LC/MS/MS Analysis of Steroid Hormones in Plasma on Ascentis® Express C18 after Sample Prep using HybridSPE®-Phospholipid
Materials
SPE tube or plate
standard
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