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biomarkers analysis is carried out concurrently to the active compound.
Figure 2: Chromatographic resolution andMS/MS detection of an active pharmaceutical ingredient (API) and its metabolites (1&
2.Metabolites; 3. Proprietary API; 4. Internal standard) on a C18 Zorbax Extend column (150 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 from human plasma.
New off-line robotic sample handlers (96- and 384-well format) coupled to short LC- MS/MS methods (typically 1.0 – 2.0 min) are nowadays the standard platforms to analyse a very large number of samples for high throughput screening, pre-clinical and clinical bioanalysis. Therefore, for on-line techniques to compete, system robustness is essential to bring down cost without compromising quality. To achieve this, the chemistry of TurboFlow extraction columns has evolved over time. More than a thousand plasma samples (15 L injection) are routinely analysed in our labs using the same polymeric column (Cyclone 50 x 0.5 mm i.d.) without observing any changes in peak shape or retention time. This is a significant cost reduction when compared with an off-line technique where ten 96- well SPE or protein precipitation blocks would have been needed. In addition to a significant increase in robustness, the polymeric sorbent used is very retentive for a wide range of chemistries, therefore reducing considerably the need for method development from one compound to the other.
In summary, the TurboFlow platform has reached a level of maturity through a step by step approach in addressing the issues highlighted throughout the years by the analytical scientists using this technique for day-to-day samples analysis. As a result, TurboFlow has now become in its own right the tool of choice for modern bioanalysis.
Figure 3:Multiplexing with Turboflow chromatography allows up to four LC systems to run into oneMS.
reduced the solvent usage which was considered as a main drawback with initial 1.0 mm id columns.
Reduction of both flow rate and column i.d. had its limitations though, particularly for the injection volume. Typically 15 L of crude plasma (or 30 L diluted 1:1 with internal standard) could be loaded onto the system. This is a very small volume compared to the typical 500 L of plasma originally used for off-line extraction (LLE, SPE or protein precipitation) of early clinical samples where high assay sensitivity is key. Hence initial TurboFlow usage in our lab was mainly directed towards analytical support of toxicology studies where high systemic exposures only require average assay sensitivity (1 - 5
ng/mL).With the development of new generations of mass spectrometers over the past decades, sensitivity is now rarely an issue when using the TurboFlow platform. For a typical set of pharmaceutical compounds designed for oral administration (e.g. following the rule of 5 [3]), we routinely reached LLOQ values in the 0.1 - 0.5 ng/mL range with 15 L injection volume [4]. The possibility of extracting very small sample volumes allows the development of more robust pre-clinical pharmacokinetic and pharmacodynamic (PK and PKPD) models. On-line extraction also reduces considerably sample preparation and could prevent sample degradation. This is the case when labile metabolites and/or
Applications Once generic, validated TurboFlow, or indeed ‘regular’ analytical LC methods, are in place within a high throughput screening laboratory there can be two bottlenecks for sample throughput. The first is the time of the LC method gradient, where ironically, the more costly part of the system, the MS, is idle during the loading and wash stages of analysis. A solution that has gained wide use, particularly in the clinical field, to increase throughput on such systems is to stagger and channel multiple LC flows through the valve system to a single mass spectrometer and hence ‘multiplex’ the system. A schematic for such a system is presented in Figure 3. A practical example is provided in Figure 4. An example of pharmaceutical compounds that have been analysed via 4 separate LC channels onto a single MS is described by Berube [5] and for two LC channels by Chassaing et al [6]. In the work described by Berube, the sample batch would normally take 48 hours to analyse using one LC system, however,
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