Kaylee Mastrianni (Presenter)
University of South Carolina
Bio: I am a PhD candidate at the University of South Carolina in Dr. Stephen Morgan’s analytical chemistry lab. My research focuses on optimizing sample preparation methods for complex biological matrices for sensitivity, speed, and robustness using automated platforms, DPX technologies, and LC-MS/MS analysis.
Authorship: Kaylee R. Mastrianni (*1), Evan S. DiVirgilio 2, William E. Brewer (1,2), Stephen L. Morgan(1)
(1)Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208; (2) DPX Labs, LLC, 151 Powell Rd, Suite 116, Columbia, SC 29203
| Short Abstract A high throughput sample preparation method for drugs of abuse (38 compounds) in urine was developed using DPX tip technology coupled with the Hamilton NIMBUS96 robotics system. The sample preparation of two well plates of 96 hydrolyzed samples takes approximately 15 minutes to complete for LC-MS/MS analysis. 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
Urine analysis is a common method for drug testing because collection is easy, non-invasive, and it’s generally available in large volumes. While urine is less complex than other biological matrices such as blood and serum, the presence of endogenous compounds like urea, creatinine, proteins, and other organic and inorganic compounds interfere with the analysis of drugs and metabolites. Although sample preparation like solid-phase extraction (SPE) can remove the interferences, SPE is time consuming and can be the rate limiting step in urine analysis. The implementation of automated SPE in pipette tips is the key to rapid urine analysis. Dispersive Pipette Extraction (DPX) is a dispersive SPE technique that is performed within pipette tips. DPX tips can be used on the Hamilton NIMBUS96 platform in order to simultaneously extract 96 samples in about 8 minutes. For increased workflow, the platform can be designed to extract 192 samples in approximately 15 minutes.
Methods
Well plates containing hydrolyzed urine and reservoirs containing water, 30% methanol and 1% formic acid (FA) in methanol are loaded on to the NIMBUS system. The NIMBUS system fills a well plate with 200 uL of water and a well plate with 150 uL of 1% FA in methanol. 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 three times in order to bind the drugs of abuse. Water is then aspirated and dispensed to remove any free salts, urea and creatinine. The analytes of interest are eluted by aspirating and dispensing 1% FA in methanol three times. The eluent is simply diluted until an appropriate percentage of methanol is reached for injection (which is dependent on the LC conditions). In this case, 1050uL of water was added for a total volume of 1200 uL (12.5% methanol). 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
From start to finish, this fully automated DPX method takes approximately 15 minutes to prepare two 96 well plates of hydrolyzed samples. Recoveries were above 75% for most compounds, with only pregabalin and gabapentin being less than 50%. Results from this method are linear, accurate, and reproducible. All correlation coefficients were greater than 0.99 for at least 12.5-400 ng/mL, but most were linear from 6.25-800 ng/mL. Relative standard deviation was calculated using 7 replicate extractions at 400 ng/mL and ranged from 1.6 to 8.0. Limits of detection (LOD) 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 (LOQ) is 10*σ/m. Limits of detection ranged from 0.50 to 18 ng/mL and limits of quantitation ranged from 1.5 to 54 ng/mL.
Lower detection limits can also be obtained by using an alternate solvent evaporation procedure. The elution volume is increased to 500 uL to increase recoveries, and the eluent is then solvent evaporated and reconstituted in as little as 150 uL of 10% methanol.
Conclusion
The method described herein provides the necessary recoveries, sensitivity, and reproducibility for a reliable sample preparation method in a high throughput setting. A fully automated procedure minimizes human error and maximizes laboratory efficiency. Most importantly, with high quality data, turnaround time is improved by making data review easier to accomplish.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | yes | DPX Labs, LLC |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | yes | DPX Labs, LLC |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |