Callee Walsh (Presenter)
Protea Biosciences
Authorship: Callee M. Walsh (1), Kevin Girvan (2), Kim Christensen (3), Haddon Goodman (1)
(1) Protea Biosciences, Morgantown, WV, (2) Elithea Sciences, Fort Lauderdale, FL, (3) Markertest, Scottsdale, AZ
| Short Abstract Laser ablation electrospray ionization mass spectrometry (LAESI-MS) is a technique for high throughput screening from complex liquid matrices. LAESI-MS was used in high throughput screening of urine containing polyethylene glycol markers, which are used in drug screening to assure urine identity. Detectability of polyethylene glycol markers in urine indicate utility for expected concentrations within patient samples. For high throughput screening, data acquisition time was 2.3 seconds per well and 13 minutes for 96-well plate, considerably increasing efficiency. |
Long Abstract
High throughput screening of targeted compounds in complex matrices is a task that is traditionally been performed using LC-MS. However, even with the most optimized methodology, chromatographic separations typically require several minutes in addition to sample preparation processes, which also add time and material costs. Alternatives to LC-MS for certain applications would be beneficial to provide higher throughput screening of biological samples to reduce instrument usage and time. Laser ablation electrospray ionization (LAESI) is a direct analysis, ambient ionization source that interfaces to a variety of types of mass spectrometers. The process of LAESI uses a mid-infrared laser to produce a fine mist of droplets from liquid samples that is then ionized by electrospray. Using this rapid sample introduction method, LAESI-MS has been employed in qualitative and quantitative applications in neat and complex matrices with minimal to no sample preparation (1,2).
In this work, LAESI-MS was used to evaluate, in a qualitative manner, the presence or absence of polyethylene glycol markers (PEG; Marker Test UR Code™) within urine matrix, both native and diluted. UR Codes are oral capsules providing a patient with polyethylene glycol molecules in a specific combination. The subsequent urine “codes” can then be evaluated to assure urine samples have not been tampered with or substituted and, in fact, originate from the patient providing the sample. This technology could be especially useful in the drug testing industry where the PEG markers provided successful identity to urine provided by patients being monitored for drug use (3).
The current experiments described determine the utility of LAESI-MS qualitative measurement of urine samples containing biologically relevant concentrations of PEG markers. In the current experiments, octaethylene and nonaethylene glycol standards were diluted serially (1x10-5 to 1x10-8) in urine and diluted urine (1:10 with HPLC grade water). Urine standard preparations were evaluated with the LAESI® DP-1000 Direct Ionization System™ interfaced to a Thermo Scientific LTQ Velos™ and a Thermo Scientific Q Exactive™ to determine the minimum detected concentration from each of the dilution series. Signal-to-noise ratios were determined for each of the minimum detected concentrations. Using direct analysis without sample clean-up, the majority of each of the PEG standards was detected as sodium and potassium adducts, although molecular ions were detectable. Approaches were focused on using full scan modes as well as SIM modes for salt adducts for further work. Diluted urine was assessed in order to determine whether greater sensitivity could be achieved by dilution of matrix effects; however, it was determined that the dilution provided no advantage to the sensitivity of the assay for the PEG markers of interest. Overall, for nonaethylene glycol, sensitivity was comparable between the LTQ Velos in SIM mode versus the Q Exactive in full scan mode (R=35,000) where the lowest detected amount for each was the 1x10-5 dilution (11,280 ng/ml). The Q Exactive in SIM mode at R=140,000 had the greatest sensitivity (1x10-6 dilution; 1,128 ng/ml). Similar results were observed for octaethylene glycol, and all results indicate that LAESI-MS provides sufficient sensitivity for the range of concentrations expected in clinical samples.
A high throughput screening simulation was performed using LAESI-MS with urine samples analyzed in 96-well plate format. Samples consisted of blank, diluted urine (1:10 dilution in water), 5,640 ng/ml nonaethylene glycol in diluted urine, and 11,280 ng/ml nonaethylene glycol in diluted urine loaded in a 96-well plate and analyzed in SIM mode with three technical replicate analyses of the plate. In two technical replicates, all wells were correctly identified as exhibiting the presence or absence of the single PEG marker. In one analytical replicate plate, there were five incorrect assignments. Data acquisition required 2.3 seconds per well and approximately 13.5 minutes for a 96-well plate.
The data acquired by the LAESI DP-1000 are collected within the same raw mass spectrometry file, which can be difficult to process in base data system software packages. For high throughput data processing, we have developed a software package called LAESI Bridge (Gubbs, Inc.). In customized LAESI Bridge reports, targeted masses of the PEG markers were extracted from mass spectrometer raw files on a well-by-well basis automatically. Data are populated into the report, which is a Microsoft® Excel application for further data processing and statistics to identify samples containing PEG markers.
Ongoing work is evaluating the efficacy for use of full scan analyses for detection of multiple PEG markers simultaneously and further optimization of the assay focusing on robustness.
Results from the LAESI-MS experiments demonstrate that this technology can rapidly, with precision provide efficacy as a screening tool for UR Codes within a urine matrix.
References & Acknowledgements:
(1) Deimler RE, Razunguzwa TT, Reschke B, Walsh C, Powell M, Jackson GP. 2014. Analytical Methods. 6:4810-3817.
(2) Beach DG, Walsh CM, McCarron P. 2014. Toxicon. 92:75-80.
(3) Jones JD, Atchison JJ, Madera G, Metz VE, Comer SD. 2015. Drug and Alcohol Dependence. 153: 201-206.
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Protea Biosciences |
| Board Member | no | |
| Stock | yes | Protea Biosciences |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |