Paul Roberts (Presenter)
Biotage GB Limited
Authorship: Rhys Jones1, Helen Lodder1, Lee Williams1, Alan Edgington1, Geoff Davies1, Paul Roberts1, Claire Desbrow1, Adam Senior1, Steve Jordan1, Victor Vandell2 & Elena Gairloch2.
1Biotage GB Limited, Distribution Way, Dyffryn Business Park, Cardiff, CF82 7TS, UK.
| Short Abstract In all areas of analytical testing it is vital to ensure proper measures are in place to reduce or eliminate cross contamination between samples, which could result in false positive and/or false negative results. Sample carryover in the LC/MS system is usually monitored early in the method development process. However, one area often overlooked is sample preparation. This involves multiple aspects: pipetting, sample transfer, extraction, evaporation and mixing steps. This poster will discuss various stages of the sample preparation process to determine the potential for cross contamination and present approaches to minimize and or eliminate the effect. |
Long Abstract
Introduction
In all areas of analytical testing it is vital to ensure proper measures are in place to reduce or eliminate cross contamination between samples, which could subsequently result in false positive and/or false negative results. Sample carryover in the LC/MS system is usually investigated early in the method development process. However, one area often overlooked is sample preparation. This involves multiple aspects: pipetting, sample transfer, extraction, evaporation and mixing steps. This poster will discuss various stages of the sample preparation process to determine and assess the potential for cross contamination and present approaches to minimize and or eliminate the effect.
Methodology
All work was performed using 96-well plates f due to the close proximity of samples and consequently increased potential for cross contamination in this format. Initial experiments utilised a dye, rhodamine B dissolved in multiple solvents with differing characteristics; MTBE, DCM and MeOH, as a visual marker of cross contamination. Occurrence of cross contamination was investigated in the pipetting, sample transfer steps, extraction protocol, evaporation and mixing steps. Dye evaporation experiments were repeated using various analyte suites spiked at high concentrations; typically 2 µg. All surrounding wells were spiked at the assay’s limit of quantitation. Evaporated samples were reconstituted in mobile phase prior to analysis. Samples were analyzed using a Waters ACQUITY IClass UPLC coupled to a XEVO TQS triple quadrupole mass spectrometer.
Results
Dye experiments highlighted multiple potential causes of cross contamination if experiments are not structured correctly. We investigated the difference between positive pressure and vacuum processing. The former demonstrated less potential for cross contamination due to increased penetration of the luer tips (outlet nozzles) into the collection plate. This was the case for semi-automated processing using a positive pressure system and the automation platform Biotage® Extrahera™. For vacuum processing it is important to ensure adequate penetration of the luer tips into the collection plate, due to different manifold spacing and SPE plate design in terms of tip length.
Mixing was investigated using square and round well collection plates. Subtle differences were observed between the plates and care should be taken when selecting vortex speeds combined with solvent volumes for each.
Evaporation experiments used dye spiked into multiple positions in 96-well plates, while all surrounding wells were populated with blank solvent. The parameters investigated for evaporation studies were: gas flow rate, temperature, needle height position and volume of solvent in the wells. All parameters were demonstrated to be important and need to be considered depending on experimental set up. Evaporation experiments were confirmed when investigating different analyte suites combined with LC-MS/MS analysis. Analyte suites were selected based on their varying degree of volatility and hydrophobicity; amphetamines, opiates and benzodiazepines were investigated. Experiments were performed under “normal operating” conditions and replicated using the Biotage® ACT plate adapter. The adaptor is designed to fit on top of square 96-well collection plates, while the needles of the evaporator penetrate through the chimneys during evaporation. This design reduces the open surface area of the top of the wells, directing evaporated solvent and gases away from adjacent well therefore substantially reducing the likelihood of cross contamination.
Conclusion
This poster illustrates the potential for cross contamination during various stages of the method development process. All aspects from pipetting, solvent properties, analyte volatility can influence cross contamination. One of the most difficult to eliminate was during the evaporation process. We demonstrate the effective use of a novel plate cap mat for reducing or eliminating evaporative cross contamination.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| 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: | no |