Kristin Jones (Presenter)
Biotage
Authorship: Kristin Jones±, Tifanie Vansach¥, Kristyn Astern¥, Victor Vandell±
±Biotage, 10430 Harris Oaks Blvd., Charlotte, North Carolina, 28269, USA ¥Physicians Lab, 4950 Communications Ave, Boca Raton, FL 33431
| Short Abstract Estrogen, androgen, and glucocorticoid metabolism can be used to assess overall hormonal balance during hormone therapy. Urine is the recommended testing matrix for quantification of primary estrogen levels as well as secondary estrogen metabolites when monitoring overall hormone balance, therapy, and detoxification; non-invasive collection allows for sampling over a 24-hour period, providing insight to circadian rhythm. Since many samples are generated for a single patient in one day, a fast and robust testing protocol is needed during clinical testing. Here we demonstrate a rapid and reliable sample preparation method using SLE+ to extract a suite of 30 hormone analytes from a hydrolyzed urine matrix. LC-MS/MS analysis in a single injection shows that matrix effects are eliminated by the SLE+ protocol and that analyte recovery and sensitivity are adequate for clinical interpretation. |
Long Abstract
Introduction:
Estrogen, androgen, and glucocorticoid metabolite concentrations and ratios can be used to assess overall hormonal balance during hormone replacement therapy. Urine is the recommended testing matrix for accurate quantification of primary estrogen levels as well as secondary estrogen metabolites when monitoring overall hormone balance, estrogen therapies, and estrogen detoxification; the non-invasive collection allows for sampling over a 24-hour period, providing insight to a patient’s circadian rhythm. Since many samples are generated for a single patient in one day, a fast and robust testing protocol is needed for extraction and analysis during clinical testing. Here, we demonstrate a rapid and reliable sample preparation method using Support Liquid Extraction (SLE+) to extract a suite of 30 hormone analytes from a hydrolyzed urine matrix. LC-MS/MS analysis in a single injection shows that matrix effects are eliminated by the SLE+ protocol and that analyte recovery and sensitivity are adequate for clinical interpretation.
Aim:
This poster details the use of Supported Liquid Extraction (SLE+) sample preparation to extract a suite of 24 hormone metabolites from a complex biological matrix.
Method:
Supported Liquid Extraction in a 96 fixed well plate format was used to extract estrogen, androgen, and glucocorticoid metabolites spiked at concentrations from 0.3 -100 ng/mL. Urine samples (500µL) were hydrolysed with β-Glucuronidase (H. pomatia, 3000 units/mL) in sodium acetate buffer (150mM, pH 4.0) at 38°C for 16 hours. Sample pretreatment conditions were screened and deemed unnecessary prior to loading 380µL of sample onto the sorbent bed. Extraction solvents were screened and the optimal combination was determined to be dichloromethane followed by ethyl acetate. Extracts were evaporated to dryness and then re-constituted in 100µL 100mM sodium bicarbonate, 100µL 1mg/mL dansyl chloride mobile phase and incubated at 64°C for 5 minutes in order to selectively dansylate estrogen metabolites. After dansylation, each sample was diluted with 25uL deionized water prior to injection onto a Thermo Scientific C-8 XL (50x0.5 mm) TurboFlow column. Samples were eluted from TurboFlow onto a Thermo Scientific Accucore C18 column (100x3.0mm, 2.6µm) using an Agilent 1200 Series Pump in line with a Thermo Scientific TSQ Quantiva triple quadrupole mass spectrometer operating in positive ESI mode.
Results:
Steroid species are generally neutral at pH 4-8, and pretreatment screening showed that no pH modification was necessary for optimal recovery using SLE+. The optimal elution solvent combination was 900µL dichloromethane followed by 900µL ethyl acetate. Average recoveries of greater than 75% were observed for the target analytes in urine. All isobars were resolved in a single injection, 13 minute LC method. Clinically significant LLOQs ranged from 0.3-3ng/mL, with observed S/N of at least 10 for each analyte. Calibration curves ranging from 0.3-500ng/mL were generated for all 30 analytes in extracted, dansylated urine matrix. More work is needed to validate the method.
Conclusion:
We present a fast, simplified approach for extraction of 30 estrogen, androgen, and glucocorticoid metabolites using a non-invasive matrix (urine) in a clinical hormone monitoring application.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Biotage |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |