Cortisol Everywhere! Neus Fabregat-Cabello (Presenter) University of Liège, CHU de Liège >> POSTER (PDF) Presenter Bio: Neus Fabregat Cabello is Post-Doc at the Department of Clinical Chemistry, University Hospital of Liège (CHU de Liège, Belgium) under the supervision of Prof. Etienne Cavalier since November 2015. At the moment she is responsible for the development of new mass spectrometry applications based on LC-MS/MS for endocrinology research dealing with small molecules, most of them related to steroids and vitamin D metabolites.

Authors: Benjamin Arias-Carnero(1), Stéphanie Peeters(1), Marine Deville(2), Caroline Le Goff(1), Etienne Cavalier(1), Neus Fabregat-Cabello(1).
(1) Department of Clinical Chemistry, CIRM, University of Liège, CHU Sart-Tilman, Liège, Belgium (2) Laboratory of Clinical, Forensic and Environmental Toxicology, CIRM, University of Liège , CHU Sart-Tilman, Liège, Belgium

Our problem with a routine LC-MS/MS system is related to the presence of an isobaric interference of cortisol from an unknown source. After cleaning and checking the proper function of the LC system, we performed an exhaustive study of all the possible contamination sources that can affect the method performance. Bovine Serum Albumin (BSA) seems the critical reactive that contained the highest amount of this interference, among other sources tested. Identity of this compound was obtained by QTOF analysis and resulted to be cortisol.

Problem

On a Friday afternoon, when running the first solvent blanks on our instrument and before the System Suitability Test (SST), we observed a huge contamination of cortisol, leading to peaks with an intensity between 10E6-10E7 cps. Our field service engineer checked the proper function of the instrument and removed all contamination traces. However next Monday, contamination reappeared.

Method Information

-100 µL Urine with 10 µL Internal Standard(IS) and 400 µL ammonium acetate extraction with ethyl acetate or 50 µL of serum with 20µL and 1600 µL of MTBE for the extraction

-Shimadzu Prominence UFLCXR

-Sciex QT5500

-MP-A : H2O 0.1% formic acid + ammonium acetate 2mM

-MP-B : MeOH

-6.10 min gradient LC program, 0.325mL/ min total flow rate

-Column : 50 x 2mm, 2.7µm C 18, with guard cartridge

-Column oven 40 °C

-Injection volume 5µL

-Quantitative MRM acquisition

Troubleshooting Steps

In a first approach, a thorough cleaning of the instrument was performed, which included the following steps:

1.Replacement of the rinse port cap of the injector

2.Cleaning the outside of the injector needle and needle seal with isopropanol

3.Wash settings from the autosampler were modified by increasing the Rinse dip time to 5 seconds.

4.Injection of several acetonitrile blanks in order to clean the system

These approaches permitted to completely clean the instrument. When we observed again the contamination we studied all the possible contamination sources (after decontamination of the system) by individual injection of the suspect solutions in LC-MS glass vials.

1.Ammonium acetate at 10mM and 1N used for sample and mobile phase preparation

2.Mobile phases A and B

3.Injection of water and methanol from different recipients used at the lab (glass/plastic).

4.Internal standard

5.Phosphate Bufffer Saline (PBS) with 0.1% of Bovine Serum Albumin (BSA) from different sources

6.Injection of air from empty vials using another C18 colum

7.Study of the identity of the contamination source in the reagents by QTOF

Outcome

Contamination coming from the instrument was solved by increasing the rinse dip time from 0 to 5 seconds and by changing the rinse port cap diameter. In the second study, two contamination sources were found, being the ammonium acetate solutions and the BSA. The identity of the contaminant on the BSA was confirmed by QTOF as cortisol even though we suspected from a plasticizer. We still do not know how cortisol (or hydrocortisone) reached these materials.

We acknowledge the help given by Christophe Bries.