MSACL 2023 Abstract

Introduction
Miniaturized sample preparation, particularly solid phase extraction (SPE), has gained popularity in recent years with perceived benefits such as lower solvent consumption, sample processing and turnaround time improvements. However, implementation rarely results in total workflow solution with multiple pain points still evident. One of the greatest workflow compatibility issues is related to the SPE elution solvents and LC/MS mobile phases. This incompatibility often results in continued necessity of an evaporation step. While elution solvent volumes are much lower this can still be somewhat of a bottleneck. Here we investigate alternative solid phase extraction chemistries to provide a more complete workflow solution resulting in elimination of the evaporation step prior to LC-MS/MS analysis.

Objectives
This poster will present strategies for automating drugs of abuse extraction, eliminating evaporation steps while maintaining sensitivity using a dedicated low volume sample preparation format.

Methods
A typical drugs of abuse target analyte panel was spiked at various concentrations and extracted from non-hydrolysed and enzymatically hydrolysed human urine. Sample extractions were investigated using polymer-based solid phase extraction: comparing traditional reversed-phase and mixed-mode weak cation exchange chemistries. For increased sensitivity assay miniaturisation was performed using the Biotage® Mikro 2 mg (low volume plate) SPE format. Final low volume extraction protocols were transferred to the Extrahera LV-200 automated sample preparation workstation. UHPLC-MS/MS analysis was performed using a Shimadzu Nexera UHPLC coupled to an 8060 triple quadrupole MS system. Chromatography utilised traditional mobile phases of ammonium formate and formic acid in water and MeOH and separation afforded by a Restek Raptor Biphenyl analytical column.

Results
A drugs of abuse panel including amphetamines, opiates, benzodiazepines, cocaine and other regularly analysed drugs were extracted from urine using polymer-based reversed phase and the corresponding mixed-mode weak cation exchange SPE chemistries in 10 mg 96-well plate format. Strong cation exchange SPE chemistry was ruled out due to necessity of high pH elution conditions which were incompatible with LC mobile phases. Initial evaluations of the polymeric reversed phase chemistry delivered reproducible analyte recoveries, typically greater than 80% depending on drug polarity and wash solvent composition. Further investigation with respect to matrix factors demonstrated very different results. High matrix effects were observed on many mid to non-polar analytes such as the benzodiazepines. Wash solvent, pH and elution solvent optimisation did not adequately remove the matrix effects ultimately ruling out this chemistry option. Weak cation exchange SPE optimisation delivered recoveries great than 80% for all but a few target analytes such as benzoylecgonine and the 7-amino-benzodiazepine metabolites. Matrix factors were substantially better compared to the corresponding reversed-phase chemistry data. Miniaturisation to the Biotage® Mikro 2 mg WCX (low volume plate) format demonstrated excellent scalability. Further optimisation was performed for all steps in terms of pH control, ionic strength and volumes. Final elution volumes of 30 µL were achieved using the optimised method. Comparison of direct injection of the elution solvent to pre-dilution prior to injection for chromatographic peak shapes and injector reproducibility demonstrated the latter approach to be our preferred option. Ultimately, either approach could be used order to allow the elimination of evaporation steps. The extraction protocol was transferred and optimised using the Extrahera™ LV-200 automated sample preparation platform, demonstrating excellent correlation to manual PPM-96 processing. Final method performance and calibration curves demonstrated excellent linearity and coefficients of determination, r2 > 0.99 for all analytes while delivering sub ng/mL LOQs.

Conclusion
The WCX chemistry provided a better approach to drugs of abuse testing while allowing the elimination of evaporation steps for improved workflow. Good correlation was observed scaling chemistry from 10 mg to 2 mg plate options and also between manual and automated processing of the final method.