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Extraction of Amphetamines from Urine using Strata-X-C Microelution & Kinetex 2.6u Biphenyl, 50x2.1

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Sample Preparation Details

Part Number :8E-S138-FGA

Description :Novum SLE, MINI 96-Well Plate, 1/Pk

Load :Load 150.00 µL Sample diluted in 150

Elution 1 :1000 mL Ethyl acetate

Evaporate: :Evaporate to dryness

Reconstitute :150 µL 10mM ammonium acetate in water:methanol(50:50)

Notes :Sample pre-treatment Dilute 150 uL of human plasma (spiked with 25 ng/mL and 125 ng/mL of cortisone and prednisolone respectively) with 150 uL of 50 mM sodium phosphate dibasic heptahydrate, pH unadjusted. Mix briefly (3-5 sec). Sample loading: Load the sample from pre-treatment step above onto the Novum plate and apply a short and gentle pulse of vacuum (~ 10" of Hg for 20 secs) until the sample has completely entered the media. Wait for 5 minutes. Elution: Dispense 1000 uL of ethyl acetate onto the SLE media and allow the solvent to elute by gravity (~ 5 min elution time) and collect the eluant. Apply vacuum at 5" of Hg for 45 secs to complete the extraction. NOTE: Prolonged application of vacuum will result in elution of plasma out of the SLE media and into the final extracted solvent. Dry down: Evaporate the final extract to complete dryness under slow stream of N2 at 40°C Reconstitute: Reconstitute the dry residue in 150 uL of initial mobile phase fortified with cortisol-D4.

Application Detail (App ID: 22986)

Column: Kinetex® 2.6 µm Biphenyl 100 Å, LC Column 50 x 2.1 mm, Ea

Part No:00B-4622-AN

Dimensions:50 x 2.1 mm ID

Elution Type:Gradient

Elution A:0.1% formic acid in water

Elution B:0.1% formic acid in methanol

Gradient Profile:

Step No.	Time (min)	Pct A	Pct B
1	0	90	10
2	3	5	95
3	4	5	95
4	4.01	90	10
5	6	90	10

Flow Rate:0.5 mL/min

Col. Temp: 25 °C

Detection: Tandem Mass Spec (MS-MS) @ (22°C)

Note:Extraction of Amphetamines from Human Urine Using Strata-X-C u-Elution Plate

Sorbent: Strata-X-C µ Elution 96-well Plate

Condition: 200 uL methanol
Equilibrate: 200 uL water
Load: 400 uL diluted human urine (200 uL sample diluted 1:1 with water)
Wash 1: 200 uL 2% formic acid/water
Wash 2: 200 uL methanol
Elute: 2x25uL 5% ammonium hydroxide/acetonitrile: methanol (60:40)
Direct injection: injection of 2 uL sample directly

HPLC:
Kinetex Biphenyl, 2.6u, 50x2.1; A=0.1% FA/water, B=0.1% FA/methanol;
Flow rate=0.5 mL/min; Inj. vol=2uL.

Step Total Time(min) Flow Rate (µl/min) A (%) B (%)
0 0.00 500 90.0 10.0
1 3.00 500 5.0 95.0
2 4.0 500 5.0 95.0
3 4.01 500 90.0 10.0
4 6.0 500 90.0 10.0

Analyte Details

Analyte Detail not available

Amphetamine

Methamphetamine

MDMA

MDA

MDEA