Applications
Pain Panel using Strata-X-Drug B and Kinetex Phenyl-Hexyl 2.6u 50x4.6mm
20847
Separation Mode: Reversed Phase
Reversed Phase
Phenyl-Hexyl
HPLC
Adrenergic Agents
Clinical
Forensics/Toxicology
Therapeutic / Clinical
Chemical Compound
Therapeutic / Clinical

Applications

Pain Panel using Strata-X-Drug B and Kinetex Phenyl-Hexyl 2.6u 50x4.6mm
Analytes
1
2
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10
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12
13
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55
Compound Name
Morphine
Details
LC Conditions (App ID: 20847)
Column:
Brand Name:
Part No:
Elution Type:
Gradient
Mobile Phase:
A: 10mM ammonium formate
B: 0.1% Formic acid/methanol
Gradient Profile:
Step No.Time(min)%A%B
10955
240100
350100
45.1955
57955
Flow Rate:
0.6 mL/min
Temperature:
22°C
Detection:
Mass Spectrometer (MS) amu @ 22°C
Sample Preparation Details
Description:
Strata-X-Drug B 33 µm Polymeric Strong Cation, 30 mg / 3 mL, Tubes, 50/Pk
Part Number:
Pre Treatment Note:
Load: Samples from hydrolysis Using b-Glucoronidase as below To 200 µL Urine (calibrator, control) in a 1.5 mL micro centrifuge tube, add 20 µL combined IS (Internal Standards) spiking solution. Vortex 10-15 seconds followed by adding 100 µL 0.1 M Ammonium acetate buffer (pH 4.0) to each tube, mix/vortex another 15 seconds. Add 40 µL ß-Glucoronidase solution (100,000 units/mL). Vortex 10-15 seconds. Incubate for 2 hours in a shaker at 55 °C to complete hydrolysis of the glucuronides. Add 400 µl of 0.1% Formic acid and vortex for another 60 seconds. Centrifuge the hydrolyzed samples at 21,000 g for 10 minutes (to separate any proteins). Collect supernatant for further cleanup via Solid Phase Extraction (SPE). Wash 1: 1 mL 0.1% FA Wash2: 1 mL 30% methanol Dry: 3 to 4 mins at high vacuum (~10” of Hg) Elute: 2x500 uL 2% ammonium hydroxide/methanol: acetonitrile (1:1) Blow Down: To dryness under nitrogen at 40-45o C Reconstitute: With 200 uL of 85/15 (A/B) of LC mobile phase (Note: 1. No conditioning or equilibration needed in extraction) 2.Total of 15 internal standard used for the quantitation of 41 analytes.
SPE Condition 1:
1 at 1.0
SPE Condition 2:
1 at 1.0
SPE Load:
Load 0.20 mL Sample diluted in 0.1% Formic acid/water
SPE Wash 1:
1 mL 0.1% Formic acid/water at 1.0 mL/min
SPE Wash 2:
1 mL 30% methanol at 1.0 mL/min
SPE Dry:
3 to 4 mins at high vacuum (10" of Hg)
SPE Elution 1:
0.5 mL 2% ammonium hydroxide/methanol: acetonitrile (1:1) at 1.0 mL/min
SPE Elution 2:
0.5 mL 2% ammonium hydroxide/methanol: acetonitrile (1:1) at 1.0 mL/min
SPE Evaporation:
Evaporate to dryness
SPE Reconstitute:
200 µL HPLC mobile phase A/B=85/15

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