William Brewer (Presenter)
DPX Labs, LLC
Bio: William E. Brewer, PhD, is a Research Associate at the University of South Carolina. He is also the founder and Chief Scientific Officer of DPX Labs.
Authorship: William E. Brewer(*1,2), Kaylee R. Mastrianni (1), Evan S. DiVirgilio (2), and Stephen L. Morgan (1)
(1) Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208; (2) DPX Labs, LLC, 541 Main St. Suite 116, Columbia, SC 29201
| Short Abstract A high throughput sample preparation method for drugs of abuse (39 compounds) in oral fluid was developed using DPX tip technology coupled with the Hamilton NIMBUS96 robotics system. A well plate of 96 samples takes 10 minutes to complete sample preparation followed by solvent evaporation, reconstitution and injection into the LC-MS/MS system. The method was evaluated for linearity, precision, extraction efficiency, and limits of detection and quantitation. The method established herein is highly reproducible and provides the necessary sensitivity for forensic and clinical purposes. |
Long Abstract
Introduction
Oral fluid testing is gaining momentum as an alternative method for determining drug use in many laboratory settings, including forensic, workplace, clinical and drug treatment. Oral fluid is ideal because it’s minimally invasive, observable, and easy to collect. Oral fluid and the buffer preservative used in collection generally complicate analysis due to large matrix effects. Therefore, sample preparation is generally required and preferred. Dispersive Pipette Extraction (DPX) tips are a dispersive solid phase extraction (SPE) product that is readily adaptable to automation for rapid and efficient sample preparation. A mixed mode DPX tip (10 mg), with both weak anion exchange and reversed phase properties, was used on the Hamilton NIMBUS96 robotics system to enable robust processing of 96 samples in approximately 10 minutes.
Methods
Well plates containing oral fluid (QuantisalTM, 250 uL) and internal standard were loaded on to the NIMBUS system, along with reservoirs of water, 30% methanol, and 0.1% formic acid in acetonitrile. The NIMBUS system fills a well plate with 200 uL of water, a well plate with 500 uL of 0.1% FA in acetonitrile (eluent 1), and a well plate with 250 uL of 0.1% FA in acetonitrile (eluent 2, which provides a 2nd elution step to improve recoveries). The DPX Mixed Mode tips are then picked up and conditioned in a solvent reservoir with 30% methanol. After conditioning, the DPX tips aspirate and dispense the samples five times in order to bind the drugs of abuse. Water is then aspirated and dispensed to remove free salts. The analytes of interest are eluted by aspirating and dispensing 500 uL of 0.1% FA in acetonitrile (eluent 1) three times. Subsequently, a second elution is performed by aspirating the 250 uL of 0.1% FA in acetonitrile and dispensing into the well that contains the original eluent 1. The total eluent (750 uL) is solvent evaporated to dryness and reconstituted in 125 uL of 10% methanol in water, for a 2-fold saliva dilution factor. Analysis was performed on a Thermo TSQ Vantage triple quadrupole instrument with an Agilent 1260 HPLC using an Agilent Poroshell EC-C18 column (3.0 x 50mm, 2.7um) with a 10 uL injection.
Results
The extraction procedure had recoveries above 80% for most compounds. The only compounds with less than 50% recoveries were the highly polar gabapentin and pregabalin. Results from this method were linear, sensitive, and reproducible. All correlation coefficients were greater than 0.99 for at least 2.5-500 ng/mL, but most were linear from 0.625-500 ng/mL. Relative standard deviations were calculated using 6 replicate extractions at 100 ng/mL and ranged from 1.6 to 8.0. Limits of detection were calculated as 3.3*σ/m, where σ is the standard deviation of the lowest non-zero calibrator and m is the slope of the calibration line. Limit of quantitation is 10*σ/m. Limits of detection ranged from 0.023 to 4.3 ng/mL and limits of quantitation ranged from 0.069 to 13 ng/mL.
Conclusion
The method described herein provides the necessary recoveries, sensitivity, and reproducibility for an easy and reliable sample preparation method in a high throughput setting. The main advantages of this automated DPX method are the methods are non-tedious and rapid to perform.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | yes | DPX Labs, LLC |
| Salary | no | |
| Board Member | yes | DPX Labs, LLC |
| Stock | yes | DPX Labs, LLC |
| Expenses | yes | DPX Labs, LLC |
IP Royalty: yes
IP Desc:DPX Labs, LLC
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |