T3/T4 Plasma Extract by LC/MS/MS on Kinetex C18, 2.6 um, 75x2.1 mm

LC Conditions

Column

Kinetex 2.6 µm C18 100 Å, LC Column 75 x 2.1 mm, Ea

Brand

Kinetex

Part No

00C-4462-AN

Phase Name

C18

Elution Type

Gradient

Mobile Phase

A: 0.05% Formic Acid in DI H2O

B: 50:50 MeOH:ACN

Gradient Profile

Step No.

Time(min)

%A

%B

1

0

70

30

2

2.5

30

70

3

3

30

70

4

3.01

10

90

5

4

10

90

6

4.01

70

30

7

5.5

70

30

Flow Rate

600.00 mL/min

Temperature

22°C

Detection

Mass Spectrometer (MS) @ (0°C)

Detection Info

Sample Preparation Details

Description

Strata-X-C 33 µm Polymeric Strong Cation, 60 mg / 3 mL, Tubes, 50/Pk

Part No

8B-S029-UBJ

Condition 1

1 mL 100% MeOh at 1 mL/min

Condition 2

1 mL 0.1 N HCl at 1 mL/min

Sample Pretreatment

Extraction Procedure: Activate with 1 mL MeOH Equilibrate the SPE cartridge with 1 mL 0.1 M HCl Load sample (0.4 mL serum/plasma + 1 mL 0.1 M HCl) Wash SPE with 1 mL 0.1 M HCl Wash SPE with 1 mL 100% MeOH Dry the SPE bed under maximum vacuum for 5 min Elute the analytes with 2x1 mL 2.5% NH4OH in MeOH Evaporate the solvent to dryness under gentle N2 stream at 40-45°C Reconstitute the dry residue in 200 uL initial MP (30%MeOH) solvent

Load

Load 0.10 mL Sample diluted in 0.1 1N HCl

Wash 1

1 mL 0.1 N HCl at 1 mL/min

Wash 2

1 mL 100% MeOH at 1 mL/min

Dry

5 min under max vac

Elution 1

1 mL 2.5% NH4OH in MeOH at 1 mL/min

Elution 2

1 mL 2.5% NH4OH in MeOH at 1 mL/min

Evaporation

Evaporate to dryness

Reconstitute

200 µL 30% MeOH in 0.05% HCOOH