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LC/MS/MS Analysis of Steroid Hormones in Plasma on Ascentis® Express C18 after Sample Prep using HybridSPE®-Phospholipid

LC/MS/MS Analysis of Steroid Hormones in Plasma on Ascentis® Express C18 after Sample Prep using HybridSPE®-Phospholipid application for HPLC

Materials

analytical column

Product No.
Description
Pricing

Ascentis® Express C18, 2.7 μm HPLC Column

2.7 μm particle size, L × I.D. 10 cm × 2.1 mm

SPE tube or plate

Product No.
Description
Pricing

HybridSPE®-Phospholipid

96-well Plate, bed wt. 50 mg, volume 2 mL, pk of 1

Standard

Product No.
Description
Pricing

Aldosterone solution

100 μg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®

Corticosterone solution

1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®

17α-Methyltestosterone solution

1.0 mg/mL in 1,2-dimethoxyethane, ampule of 1 mL, certified reference material, Cerilliant®

Progesterone solution

1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®

Testosterone solution

1.0 mg/mL in acetonitrile, ampule of 1 mL, certified reference material, Cerilliant®

CONDITIONS

SPE tube/cartridge

HybridSPE®-Phospholipid, 96-well plate, 50 mg/well (575656-U)

sample addition

To each well add 100 μL of plasma followed by 300 μL of precipitation solvent (1% formic acid or 0.5% citric acid in acetonitrile).  (Agitate via vortex for 4 minute, place on vacuum manifold and apply 10" Hg vacuum for 4 minutes. Collect filtrate and analyze directly.)

column

Ascentis® Express C18, 10 cm x 2.1 mm I.D., 2.7 μm particles (53823-U)

gradient

60% B for 3 min, 60% B to 95% B in 5 min, held at 95% B for 2 min

mobile phase

[A] 5 mM ammonium formate pH 4.0 with formic acid; [B] methanol

flow rate

0.3 mL/min

column temp.

50 °C

detector

ESI+, MRM

injection

2 μL

Description

Analysis Note

This application shows the separation of steroid hormones in plasma using LC/MS/MS. HybridSPE-Phospholipid removed endogenous phospholipids and precipitated proteins, thereby allowing rapid, sensitive analysis by LC/MS/MS using an Ascentis Express C18 column. The highest grade LC-MS solvents were used to supply low background interference and low particulate contaminants for robust, trouble-free operation. Cerilliant CRMs provided reliable identification and quantification.

Legal Information

Ascentis is a registered trademark of Merck KGaA, Darmstadt, Germany
HybridSPE is a registered trademark of Merck KGaA, Darmstadt, Germany