David Hall (Presenter)
SPEware Corporation
Bio: I have over 30 years of experience in sample preparation of biological matrices for analysis for drugs of abuse and pharmaceuticals, including 15 years of experience with LC-MS/MS method development and analysis. My professional career includes positions with HFL Sport Science, Inc. (now LGC Limited), the University of Florida Racing Laboratory, the Ohio Department of Agriculture Analytical Toxicology Laboratory, the Ohio State University Analytical Toxicology Laboratory, ANSYS Diagnostics, Inc., and the Truesdail Laboratories Racing Laboratory.
Authorship: David Hall, Karsten Liegmann, John Laycock, Ph.D., Phil Dimson
SPEware Corp., 14180 Live Oak Ave, Suite I, Baldwin Park CA. 91706
| Short Abstract Increasingly, clinical and toxicology laboratories are asked to analyze oral fluid samples. The ease and non-invasive nature of oral fluid collection makes it an attractive alternative to more traditional collection of urine or blood. Here, we present a process for robotic pipetting of oral fluid samples, automated extraction of the samples, online evaporation and reconstitution of the extracts (“selective elute and shoot”) and subsequent analysis of the extracts for the presence of a representative list of drugs and drug metabolites. Analyses of the extracts were performed on an AB Sciex 5000 LCMS system with focus on linearity and LLOQ measurements. |
Long Abstract
Increasingly, clinical and toxicology laboratories are asked to analyze oral fluid samples for the presence of both prescription medications and illicit drug substances. The ease and non-invasive nature of oral fluid collection makes it an attractive alternative to the more traditional collection of urine or blood. Oral fluid, however, poses challenges to the laboratory. Typical collection devices collect only ~1 mL of oral fluid, which is then diluted with a buffer already in the device, resulting in a total specimen volume of no more than 4 mL. Additionally, the concentrations of analytes in oral fluid are significantly lower than those found in urine.
The combination of these factors produces an analytical challenge that requires the use of efficient sample preparation techniques and more sensitive mass spectrometers.
Sample preparation in laboratories has historically been very labor intensive. Integration of sample accessioning and of instrumental results into LIMS software have been streamlined and have improved the reliability of sample identification, but the intermediate steps of preparation of the samples for instrumental analysis have remained, to one degree or another, manual in nature.
In recent years, the use of robotic liquid handlers has done much to address the issues associated with the transfer of samples, buffers, internal standards, and other reagents, in preparation for extraction and purification. The use of barcode readers has been used to create worklists for transfer of sample batch information from LIMS to liquid handlers to GCMS and LCMS systems.
Similarly, automated solid phase extraction devices have addressed the purification steps used to prepare the samples for instrumental analysis. What were once purely multi-step manual processes on vacuum boxes or positive pressure manifolds (column conditioning, sample application, sorbent washing, sorbent drying, and elution) are now performed by devices built specifically for these purposes.
Here, we present data for the extraction of oral fluid samples, collected using 3 commercially available devices, with a fully automated platform. The system runs unattended from the time the oral fluid samples are placed on the deck until the sample extracts are ready for transfer to the LCMS autosampler.
The system consists of a SPEware IP8 Integrated Positive Pressure Plate Processor coupled to an 8-probe Hamilton Microlab® Starlet liquid robotic handler. The Starlet is equipped with a barcode reader and gripper arm capability. Samples in barcode-labelled test tubes are placed on the deck in strip racks. The samples (volume 0.5 mL) are transferred via disposable tips to a 96 well SPE plate, along with buffer and internal standard. The samples in the SPE plate are then transferred by the gripper arm to the IP8 for processing.
The IP8 SPE platform features dual 96-well SPE capability, with microprocessor controlled positive pressure profiles for liquid application and switching valves for access to up to 11 solvents. Column conditioning steps (when required), sample application, column washing, sorbent drying, extract collection and drying, and extract reconstitution are all performed by the IP8 without user intervention.
A suite of analytes were chosen as model compounds to demonstrate this process. Analyses for the selected compounds are frequently required by pain management and rehabilitation management laboratories. Clean sample extracts provide improved chromatographic performance and signal to noise ratios as well as decreased frequency of instrument maintenance.
SPEware Narrow Bore OFX 96-well plates were used for the extractions. These plates feature reduced dead-volume SPE columns containing a narrow particle size distribution sorbent optimized for use with oral fluid samples. For this work, selective elution solvents were used in order to improve extract cleanliness. Elution solvent volumes were optimized for maximum recovery. The resulting extracts were clear and colorless.
Selective elution solvents typically contain water immiscible, non-HPLC compatible components. Such solvents must be evaporated before dissolving the extracts in HPLC mobile phase for analysis. This step requires transfer of the collection tubes or plates to a separate device designed for this purpose. The use of Narrow Bore columns, however, permits the use of very low elution volumes (25-200uL, depending on bed mass and the chemistry of the elution). These low volumes are evaporated using the positive pressure manifold on the IP8 – transfer to a separate device is not required. The resulting dried residues are dissolved in mobile phase, delivered by the IP8, in preparation for transfer to the autosampler.
In this work, the sample extracts were analyzed in MRM mode using an AB Sciex 5000 triple-quadrupole mass spectrometer interfaced with a Shimadzu liquid chromatograph. Data are presented showing limit of detection, limit of quantification, and linearity of the total process.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | SPEware Corporation |
| 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 |