Extraction of a Comprehensive Steroid Panel from Human Serum Using Biotage® Mikro ABN SPE Microelution Plates Prior to LC/MS-MS Analysis

This application note describes the extraction of steroid hormones from human serum using Biotage^® Mikro ABN microelution plates prior to LC/MS-MS analysis. The simple sample preparation procedure delivers clean extracts and analyte recoveries mostly greater than 70% with RSDs lower than 5% for most analytes. Linearity of greater than 0.99 is achieved for all analytes from 1–1000 pg/mL. An elution volume of 30 µL is used, which is simply diluted prior to analysis, avoiding a time consuming evaporation step.

Mikro plate extraction allows for very low elution volumes and enhanced workflow efficiency.

Cortisol, 18-OH-Corticosterone, 21-Deoxycortisol, Cortisone, Estradiol, 17-OH-Pregnenolone, Aldosterone, 11-Deoxycortisol, Corticosterone, Estrone, Dehydroepiandrosterone (DHEA), 17-OH-Progesterone, Dehydroepiandrosterone sulfate (DHEA-S), Testosterone, Dihydrotestosterone (DHT), Pregnenolone, Androstenedione, 11-deoxycorticosterone, Progesterone

Internal Standards

Dihydrotestosterone- D₃ (DHT-D₃) and Aldosterone-D₄

Format Biotage^® Mikro ABN Plate, 2 mg, part number 600-0002-LVP

Sample Pre-Treatment

Spike serum (200 µL) with internal standard solution and allow to equilibrate for 1 hour. Dilute with 1% formic acid (1:1, v/v). Mix.

Internal standard solution consisted of 10 pg/µL methanolic solution. 20 µL was added to 200 µL serum to give a 1 ng/mL spike concentration.

Conditioning

Condition wells with methanol (100 µL)

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