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Extraction of COOH-THC from Urine Using Novum SLE & Kinetex 2.6u Biphenyl

Sample Preparation Details

Part No:8E-S138-5GA
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a 400 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Load:Enzymatic Hydrolysis: To 300 uL urine add 75 uL of 300 mM ammonium acetate (pH 4.0) and 25 uL of
-Glucoronidase solution (100,000 units/mL (www.campbellscience.com, DR2100).

Mix and vortex for 30 secs.
Incubate at 37- 40°C for 60 minutes. (Gentle shaking during this step is recommended).
After incubation, bring samples to room temperature prior to extraction.
Final Prep and Analysis: Sample Pretreatment Step
Enzymatic Hydrolysis: To 300 uL urine add 75 uL of 300 mM ammonium acetate (pH 4.0) and 25 uL of -Glucoronidase solution (100,000 units/mL (www.campbellscience.com, DR2100).
Mix and vortex for 30 secs. Incubate at 37- 40°C for 60 minutes. (Gentle shaking during this step is recommended). After incubation, bring samples to room temperature prior to extraction.
Extraction Procedure:
Sample Loading: Load the sample (~400 uL) from pretreatment step onto the Novum plate and apply a short and gentle pulse of vacuum (~5-10 sec and 5"or less of Hg) until the sample has completely entered in the media.
NOTE: Avoid prolonged or excessive vacuum. The sample must not leave the Novum bed.
Wait for 5-6 minutes. NOTE: Inadequate or excessive wait period will lead to variable recoveries and poor precision.
Elution: Dispense 900 uL of ethyl acetate onto the Novum plate and collect the solvent under force of gravity (approximately 5 min).
Repeat with another 900 uL addition of ethyl acetate and collect the solvent under gravity.
Apply vacuum at 5" of Hg (or lower) for 20-30 secs to complete the extraction.
NOTE: Excessive/Prolong application of the vacuum may force the elution of plasma and cause dirty extraction
Dry down: Evaporate extracted samples to complete dryness under slow stream of N2 and room temperature.
Reconstitute the dry residue in 200 uL of initial mobile phase containing internal standard
Inject: 20 µL on HPLC / Tandem Mass Spec (MS-MS) @ (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)
1 11-nor-9-carboxy-delta9-THC 20  83
2 11-nor-9-carboxy-delta9-THC-d3 (IS) 50 

Application Detail (App ID: 22786)

Part No:00B-4497-AN
Dimensions:50 x 2.1 mm ID
Elution Type:Gradient
Elution A:0.1% formic acid
Elution B:Methanol/acetonitrile(50:50)
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 50 50
2 2.5 5 95
3 3.5 5 95
4 5 50 50

Flow Rate:0.5 mL/min
Col. Temp: 25 °C
Detection: Tandem Mass Spec (MS-MS) @ (22°C)
Detector Info: AB Sciex API 5000

Analyte Details

Analyte Detail not available

1
11-nor-9-carboxy-delta9-THC

2
11-nor-9-carboxy-delta9-THC-d3 (IS)