THC in Oral Fluid by LC-MS/MS Kristine Van Natta 24 Sep 2010

2 Proprietary & Confidential Background Oral fluid is less invasive and embarrassing to collect than blood or urine. THC in oral fluid is in equilibrium with blood plasma at a nearly 1:1 ratio. Oral fluid is collected on expanding foam pads and shipped in preservative solution. Developed LC-MS method for THC in oral fluid using immunoassay kit. CEDIA® THC OFT Calibrators, Controls and Thermo Scientific Oral Fluid Device.

3 Proprietary & Confidential THC Extraction Protocol Measure 0.2 mL of CEDIA THC OFT Kit Calibrator or Control into a 16 x100 mm silianized glass screw-top culture tube. Add 10 µL of IS spiking solution ng/mL in methanol) and vortex to mix. Add 400 µL of 1M Na 2 CO 3 solution and vortex to mix. Add 2.0 mL of ethyl acetate. Cap tubes, mix using multi-tube vortexer for 15 minutes Centrifuge at 3500 rpm for 10 minutes. Place tubes in freezer until aqueous layer has frozen (~60 min). Transfer the upper (organic) layer to a clean 16 x100 mm silianized glass tube. Evaporate to dryness under N 2 at 37°C in Turbo Vap (~15-20 minutes). Reconstitute the samples with 50 µL of mobile phase and vortex briefly. Transfer to a limited volume inset HPLC vial and cap.

4 Proprietary & Confidential HPLC Conditions Mobile Phase: A: 5 mM ammomium acetate with 5% methanol B: 90% methanol,10% 5 mM ammonium formate Flow Rate: 500 µL/min Column: Thermo Hypersil Gold, 50x2 mm, 5µ Injection Volume: 15 µL Total Run Time: 5 minutes Gradient

5 Proprietary & Confidential TSQ Quantum Vantage Method

6 Proprietary & Confidential 0.5 ng/mL THC in Saliva THC-315/123THC-315/193

7 Proprietary & Confidential Internal Standard (d3-THC) d3-THC-318/123d3-THC-318/196

8 Proprietary & Confidential Calibration Curves THC-315/123THC-315/193

9 Proprietary & Confidential Results THC-315/123 Transition Sample Nominal Conc. (ng/mL) Calc. Conc. (ng/mL) % Diff.%CV LQC¹ / %/ -7.61% 13.6%/ 5.57% 1 ng/mL %3.27% HQC %²2.56% 10 ng/mL %1.07% THC-315/193 Transition Sample Nominal Conc. (ng/mL) Calc. Conc. (ng/mL) % Diff.%CV LQC %2.53% 1 ng/mL %1.82% HQC %²1.67% 10 ng/mL %0.724% ¹ Data with and without one outlier ² There is a known stability issue with this lot of QCs.

10 Proprietary & Confidential CEDIA Oral Fluid Calibrators and Controls

11 Proprietary & Confidential Conclusion and Further Development Immunoassay calibrators and controls can be used directly for confirmation of cutoff on LC-MS. No need to prepare separate standard curve. Looking into developing UPLC method.