MSACL 2016 EU Abstract

Integration of Steroids Analysis in Serum Using LC-MS/MS with Full-automated Sample Preparation

Stephane Moreau (Presenter)
SHIMADZU Europa GmbH

Authorship: Stéphane Moreau (1): Daisuke Kawakami (2): Toshikazu Minohata (2)
(1) Shimadzu Europe GmbH, Duisburg, Germany: (2) Shimadzu Corporation, Kyoto, Japan

Short Abstract Currently sample preparation for the detection of steroids in serum by LC-MS/MS involves complex offline extraction methods such as solid phase extraction or liquid/liquid extraction, all of which require additional sample concentration and reconstitution in an appropriate solvent. These sample preparation methods are time-consuming, often taking 1 hour or more per sample, and are more vulnerable to variability due to errors in manual preparation. Our approach to offering a high sensitivity steroid detection method and timely, automated analysis of multiple samples is to use the automated sample preparation system coupled to the detection capabilities of a high-sensitivity LC-MS/MS.

Long Abstract

Introduction

Currently sample preparation for the detection of steroids in serum by liquid chromatography-mass spectrometry (LC-MS/MS) involves complex offline extraction methods such as solid phase extraction or liquid/liquid extraction, all of which require additional sample concentration and reconstitution in an appropriate solvent. These sample preparation methods are time-consuming, often taking 1 hour or more per sample, and are more vulnerable to variability due to errors in manual preparation. Our approach to offering a high sensitivity steroid detection method and timely, automated analysis of multiple samples is to use the automated sample preparation system coupled to the detection capabilities of a high-sensitivity triple stage quadrupole mass spectrometer.

Method

10 steroid hormones (cortisol, aldosterone, 11-deoxycortisol, corticosterone, 17-alpha-hydroxyprogesterone (17-OHP), 4-androstene-3,17-dione (androstenedione), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEAS), progesterone and testosterone) in serum were verified using CHS™ MSMS Steroids Kit (PerkinElmer, USA).

Serum sample was loaded directly into the automated sample preparation system (CLAM-2000 Shimadzu, Japan). The CLAM-2000 was programmed to perform protein precipitation using acetonitrile followed by filtration and sample collection. The sample is then transported using an arm from the CLAM-2000 to the HPLC without human intervention for LC-MS/MS analysis.

The treated samples were trapped using a MAYI-ODS column (2mm x 5mm) and then separated by Core-Shell Biphenyl HPLC column (Kinetex Biphenyl, 50mm x 2mm, 2.6μm, Phenomenex) at 40°C with a binary gradient system at a flow rate of 0.3 ml/min in 11 min.

Results

We evaluated this system using calibrator and control serum spiked with 10 steroids in Kit and carried out concurrent analysis over a range of concentrations for each steroid: cortisol (1.51-320 ng/mL), aldosterone (0.03-7.05 ng/mL), 11-deoxycortisol (0.08-18 ng/mL), corticosterone (0.29-62 ng/mL), 17-OHP (0.12-26 ng/mL), androstenedione (0.08-18 ng/mL), DHEA (0.31-65 ng/mL), DHEAS (12.9-2750 ng/mL), progesterone (0.12-26.5 ng/mL) and testosterone (0.03-7.2 ng/mL). The calibration curves that were generated had linear regression values of r2 >0.997 for each curve. The reproducibility (N=3) at seven concentrations, including LLOQ of each compounds was excellent (CV<10%).

We found that the sample preparation time was reduced from 60 minutes to 10 minutes by the automated system.

Conclusion

We completed steroid analysis using the automated sample preparation system coupled to LC-MS/MS. The results shows the capability of the system for large sample set analyses with improved accuracy and precision by eliminating human error associated with manual sample handling.

Novel Aspect

Automated sample preparation which interfaces directly to a fast scanning LCMS triple quadrupole improved reproducibility while achieving increased sample prep efficiency.

References & Acknowledgements:

Financial Disclosure

IP Royalty: no

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