The International Association of Forensic Toxicologists (TIAFT 2023)
27-31 August, Rome, Italy
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RESTEK TECHNICAL POSTERS:
Consolidating LC-MS/MS Method Conditions for the Analysis of Alcohol Metabolites, Barbiturates, and Drugs of Abuse
28 August, Monday
Emanuele Ceccon (presenter), Jamie York, Justin Steimling, Connor Flannery
Restek Corporation
A panel of 136 ESI+/ESI- therapeutic drugs; drugs of abuse and their metabolites; and biomarkers of alcohol consumption were analyzed using a Force Biphenyl analytical column equipped with a guard column and an online filter. Mobile phase A consisted of 0.1% formic acid in water, mobile phase B consisted of 0.1% formic acid in methanol, the temperature was 30°C, and the ESI+ isobars utilized gradient conditions with a 10 minute cycle time. Urine samples underwent glucuronide hydrolysis. Barbiturates, THCA-A, and THC-COOH were analyzed in ESI- and had a total run time of 5 minutes. They were spiked into urine and diluted at a 1:10 ratio with water. Finally, alcohol metabolites were monitored in ESI- with a 5 minute analysis time. Samples were prepared by diluting with water at a 1:10 ratio and injecting 10 μL.
The Biphenyl stationary phase showed an improved resolution of isobars thanks to its unique selectivity due to the pi-pi interactions that occur between the phase and most drugs and drug metabolites. A demonstration of this powerful selectivity is exemplified for seven isobaric compounds sharing the m/z 286 (morphine, hydromorphone, norcodeine, norhydrocodone, 7-aminoclonazepam, pentazocine, and asenapine), which are all baseline resolved. The problematic urinary interferences in alcohol metabolites analysis were resolved without the use of buffer or additional mobile phases. The ESI- panel with barbiturates achieved partial resolution of amobarbital and pentobarbital, which may eliminate the need for confirmatory testing.
This work demonstrates that one LC-MS/MS setup is possible for the analysis of multiple panels, which simplifies testing procedures, saves time, and ultimately reduces costs.
One Column, One Setup: Consolidating LC-MS/MS Method Conditions for the Analysis of Alcohol Metabolites, Barbiturates, and Drugs of Abuse
30 August, Wednesday
Emanuele Ceccon (presenter), Jamie York, Justin Steimling, Connor Flannery
Restek Corporation
A panel of 136 ESI+/ESI- therapeutic drugs; drugs of abuse and their metabolites; and biomarkers of alcohol consumption were analyzed using a Force Biphenyl analytical column equipped with a guard column and an online filter. Mobile phase A consisted of 0.1% formic acid in water, mobile phase B consisted of 0.1% formic acid in methanol, the temperature was 30°C, and the ESI+ isobars utilized gradient conditions with a 10 minute cycle time. Urine samples underwent glucuronide hydrolysis. Barbiturates, THCA-A, and THC-COOH were analyzed in ESI- and had a total run time of 5 minutes. They were spiked into urine and diluted at a 1:10 ratio with water. Finally, alcohol metabolites were monitored in ESI- with a 5 minute analysis time. Samples were prepared by diluting with water at a 1:10 ratio and injecting 10 μL.
The Biphenyl stationary phase showed an improved resolution of isobars thanks to its unique selectivity due to the pi-pi interactions that occur between the phase and most drugs and drug metabolites. A demonstration of this powerful selectivity is exemplified for seven isobaric compounds sharing the m/z 286 (morphine, hydromorphone, norcodeine, norhydrocodone, 7-aminoclonazepam, pentazocine, and asenapine), which are all baseline resolved. The problematic urinary interferences in alcohol metabolites analysis were resolved without the use of buffer or additional mobile phases. The ESI- panel with barbiturates achieved partial resolution of amobarbital and pentobarbital, which may eliminate the need for confirmatory testing.
This work demonstrates that one LC-MS/MS setup is possible for the analysis of multiple panels, which simplifies testing procedures, saves time, and ultimately reduces costs.
The Development of a Virtual Liquid Chromatography Method Development Tool
31 August, Thursday
Cyrille Lamboley (presenter), Melinda Urich, Justin Steimling, Jamie York, Chris Nelson, Tim Yosca, John Garrett
Restek Corporation
The development of LC separations can be time-consuming and costly, with numerous steps, including literature research, column selection, method scouting development and optimization. To eliminate these steps, a software modeling tool that instantly models a separation on different column phases of preselected compounds was developed. Optimization can be performed while maintaining critical pair separations by adjusting for volume effects, mobile phases, gradient steps, and more. The modeler delivers a fast, no-cost starting point. The initial database includes a Drugs of Abuse (DoA) library and a Nitrosamines library with plans to continually expand the utility.
To build the modeler, a DoA library of 250 compounds was created. Retention times were collected using a fast/slow gradient, 30°C/60°C temperatures, and ACN/MeOH mobile phases on a single column dimension. Some additional data points outside of these runs were also collected for the development of a semiempirical correction factor that was used to improve modeling accuracy.
To assess the modeler’s accuracy, experiments comparing retention time values between wet-lab and modeled data were conducted. During development, the acceptance criteria for retention time agreement was set at +/- 15 seconds because it represents a typical MRM window. In the last stage of verification, only 13 retention time datapoints out of 704 collected exceeded the +/- 15 seconds window, giving a pass rate of 98.2%.
Once the initial library built, the modeler was evaluated over four increasingly more complex stages of verification. In the final stage, to assess the viability of adding future compounds to existing libraries, new compounds not present in the initial DoA library were added and then compared by testing different column dimensions and lengths, mobile phases, stationary phases, gradients programs, and temperatures against modeled retention time values.
This free virtual method development software can be used to deliver a fast, no-cost starting point for method development and optimization to LC users, no matter their expertise, via an on-demand consultative user experience that instantly generates a chromatogram and instrument-ready conditions (that can be further optimized by users) for the separation of preselected compounds.