23


Figure 4: 2-fold increase in throughput using a parallel TurboFlow platform for the analysis of a lipophilic compound in human plasma (1.Metabolite; 2. Proprietary API); chromatographic resolution andMS/MS detection on a C18 Zorbax Extend column (50 x 4.6 mm i.d.; 5 micron particles; flow rate: 0.8 mL/min; mobile phase (isocratic): 50/50 v/v water/acetonitrile containing 0.01% trifluoroacetic acid) after turbulent flow extraction.


by channelling additional LCs the throughput is quadrupled taking just 12 hours to run the same batch. The second area of improvement comes in the compound optimisation step where an analyst would re-plumb the system in order to infuse each compound of interest and determine the optimum transitions, collision energy, and tuning before manually creating a new instrument method. This process can also be automated now using an algorithm in the software named QuickQuan. Instead of manual infusion of individual components a group of compounds is added to a database and the software selects an LC and autosampler method in order to submit a batch to the system. The autosampler can then pick up each solution, inject through the system, and hence, infuse into the MS so that optimal settings per compound can be saved and a report generated [5]. Manual compound optimisation for the ten compounds analysed was estimated at ~ 2 hours as compared to an approximate ten-fold time saving of thirteen minutes when utilising the automated optimisation algorithm.


In addition to the time savings achieved using automated sample optimization and multiplexing, further utilisation of the small


injection volume required on the 0.5mmi.d. columns has been exploited. For example a number of investigators have now reported the successful analysis of drug compounds such as immunosuppressants and antibiotics fromlow volume samples such as ocular fluid (tears) [7, 8]. Research laboratories are reporting the use of TurboFlow technology in their analyses of extremely complex matrices such as hemodialysates [9] and edible animal tissues [10]. Perhaps ofmore interest to the pharma industry is the emerging use of the TurboFlow technology in protein-ligand screening/affinity ranking experiments [11]. So far thismethod has been utilised to demonstrate the affinity selection of a small steroidal alkaloid library with the acetylcholinesterase and butyrylcholinesterase proteins. The fast isolation and generic retention of the protein/ligand complexes would suggest that itmay become useful in the high- throughput screening of such compound mixtures in the future. Several other scientific fields not discussed here could benefit fromTurboFlow technology. This is certainly the case within pharmaceutical development where the recovery of a drug substance from a complex drug product formulation can sometimes be very challenging.


TFC: The Future Bioanalysis of oral drugs, e.g. generally defined by a set of physicochemical properties following the rule of 5 [3], has become a well understood field where most challenges have already been identified and dealt with successfully over the past ten years. However, the increased emphasis on both drug safety and translational biology, e.g. the need to understand how pre-clinical efficacy models are representative of human pharmacology, has considerably modified the expectations for what needs to be measured routinely in biological samples. Therefore, the historical design of bioassays is gradually evolving towards a higher degree of complexity. Nowadays, it is not unexpected to monitor, sometimes in the same sample, not only the drug levels but also its potential active/reactive metabolites as well as the biomarkers associated with the mechanism of action of the drug. Metabolites are obviously more polar and/or generally smaller than the drug itself and could present stability issues (e.g. acyl glucuronides, N-oxides). As for the chemical space for biomarkers, it could span from a very small and polar compound such as a neurotransmitter to a very large and hydrophobic entity like fatty acids. Amongst the key analytical challenges with biomarkers are generally the sampling procedure, the sample volume available and, again, the potential stability issues.


Adventuring outside the boundaries of a well defined ‘rule of 5’ box is requiring further thinking in the development process for bioanalytical assays. The TurboFlow approach already has some intrinsic capabilities that facilitate the analysis of biomarkers and metabolites. First, it provides high sensitivity assays without the need for high sample volume, typically 15 L injection volume could suffice. In addition, the on-line extraction approach removes the need for lengthy sample preparation procedure, hence reducing sample degradation issues frequently observed with biomarker analysis. Over the years, different types of chemistries have become available for the TurboFlow extraction columns, from polymer-based weak ion-exchangers to hydrophobic silica-based sorbents. Therefore, the need to extract a wide range of compounds from a wide range a matrices has been addressed through the diversity of extraction sorbents commercially available. A method development module is also available to provide a quick screen of columns and mobile phases best suited for a target application.


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