This document introduces the use of the Agilent 1200 Series liquid chromatography (LC) system coupled to the 6410 Triple Quadrupole Mass Spectrometer (QQQ) by way of the G1948B electrospray ionization (ESI) source, for the analysis of anabolic substances in urine.
The document describes a method for detecting and confirming anabolic substances in urine to meet World Anti-Doping Agency (WADA) Minimum Required Performance Levels (MRPLs). The method utilizes the Agilent 1200 Series liquid chromatography system coupled to a 6410 Triple Quadrupole Mass Spectrometer (QQQ) with an electrospray ionization (ESI) source. A 2.1 mm id C18 column with a 3.5-um particle size is employed to achieve high chromatographic resolution and speed, with all seven compounds eluting within 10 minutes at a flow rate of 0.4 mL/min. The analysis targets compounds including clenbuterol, 19-norandrosterone, 4B-OH-stanozolol, and others, with specific attention to the use of Girard's Reagent P for derivatization of 19-norandrosterone to improve sensitivity.
| Column dimensions | 2.1 x 100 mm |
|---|---|
| Column particle size | 3.5 um |
| Column temperature | 50 C |
| Injection volume | 2 uL |
| Q1 resolution | 0.7 amu |
| Q2 resolution | 0.7 amu |
| Drying gas temp | 350 C |
The analysis uses the Agilent 1200 Series liquid chromatography system coupled to the 6410 Triple Quadrupole Mass Spectrometer.
The limit of detection for clenbuterol is approximately 3 pg on-column, which corresponds to 1/40th of the Minimum Required Performance Level.
The sample mixture is incubated at 50 °C for one hour before extraction.
The analysis is performed at a flow rate of 0.4 mL/min.