Alexander Gaudl(1), Yoon Ju Bae(1), Jürgen Kratzsch(1), Joachim Thiery(1), Uta Ceglarek(1)
(1)Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Germany
The quantitative analysis of endogenous steroid hormones cuts across plasma, serum, saliva, urine, dried blood and hair rendering immunological analysis laborious due to the need of different methods. We developed a unifying, highly adaptive LC-MS/MS-method covering the most important endogenous steroid hormones. With excellent inter day imprecision (3 15%), very high recovery (89 103%) and a total runtime of 5.0 min we enabled rapid, multi-matrix analysis utilizing MRM and MS³ in modified, positive and negative electrospray ionization. Online sample clean-up via automatic column switching combined with an extremely simple dilute-and-shoot sample preparation allows high throughput analysis in clinical routine diagnostics and epidemiological studies.
Introduction: For a fairly long time now quantitative analysis of endogenous steroid hormones goes way beyond the matrices plasma or serum. Glucocorticoids are analysed in saliva and hair for short- and long-term stress evaluation. Newborn screening is done in dried blood spots and urine for example is used for the assessment of drug abuse. To meet the particular needs of different matrices, different analytical methods have to be used, complicating a routine work flow. We present a unifying method that can be applied to the aforementioned matrices simplifying the everyday procedures in high throughput analysis.
Methods: Plasma, serum, saliva and urine samples as well as extracts of hair and dried blood (100 µl each) were prepared by dilution with 200 µl precipitating agent (methanol/aqueous ZnSO4 80/20 v/v including deuterated internal standards) prior to tandem mass spectrometric analysis. Whole hair (10-30 mg) was washed with water, 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 clean-up 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.
Results: Online sample clean-up, chromatographic separation and quantitative detection of 17 OHP, aldosterone (A), androstenedione (AE), cortisol (F), cortisone (E), DHEAS, estradiol (E2), progesterone (P) and testosterone (T) was achieved within a total run time of 5.0 min. The following lower limits of quantification were determined; for plasma/serum: 100 pg/ml for 17 OHP, 30 pg/ml for A, 30 pg/ml for AE, 20 pg/ml for F, 1 ng/ml for DHEAS, 10 pg/ml for E2, 50 pg/ml for P and 10 pg/ml for T; for saliva: 100 pg/ml for 17 OHP, 20 pg/ml for F, 40 pg/ml for E. The overall imprecision was 3.9-10.1% for 17 OHP, 6.0 10.6% for A, 3.1-5.1% for AE, 2.9-4.9% for F, 2.9-7.2% for E, 8.4-15.3% for DHEAS, 4.9-6.3% for E2, 3.6-5.2% for P and 3.3-5.0% for T. Recovery in plasma/serum was 96.2-100.8% for 17 OHP, A, AE, F, E2 and T, 89.1-89.7% for P and 103.5-130.1% for DHEAS. Method comparisons to commercially available immunoassays were carried out and showed good correlation, for example Elecsys® Estradiol III Assay, Roche, Mannheim, Germany (r = 0.87). For hair analysis intra and inter day imprecision (n = 10) were < 8.7% and < 11.2% for F and < 9.9% and < 13.9% for E. Impairment of chromatographic quality due to hair related pre-analytical factors could be eliminated applying MS³ in negative ion mode. The concentration altering capacity of those factors, however, remained and needed to be addressed. To that matter the influence of UV radiation, colorant, heat and length has been studied.
Conclusion: The developed tandem mass spectrometric method enables the robust and reliable quantitation of various endogenous steroid hormones in plasma, serum, saliva, urine, hair and dried blood spots adjusted to the needs of each appertaining task.