Newguard rp 18 pre column

Plasma standards were prepared in microcentrifuge tubes by spiking 5 uL of Gemcitabine and dFdU into 190 uL of plasma containing 0.125 mg/mL THU. Floxuridine (10 μL) was added as an internal standard. Gemcitabine and dFdU were isolated by protein precipitation with the addition of 600 μL of acetonitrile. Analyte extraction was carried out via protein precipitation with the addition of 600 uL of acetonitrile. The samples were centrifuged for 5 minutes at 14000 RPM at 25°C. The supernatant was collected, dried under nitrogen, reconstituted with 200 uL of water and vortex mixed. Gemcitabine and dFdU extracted from plasma were quantified by reverse-phase HPLC with UV absorbance detection on a Shimadzu 10 series HPLC system. Separation of gemcitabine, dFdU and the internal standard floxuridine was achieved on an Atlantis T3 C18 column (Waters, 100 mm × 2.1 mm i.d., 5 μm particle diameter) fitted with a Brownlee NewGuard RP-18 pre-column (Chrom Tech, 15 mm × 3.2 mm i.d., 7 μm particle diameter). The mobile phase consisted of 10 mM potassium phosphate, pH 3.0 and methanol. The run was a gradient run starting out with 100% aqueous for 6 minutes, decreasing to 95% aqueous in 7 minutes and holding at 95% aqueous for 4 minutes followed by a high organic wash. The flow rate, the injection volume and detection wavelength were 0.4 ml/min, 20 μL and 272 nm, respectively.