Extraction of COOH-THC from Urine Using Novum SLE & Kinetex 2.6u C8

LC Conditions

Column

Kinetex 2.6 µm C8 100 Å, LC Column 50 x 2.1 mm, Ea

Brand

Kinetex

Part No

00B-4497-AN

Phase Name

C8

Elution Type

Gradient

Mobile Phase

A: 0.1% formic acid

B: Methanol/acetonitrile(50:50)

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

50

50

2

2.5

5

95

3

3.5

5

95

4

5

50

50

Flow Rate

0.50 mL/min

Temperature

25°C

Detection

Tandem Mass Spec (MS-MS) @ (22°C)

Detection Info

Sample Preparation Details

Description

Novum SLE, MAX 96-Well Plate, Ea

Part No

8E-S138-5GA

Pretreatment Note

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

Load

Load 300.00 µL Sample diluted in 75

Elution 1

900 mL Ethyl acetate

Elution 2

900 mL Ethyl acetate

Evaporation

Evaporate to dryness

Reconstitute

200 µL Initial Mobile phase (A:B=50/50)