Elena Gairloch (Presenter)
Biotage
Authorship: Victor Vandell±, Dan Menasco±, James Mullins±, Lee Williams¥ Rhys Jones¥, Paul Roberts¥
±Biotage, 10430 Harris Oaks Blvd., Charlotte, North Carolina, 28269, USA; ¥Biotage GB Limited, Dyffryn Business Park, Cardiff, CF82 7TS, UK.
| Short Abstract Introduction: Buprenorphine and Norbuprenorphine are typically problematic for analysis due to analyte stability issues during sample preparation. A fast and robust testing protocol is needed to address extracting the targets out of complex matrices typically encountered during toxicological testing. The sample preparation method would also be expected to yield good analyte recovery and minimum matrix effects. Here, we demonstrate new rapid and reliable sample preparation methods that were used to extract the target analytes from small amounts of complex biological matrix. Qualitative and quantitative data demonstrates the utility of these methods prior to LC-MS/MS analysis. |
Long Abstract
Introduction:
Buprenorphine and Norbuprenorphine are typically problematic for analysis due to analyte stability issues during sample preparation. A fast and robust testing protocol is needed to address extracting the targets out of complex matrices typically encountered during toxicological testing. The sample preparation method would also be expected to yield good analyte recovery and minimum matrix effects. Here, we demonstrate new rapid and reliable sample preparation methods that were used to extract the target analytes from small amounts of complex biological matrix. Qualitative and quantitative data demonstrates the utility of these methods prior to LC-MS/MS analysis.
Aim:
This poster outlines the use of two different sample preparation techniques, specifically Solid Phase Extraction and Supported Liquid Extraction to extract buprenorphine and norbuprenorphine from complex biological matrices.
Method:
Supported Liquid Extraction in a 96 fixed well plate format was used to extract Buprenorphine and Norbuprenorphine from whole blood spiked at concentrations from 1.0 -100 ng/mL. Sample pre-treatment consisted of a 1:3 dilution of blood (100µL) with 0.1% ammonium hydroxide (300µL). An optimal extraction solvent of ethyl acetate: acetonitrile: ammonium hydroxide was identified. Cation Exchange Solid Phase Extraction in a 96 well plate format was employed for the extraction of the same target analytes from urine and oral fluid (neat and buffered) spiked at a concentration range of 0.1-100ng/mL. Sample preparation consisted of a 1:9 dilution of the matrix with 0.1% Formic Acid. Extracts were evaporated to dryness, re-constituted in mobile phase and injected onto an Agilent 1200 coupled to a Sciex 4000 Q-trap® triple quadrupole mass spectrometer for analysis.
Results:
Averaged recoveries of greater than 90% were observed for the target analytes in urine and oral fluid. Averaged recoveries greater than 70% were observed for analytes in whole blood. All averaged recovery % RSDs were calculated at less than 10%. Measured matrix effects ranged from 8%-50% for the analytes in all three matrices. Calibration curves were generated for the analytes in all three matrices with precision and linearity determined to within ±15%.
Conclusion:
We present a fast simplified approach for extraction of Buprenorphine and Norbuprenorphine with reproducible recoveries for low analyte detection levels using a minimal amount of sample (100 µL).
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | |
| Board Member | no | |
| Stock | no | |
| Expenses | yes |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |