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

Sample Preparation Details

Part No:8E-S138-FGA
Solid Phase Extraction (SPE) procedure:Note: The solvent volumes shown below are for a 200 mg bed mass. The solvent volumes will need to be adjusted for a smaller or larger bed mass.
Load: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
Final Prep and Analysis: 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.
Inject: 1 µL on HPLC / Tandem Mass Spec (MS-MS) @ (ambient)

Analytes:

Name Spiked
Conc.
Log P pKa % Rec %RSC (n=0)
1 Amphetamine 125  82 13.1
2 Methamphetamine 125  107 15.1
3 MDMA 62.25  99
4 MDA 62.25  106 4.2
5 MDEA 62.25  108 10.5

Application Detail (App ID: 22986)

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

1
Amphetamine
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2
Methamphetamine
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3
MDMA
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4
MDA
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5
MDEA
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