The system followed the basic apparatus and design outlined in the papers of Hilton [3
] and Wilson [4
], with modifications to allow for the automation of the process and to eliminate the need to start and stop pumps and the need to switch two 6-port valves [6
]. The HPLC system consists of two Waters 515 pumps, one delivering 1% acetonitrile in water to the 1–5 mL loop of a 7725i injector (IDEX Health and Science, Middleborough, MA, USA) at 1 mL/min and the other delivering the analytical mobile phase required for the separation of the compound of interest at the required flow rate and using the required mobile phase for the separation. The configuration of columns and valves, both automated and manual, used for the automated column-switch HPLC is shown in Figure 1
. A 1–5 mL sample is loaded into the injector loop S1 of a 7725i 2-position sample injection valves (V1, Rheodyne LLC, Rohnert Park, CA, USA). At the time of injection, a VXV PEEK Analytical High Pressure 2-position, 10 Port Switching Valve (V2, Analytical Sales and Services, Pompton Plains, NJ, USA) directs the plasma sample, displaced from the injector by 1% acetonitrile, to the capture column C1, while at the same time the same ten-port valve V1, directs the C1 effluent to the radioactivity detector D1 (Figure 1
). The detector contains two ~0.25 mL loops positioned between two opposing bismuth germanium oxide (BGO) detectors (Bioscan Inc., Washington, D.C., USA). One loop is used to detect the material not trapped by the trapping cartridge C1, the second loop is attached to the output of the analytical HPLC column. During the first 4 min, the detector responds to radioactive species in the plasma that are not trapped by the sorbent of the capture column, C1. Four minutes after the injection, V2 is switched to allow the analytical mobile phase to back-flush the contents of C1 onto the analytical column C2 and the effluent from the analytical column is allowed to flow past the radiation detector D1 and UV and/or Mass spectrometer D2. At the end of the HPLC elution time from the analytical column, V2 is returned to its original position, which allows the capture column C1 to be washed with 1% acetonitrile in preparation for next plasma sample. If the pressure of the analytical column exceeds a pressure limit (typically 3500 psi), the analytical column can be automatically reversed using a VXV PEEK Analytical High Pressure 2-position, 10 Port Switching Valve (V3, Analytical Sales and Services, Pompton Plains, NJ, USA). The capture column (C1, Figure 1
) measuring 4 × 20 mm (VICI part number SFEC42) is packed dry with C-18, or HLB Sep-pak sorbent (Waters Corp., Milford, MA, USA) and is packed by vibration to remove voids and is contained in a guard column housing (Part No.SFECH412, VICI) with 2µ titanium frits. The output of the HPLC column can be directed to a Mass Spectrometer D2 either directly or through a split with the remaining material going to a fraction collector F1 (Spectra/Chrom™ CF-1 Fraction Collector (SpectrumLabs, Rancho Dominguez, CA, USA). The samples from the fraction collector are then assayed for radioactive content on a Perkin Elmer/Wallac Wizard™ 1470 Automatic Gamma Counter (Perkin Elmer, Waltham, MA, USA). The signal from the coincidence detector is collected by a PowerChrom 280 system (which includes software and data acquisition software, eDAQ Pty Ltd, Denistone East, NSW, Australia) The same software is used to integrate the peak areas and to control the position of the valves.
Step 1. The plasma is injected onto the injection loop (Yellow line), either manually or by autosampler, while the trapping cartridge (Green line) is equilibrated with the wash solvent (typically 1% acetonitrile/water) and the analytical column (Blue line) is equilibrated with the elution solvent. All solvent is passed to waste and bypasses the fraction collector.
Step 2. The 6-port valve with the sample loop containing the plasma sample is transferred to the trapping cartridge (Red line). The analytical column (Blue line) continues to be equilibrated with the elution solvent. All solvent is passed to fraction collector so any material that is not trapped on the cartridge is collected in the fraction collector.
Step 3. The 10-port valve with the analytical column is switched and the plasma sample is eluted from the trapping cartridge to the analytical column (Red line). The sample loop (Green line) continues to be washed with the 1% acetonitrile/water solution to clean prior to the next sample being loaded. All solvent is passed to fraction collector so all material that is not detected by the coincidence detector can be counted using a well counter.
Step 4. If the back-pressure on the analytical column exceeds a set limit, the 10-port valve V3 changes position so that the column flow is reversed. This back-flushing cleans the column and after a set time the forward flow is restored.