Fig. 2: Schematic illustration of turbulent flow chromatography (TFC) setup in Transcend TLX-4 system coupled to TSQ Altis triple quadrupole mass spectrometer.

The TLX-4 system has four independent channels (channel 1: blue, channel 2: orange, channel 3: green and channel 4: red) that can be used simultaneously. The analytical run in each channel has three steps. The first step consists of sample injection, loading into the TFC column and cleanup. The sample is injected into the sample loop by autosampler and transferred to the valve interface module using a quaternary loading pump with high flow rate. For first dimension chromatography, the sample is loaded and washed into TFC column by a turbulent flow, flushing away unwanted matrix and large molecules while targeted analytes are retained in particles interaction sites. In the second step, the first and second columns are connected, and analytes are eluted from the TFC column by the solvent loop, transferred and focused on the analytical column head. Third step includes TFC column washing with stronger organic solvents, solvent loop refilling and ultraperformance chromatographic separation by an organic multistep linear gradient. As soon as the target analytes are eluted, the selecting valve directs the current channel flow path to the mass spectrometer (MS) to achieve data acquisition. Lastly, columns are re-equilibrated to initial conditions before starting the next run. For the present method, MS acquisition window has 2.5 min enabling multiplex analysis of up to four samples within ten minutes. Aria software coordinated multiplexed sequence logic to maximize analysis throughput.