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THC and Metabolites from Whole Blood on Strata-X-Drug B and Kinetex C18 by LC-MS-MS

Sample Preparation Details

Part No:8B-S128-UCH
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a 60 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Load:Sample Pre-Treatment (before SPE):
Protein precipitation: To 0.5 mL whole blood (spiked with analytes), add 1.0 mL cold
ACN:MeOH (85:15 v:v), vortex 1-2 mins (using a maximix II, from Barnstead) with the
maximum force possible, followed by centrifugation at 10,000 rpm, for 10 mins. Discard pellet
and dilute sample obtained from protein precipitation step with 3 mL of 1% Formic acid in
water. Load sample directly onto cartridge - NO CONDITIONING REQUIRED
Dry:5 mins at 7-15 psi Nitrogen
Final Prep and Analysis: Evaporate eluant to dryness under 50 °C nitrogen. Reconstitute in 500uL of initial mobile phase.
Extraction was automated and performed using a Gilson GX-274 ASPEC system Inject: 5 µL on HPLC / Mass Spectrometer (MS) @ amu (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)
1 THC-OH 100  100.3 3.1
2 THC-OH-d3 100  100 0.1
3 THC-COOH 100  103.9 5.4
4 THC-COOH-d3 100  100 0.1
5 THC 100  99.3 3.9
6 THC-d3 100  100 0.1

Application Detail (App ID: 19947)

Part No:00B-4462-AN
Dimensions:50 x 2.1 mm ID
Elution Type:Gradient
Elution A:1 mM ammonium formate with 0.1% FA
Elution B:0.1% FA/methanol:acetonitrile(1:1)
Gradient Profile:
Step No. Time (min) Pct A Pct B
1 0 50 50
2 3 5 95
3 4.5 5 95
4 4.51 50 50
5 6 50 50

Flow Rate:0.4 mL/min
Col. Temp: ambient
Detection: Mass Spectrometer (MS) @ amu (22°C)
Detector Info: AB Sciex API 5000™
Note:Mass spec conditions:

Polarity Analyte Q1 Q3 DP CE
(+) THC-OH (*) 331.0 193.3 55 31
(+) THC-OH 331.0 201.2 55 33
(+) THC-OH 331.0 175.3 55 31
(+) THC-OH-d3 334.0 196.3 55 31
(+) THC (*) 315.2 193.1 60 31
(+) THC 315.2 259.1 60 35
(+) THC-d3 318.2 196.1 60 25
(+) THC-COOH (*) 345.2 327.0 55 29
(+) THC-COOH 345.2 299.2 55 49
(+) THC-COOH-d3 348.0 330.1 55 29
CAD: 7 GS1: 60 GS2: 45 TEM: 600 CUR: 25 IS: 5500
* indicates quant ion

Analyte Details

Analyte Detail not available

1
THC-OH

2
THC-OH-d3

3
THC-COOH
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4
THC-COOH-d3

5
THC
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6
THC-d3