v 3mL) Extraction Procedure (continued) Reconstitute: Trifluoroacetic anhydride (TFAA, 50 L) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, 30 L) Cap, heat (70 o C/15 min) Leave at room temp for 10 min Evaporate to dryness (vacuum oven) Reconstitute in toluene (25 L) Evaporate eluent to dryness (N 2 ; 40 o C)

6 Gas chromatography Injection: Pulsed splitless mode 200 o C; 55.4 psi Purge flow: 50 ml/min; Purge Time: 1 min Volume: 2 L Oven: 100 o C for 0.5 min; ramp 50 o C/min to 200 o C; Ramp 10 o C/min to 260 o C; hold 2.9 min Cool 120 o C/min to 200 o C Post run: Oven raise to 320 o C; hold for 3 min Mass spectrometry Instrument parameters: Reagent gas: Ammonia (~7 x more sensitive than methane) Purity % with max impurities: CO 1 ppm; Methane 1 ppm; N 2 1 ppm; O 2 1 ppm; H ppm Stainless steel regulator Mode: Negative ion chemical ionization (NCI) Electron Capture Chemical Ionization (ECCI) MS (continued) Calibration curve: 0.05 pg/mg 5 pg/mg Instrument parameters: Collision gas flow: 8 x Torr MS Source: 150 o C MS Quadrupole: 106 o C Transfer line: 280 o C SIM ions: 590 (593); 422 (425)

7 Cryogenic effect Detection of 11-nor- 9-THC-9- Carboxylic Acid (THC-COOH) in Hair and Urine C.Moore, S.Rana, C.Coulter, M.Vincent, J.Soares Immunalysis Corporation, Pomona, CA W. Ross, N. Giorgi Redwood Toxicology Laboratory, Santa Rosa, CA Police collection of marijuana in KY Objective To analyze both urine and hair specimens from self-admitted marijuana smokers

8 Subjects 156 subjects: All admitted marijuana users IRB Approval Sex, age, ethnicity and hair color recorded Date of last use Frequency of use Subjects signed consent forms Hair Urine Subjects 46 subjects were positive Of these, 9 female; 37 male 56.5 % Caucasian 26 % African American 6.5 % Native American 10 % no data Criteria for positive results HAIR Positive screening result >1 pg/mg, and THCA confirmation: >0.05 pg/mg URINE Positive screening result > 50 ng/ml Results 110 were negative via both matrices 46 positive via hair, urine or both 28 positive in both matrices 8 positive urine only 9 positive hair only [Note. One specimen had no data for the urine, but was hair positive]

9 Results (n = 46) Results (continued) % positive for THCA Both Hair only Urine only Positivity rates for hair and urine were essentially the same Paired samples were not necessarily positive Mean / median hair concentration when urine positive 0.46 / 0.32 pg/mg Mean/median hair concentration when urine negative 0.25 /0.14 pg/mg Marijuana levels.v. self-reported frequency of use Summary Hair levels (pg/mg) x week 7 x week 14 x week 21 x week 28 x week 35 x week 70 x week Similar positivity rates in hair and urine No specimens quantitated between 0.05 and 0.1 pg/mg in this study No obvious correlation between self-reported intake and THCA hair concentration, however there are disadvantages to self-reporting of drug intake Hair analysis for THCA at the appropriate cutoff provides a similar positivity rate to urine

10 Application: Fentanyl in hair To date, detection of therapeutic concentrations of fentanyl in hair restricted to immunoassay a or tandem MS/MS b,c Two-dimensional chromatography offers increased sensitivity a Wang et al. Immunoassay evidence for fentanyl in hair of surgery patients. Forens Sci Int. (1993) 61(1): b Sachs et al. Analysis of fentanyl and sufentanil in hair by GC/MS/MS. Int J Legal Med (1996) 109: c Kintz et al. Evidence of addiction by anesthesiologists as documented by hair analysis. Forens Sci Int. (2005) 153(1): Fentanyl No active hydrogen groups MW = Cannot derivatize to improve sensitivity NCI not useful in this situation Analytical conditions: Fentanyl 6890 GC/ 5975 MSD; inert source, 220/240V oven Deans switch microfluidic valve Primary column: DB17 MS 15m x 0.25mm x 0.25 m Analytical column: DB-5 MS 15m x 0.25 mm, 0.25 m Injection temp: 250 o C Purge flow: 50 ml/min, 1 min

11 Issues No cryo-trap available Peak sharpening limited No high volume injector available Injection volume limited No active sites on analyte Restricts derivatization and therefore detection mode Analytical conditions: Fentanyl Injection mode: Pulsed splitless 50.4 psi for 1.5 min Oven mode: Ramped pressure 50 o C/min to 33.7psi Oven program: 70 o C for 0.5 min; 35 o C/min to 310 o C; hold 3 min Injection mode: Improve chromatography? Pulse.v. non-pulse injection No pulse: FENT004, FENT009 Pulse: FENT013, FENT ng on column

12 MS conditions: Fentanyl Electron impact mode Transfer line: 280 o C; Quadrupole: 150 o C; Ion source: 230 o C Ions monitored: deuterated fentanyl 250.2, 151.1; fentanyl 245.2, 146.1,189.1 Dwell time:70 ms Limit of quantitation No Deans switch: 50 pg/mg Deans switch: 5 pg/mg Post-mortem specimens Post-mortem hair samples received from Montgomery County, OH Blinded to toxicology results Four samples screened positively for fentanyl using ELISA Analyzed using the described procedure Fentanyl: Post mortem specimens Fentanyl in hair Hair (pg/mg) /10 Blood (ng/ml) Sample 7 Sample 5 Sample 10 Sample 6

13 Drugs in hair Some drugs incorporate well into hair, and two-dimensional GC may not be necessary Methadone (> 1000 pg/mg) Carisoprodol (> 5000 pg/mg) Tramadol (> 150,000 pg/mg) Propoxyphene Maintenance considerations 1. Simple but precise connections 2. Column maintenance causes heart-cut window to move 3. Pressures are absolutely critical 4. Incorporate back-flush into the method to remove background from subsequent injections Summation Thank you for your attention Additions to existing systems are enhancing sensitivity, improving chromatography and increasing analysis speed High volume injectors (PTV) Fast GC and pulsed injections Two-dimensional chromatography Cryo-focusing