Alexander Gaudl (Presenter)
Leipzig University
Bio: Alex, the chemist, began his master thesis in 2012 at the institute of laboratory medicine, clinical chemistry and molecular diagnostics of Leipzig University concerning the quantitation of endocannabinoids in human plasma via LC-MS/MS. Although being drawn to analytical chemistry since his bachelor thesis, it was not until finishing his master’s degree that he had finally become the analytical chemist. His subsequently beginning doctorate let him delve deeper into LC-MS/MS analysis with the quantitation of steroid hormones in human body fluids and hair being his main focus. Now, he plans to take on the neuroendocrine axis by means of LC-MS/MS for eventually helping in comprehending the development of depressive disorders.
Authorship: Alexander Gaudl(1), Jürgen Kratzsch(1,2), Wieland Kiess(2,3), Joachim Thiery(1,2), Uta Ceglarek(1,2)
(1) Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, Leipzig University, (2) LIFE – Leipzig Research Center for Civilization Diseases, Leipzig University, Germany, (3) Hospital for Children and Adolescents, University Hospital, Leipzig University, Germany
| Short Abstract Finally, LC-MS/MS assays are applicable to quantitative steroid analysis in clinical routine diagnostics substituting immunoassays due to superior selectivity and comparable sensitivity. The broadsword MRM allows massive multiplexing of analytes for maximum flexibility. Matrices like hair, dried blood and urine, however, can outplay said approach generating the need for a higher method specialization, meaning increased specificity and selectivity while sacrificing flexibility. Introducing the scalpel MS3 provides that specialization. It offers superior quantitation in the analysis of hair cortisol and dried blood 17-OHP and can even function as an emergency tool in randomly occurring problems in serum or saliva analysis. |
Long Abstract
Introduction: The cat is out of the bag. Quantitative steroid analysis via LC-MS/MS is applicable to clinical routine diagnostics substituting immunoassays due to superior selectivity and comparable sensitivity by now. Multiplexed assays covering a multitude of analytes with multiple reaction monitoring represent the gold standard in this regard. This sort of broadband method, however, might be overpowered by more complex biological matrices, which demand a higher degree of specialization, like hair and dried blood for example. Utilizing the ion trap in MS3 experiments provides said degree, increasing the methods selectivity and eliminating interfering signals. We present a LC-MS/MS(/MS) method featuring MS2 as standard tool for steroid analysis and MS3 as makeshift when encountering problems due to matrix complexity. This way, procedures in high throughput analysis can be simplified and new research pathways are opened.
Methods: Plasma, serum, saliva and urine samples as well as hair and dried blood extracts (each 100 µl) were prepared by dilution with 200 µl precipitating agent (methanol/aqueous ZNSO4 80/20 v/v including internal standard) prior to tandem mass spectrometric analysis. Whole hair (10-20 mg) was washed with water and acetone and incubated with 1.5 ml methanol for 24 hours for extraction. One dried blood spot (3 µl) was incubated with 120 µl water for 30 minutes for extraction. Online solid phase extraction for sample cleanup and analyte enrichment (Applied Biosystems POROS® R1, 30 x 2.1 mm) was combined with rapid reverse phase liquid chromatography (Chromolith® High Resolution RP-18, 25 x 4.6 mm) via automatic column switching. Detection by tandem mass spectrometry was performed on an AB SCIEX QTRAP® 6500 applying modified electrospray ionization in positive and negative ion mode, multiple reaction monitoring and MS³. Deuterium-labeled analytes were used as internal standards.
Results: With a total run time of 4.0 min the lower limits of quantification ranged from 10 pg/ml for E2 up to 1 ng/ml for DHEAS. The overall imprecision and accuracy in plasma/serum were 2.9 15.3% and 89.1-103.5%, respectively. Method comparisons to routine immunoassays showed good correlation. Impairment of chromatographic quality due to complex matrices and their pre-analytical factors could be eliminated applying MS³. Moreover, in hair analysis the potential concentration altering capacity of heat, UV radiation and artificial coloring could be proved quantitatively. Quantitation in dried blood via MS3 also proved itself superior to MS2 due to its matrix neglecting features.
Conclusion: Steroid analysis in routine and research is on the verge of being completely conquered by LC-MS/MS with MS2 analysis as its multi-purpose tool. Matrices like urine, dried blood, hair and rarely even plasma or saliva can outplay said approach generating the need for a higher method specialization. Utilizing the ion trap in MS3 experiments enables much higher selectivity for eliminating impairment due to complex matrices.
References & Acknowledgements:
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