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Cortisol and Cortisone in Extracted Female Human Urine on Raptor Biphenyl by LC-MS/MS

LC_CF0697
PeakstR (min)Precursor IonProduct IonProduct Ion
1.Cortisol-d40.75367.4121.0-
2.Endogenous cortisol0.76363.3120.991.1
3.Cortisone-d80.88369.4167.9-
4.Endogenous cortisone0.89361.3163.291.0
*Isobaric matrix peaks
ColumnRaptor Biphenyl (cat.# 9309A52)
Dimensions:50 mm x 2.1 mm ID
Particle Size:2.7 µm
Pore Size:90 Å
Guard Column:Raptor Biphenyl EXP guard column cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9309A0252)
Temp.:40 °C
Sample
Diluent:Mobile phase A
Conc.: Calculated concentration is 105.1 ng/mL and 135.9 ng/mL for cortisol and cortisone, respectively
Inj. Vol.:10 µL
Mobile Phase
A:Water, 0.1% formic acid
B:Acetonitrile, 0.1% formic acid
Time (min)Flow (mL/min)%A%B
0.000.67030
1.000.67030
1.010.60100
1.500.60100
1.510.67030
3.000.67030
Max Pressure:400 bar
DetectorMS/MS
Ion Mode:ESI+
Mode:MRM
InstrumentUHPLC
NotesSample Preparation Method:
1. Centrifuge female human urine for 5 min at 4,500 rpm, 10 °C.
2. Aliquot 380 µL supernatant. Add 20 µL each of internal standard solution (1 µg/mL in methanol).
3. Load 200 μL of sample on to ISOLUTE SLE+ 200 µL supported liquid extraction plate (part# 820-0200-P01).
4. Apply a pulse of vacuum to initiate flow.
5. Wait 5 min for sample to completely absorb.
6. Extract samples with 1 mL of MTBE. Allow solvent to flow for 5 min under gravity. Apply vacuum for 10-30 sec to complete elution.
7. Evaporate extracts to dryness under a stream of nitrogen.
8. Reconstitute in 200 µL mobile phase A prior to analysis.
9. Vortex to mix.