Estrone and Estradiol Extraction from Human Serum Using ISOLUTE® SLE+ Before LC-MS/MS

(Without Derivatisation or Fluorine Adduct Stabilization)

Introduction

Estrone (E1) and estradiol (E2) are female steroid hormones typically monitored in peri- and postmenopausal human serum samples to assess a number of conditions.

This application note describes a simple supported liquid extraction method for extraction of estrone and estradiol from human serum. An analytical HPLC-MS/MS method that avoids the use of derivatization or fluoride adduct stabilization (two approaches often used to enhance method sensitivity) is used. High, reproducible analyte recovery and low detection limits are achieved.

ISOLUTE® SLE+ Supported Liquid Extraction plates and columns offer an efficient alternative to traditional liquid-liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation, and significantly reduced sample preparation.

Analytes

Estrone (E1) and Estradiol (E2)

Sample Preparation Procedure

Format: ISOLUTE® SLE+ 400 μL Supported Liquid Extraction Plate, Part Number 820-0400-P01

Sample Pre-treatment

Add 250 μL of stripped serum to 100 μL of 25% IPA (aq) followed by 12.5 μL of 1000 ng/mL 1 stock of E1 and E2 in methanol and equilibrate for 10 minutes to simulate a 50 ng/mL sample. Dilute subsequent standards (0.001, 0.002, 0.005 and 5 ng/mL) from a 0.1 μg/mL stock in MeOH using H2O/ACN/MeOH (2:1:1, v/v).

Sample Loading

Load 350 μL of pre-treated sample into each well using a Biotage® PRESSURE+ 96 Positive Pressure Manifold (PPM-96) and allow to absorb for 5 minutes.

Analyte Extraction

Apply 600 μL of dichloromethane (DCM) and wait for 5 minutes. Apply a second aliquot of 600 μL of DCM and elute under gravity. Repeat with a third aliquot of DCM (600 μL). Apply vacuum or positive pressure (5–10 seconds) to pull through any remaining extraction solvent.

Post Elution and Reconstitution

Dry the extract to dryness in a stream of air or nitrogen using a Biotage® SPE Dry® 96 (40 °C at 80 L/min).

Reconstitute with 250 μL of H2O/ACN/MeOH (2:1:1, v/v).