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

	Peaks	t_R (min)	Precursor Ion	Product Ion	Product Ion
1.	Cortisol-d4	0.75	367.4	121.0	-
2.	Endogenous cortisol	0.76	363.3	120.9	91.1
3.	Cortisone-d8	0.88	369.4	167.9	-
4.	Endogenous cortisone	0.89	361.3	163.2	91.0

*Isobaric matrix peaks

Column

Raptor 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

Standard/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

Water, 0.1% formic acid

Acetonitrile, 0.1% formic acid

Time (min)	Flow (mL/min)	%A	%B
0.00	0.6	70	30
1.00	0.6	70	30
1.01	0.6	0	100
1.50	0.6	0	100
1.51	0.6	70	30
3.00	0.6	70	30

Max Pressure:

400 bar

Detector	MS/MS
Ion Mode:	ESI+
Mode:	MRM
Instrument	UHPLC
Sample Preparation	1. Centrifuge female human urine for 5 min at 4500 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.

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