2023 MSACL poster LLOQ peak 18 OH Corticosterone

LC Conditions

Column

Kinetex 2.6 µm C18 100 Å, LC Column 50 x 3 mm, Ea

Brand

Kinetex

Part No

00B-4462-Y0

Phase Name

C18

Elution Type

Gradient

Mobile Phase

A: 0.5 mM NH4F (aq)

B: MeOH

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

60

40

8

2

50

50

9

4.5

25

75

10

5

5

95

11

6

5

95

12

6.5

60

40

13

8

60

40

Flow Rate

800.00 mL/min

Temperature

30°C

System

Agilent Technologies SCIEX 7500 QTRAP

Detection

LC/MS/MS @ (700°C)

Detection Info

Sample Preparation Details

Description

Strata-X 33 µm Polymeric Reversed Phase, 30 mg / well, 96-Well Plates, 2/Pk

Part No

8E-S100-TGB

Condition 1

1 mL MeOH

Sample Pretreatment

Dilute 500 uL serum sample with 500 uL 1% FA.

Load

1 mL total volume of pretreated sample and spike with 10uL IS

Wash 1

1 mL 1% FA

Wash 2

1 mL 30% MeOH

Dry

Dry down under gentle nitrogen flow at 45 degrees C

Elution 1

Twice with 500 uL 1:4 MeOH:ACN

Evaporation

Evaporate to dryness

Reconstitute

100 uL 60:40 0.5mM NH4F (aq) : MeOH

Steps

0

Condition

1 mL MeOH

1

Equilibrate

1 mL Water

2

Load

1 mL total volume of pretreated sample and spike with 10uL IS

3

Wash

1 mL 1% FA

4

Vacuum

Apply vacuum to elute wash

5

Wash

1 mL 30% MeOH

6

Vacuum

Apply vacuum (15-20) for 3-4 minutes until completely dry

7

Elute

Twice with 500 uL 1:4 MeOH:ACN

8

Dry

Dry down under gentle nitrogen flow at 45 degrees C

9

Reconstitute

100 uL 60:40 0.5mM NH4F (aq) : MeOH