The plasma samples were analysed for 5-FU and DHFU concentrations by high-performance liquid chromatography (HPLC) on the day of collection. Blood samples for DPD analysis were collected 5 to 23 months after blood sampling for 5-FU pharmacokinetics, which corresponds to intervals ranging Sorafenib Tosylate manufacturer from 2 to 17 months after the last 5-FU dose. None of the patients received chemotherapy at that moment. Reversed phase HPLC analysis 5-Fluorouracil and DHFU concentrations were measured by HPLC analysis using a modification of the method described by Ackland et al (1997). Briefly, 100��l chlorouracil internal standard solution (80mgl?1 in water) was added to 1ml plasma sample, and this mixture was vortexed and subsequently deproteinated with 50��l of a 50% (wv?1) trichloracetic acid solution.

After centrifugation at 8000g for 2min the supernatant was transferred into a 20ml centrifuge tube and neutralised with 1ml 1M sodium acetate solution. Then 5ml ethylacetate was added and the mixture was vortexed during 10min. After separation of the organic and aqueous layers by centrifugation at 5000 g for 5min, the ethylacetate layer was transferred into a 10ml tube and evaporated under a stream of nitrogen at 25��C. The residue was dissolved in 100 ��l ultrapure water and 20 ��l was injected. 5-Fluorouracil and DHFU standards ranging from 0.5 to 20mgl?1 were prepared in human plasma. The chromatographic system consisted of a Waters 616 pump equipped with a Waters 717+ autosampler. The separation of 5-FU and DHFU was accomplished by gradient elution at ambient temperature on a Phenomenex Prodigy ODS 3 column (I.

D. 250��4.6mm, 5��m) equipped with a guard column (30��4.6mm) of the same material. Mobile phase A consisted of 1.5mM K3PO4 and 1% (vv?1) methanol (pH=6.0) and mobile phase B of 1.5mM K3PO4 and 5% (v v?1) methanol (pH=6.0). The gradient was programmed as follows: 100% A during 2min; 100% A��100% B in 0.5min; 100% B during 7min; 100% B��100% A in 0.5min; 100% A during 10min. Detection was performed using a Waters 996 Photo Diode Array UV detector interfaced with a Millenium 2010 Chromatography Manager Workstation. Spectra were acquired in the 201�C300nm range. 5-FU was monitored at 266nm and DHFU at 205nm. The internal standard chlorouracil was monitored at both wavelengths. Pharmacokinetic analysis The pharmacokinetic analyses Drug_discovery were performed in the ADAPT II computer program (version 4.0; USC Los Angeles). The pharmacokinetic data of both the index patient and the six control patients were tested in eight different models. In each model the patient’s data were fitted individually and for each data set the Akaike Information Criterion (AIC) was calculated. The model with the lowest summarised AIC value was selected as the better one (data not shown).