Teresa Pekol (Presenter)
Extend Consulting
Authorship: Teresa Pekol (1), Jonathan Danaceau (2), Sherri Naughton (2), Kendon Graham (2)
(1) Extend Consulting, (2) Waters Corporation
| Short Abstract Automated sample preparation improves laboratory operations by a) reducing errors in sample tracking and preparation, b) producing more consistent results free of analyst-to-analyst variation, c) allowing analysts to work more efficiently, and d) minimizing laboratory hazards in regard to solvent exposure and repetitive motions associated with manual pipetting. For labs considering automated sample preparation, the aim of this study was to compare the performance and benefits of a Tecan Freedom EVO® 100 liquid handler to manual sample preparation using a routine clinical research application. |
Long Abstract
Introduction
Automated sample preparation improves laboratory operations by a) reducing errors in sample tracking and preparation, b) producing more consistent results free of analyst-to-analyst variation, c) allowing analysts to work more efficiently, and d) minimizing laboratory hazards in regard to solvent exposure and repetitive motions associated with manual pipetting. For labs considering automated sample preparation, the aim of this study was to compare the performance and benefits of a Tecan Freedom EVO® 100 liquid handler to manual sample preparation using a routine clinical research application.
Methods
A solid-phase extraction (SPE) sample preparation method was developed for 21 opiate/opioid drugs and metabolites. An enzymatic hydrolysis step was not included in the method; rather, morphine, hydromorphone, oxymorphone, and codeine glucuronides were included as analytes. Corresponding deuterated internal standards were used for all analytes except hydromorphone-3-β-D-glucuronide.
A Waters Oasis® MCX µElution plate was used for the extraction. To 150 µL of urine, 50 µL of internal standard in stabilized Surine™ (Dyna-Tek Industries) was added followed by 200 µL of 4% phosphoric acid. Samples were loaded onto a conditioned and equilibrated SPE plate. Samples were washed with 200 µL of water followed by 200 µL of methanol and eluted with two 50 µL additions of 5% ammonium hydroxide in 60/40 methanol/acetonitrile. Samples were dried under nitrogen and reconstituted with 50 µL of 2% formic acid in 98/2 water/ACN. For analysis, a Waters ACQUITY UPLC® with BEH 2.1x100 mm, 1.7 µm column was used with a Xevo® TQD tandem quadrupole mass spectrometer. The injection volume was 5 µL and the run time was 6.2 minutes.
The EVO® 100 liquid handler has a user-configurable worktable and components to automate a variety of sample preparation operations. For this study, the liquid handler was equipped with a 4-tip liquid handling arm for sample transfers and dispensing liquids, robotic manipulator arm for moving plates, bar code reader, plate shaker, wash station, and vacuum manifold for performing the extraction. A proprietary file converter (available from Waters) interfaces the liquid handler with the mass spectrometer by automatically creating sample lists with specimen IDs, plate locations, and pre-populated method information for direct import into MassLynx™. Manual extractions were performed by an analyst with more than 10 years’ experience with SPE; a multi-channel pipette was used throughout the manual extraction.
An automated and manual sample preparation run was performed on each of three days to evaluate linearity and intra- and inter-day precision and accuracy. Each run included duplicate bracketing calibrators, prepared at 6 levels from 20-1000 ng/mL (4-200 ng/mL for fentanyl/norfentanyl), and three levels of QCs (N=6) at 30, 150, and 750 ng/mL (6, 30, and 150 ng/mL for fentanyl/norfentanyl).
Results and Conclusions
Automated sample preparation produced results similar and in many cases statistically equivalent to manual sample preparation. The time required for automated sample preparation using a 4-tip liquid handling arm was also similar to that required for manual preparation; however, automated sample preparation was overall faster when the proprietary file converter was used to automatically create a MassLynx-compatible sample list. Automated sample preparation has the additional benefits of allowing analysts to spend more time on tasks requiring human intervention while also reducing the potential for error at multiple points during sample preparation and analysis.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Waters Corporation |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | Waters Corporation |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |