Nakata et al. Journal of Experimental & Clinical Cancer Research 2010, 29:15 http://www.jeccr.com/content/29/1/15 Page 2 of 7 between the two agents have been very limited. This is the first study to compare the plasma pharmacokinetics (PK) of GEM and 5-FU after GEM+S-1 to those after single administration of individual drugs in the same patients. Methods Eligibility Patients under 80 years of age with a diagnosis of unre- sectable pancreatic cancer were eligible. Eastern Coop- erative Oncology Group performance status (PS) ≤ 2, and life expectancy ≥ 12 weeks were required. Patients were required to have measurable or assessable disease and to have had no chemotherapy or immunotherapy before enrolling. Other eligibility requirements included adequate bone marrow function (Hb ≥ 9.0 g/dl, white blood cells between 4,000 and 12,000/μl, neutrophils ≥ 2,000/μl and platelets ≥ 100,000/μl), total bilirubin ≤ 2 mg/dl, AST and ALT ≤ 100 IU/l, alkali phosphatase ≤ 2 times the upper normal level, and BUN and serum crea- tinine ≤ the upper normal level. Patients A total of six patients with unresectable pancreatic can- cer diagnosed by imaging studies including abdominal dynamic computed tomography were enrolled in this study between April and June, 2007. Mean age ± stan- dard deviation was 68 ± 4 years (range, 63-73 years). One case had liver metastasis, three had peritoneal metastasis, and two had tumors involving the celiac and/or superior mesenteric arteries. Informed consent from all participants was obtained. The institutional review board for human experimentation in our hospital approved the study protocols. Treatment S-1 (Taiho Pharmaceutical Co., Tokyo, Japan) was admi- nistered orally at a dose of 30 mg/m2 twice daily after a meal. One course consisted of consecutive administra- tion for 28 days, followed by a 14-day rest period. GEM 800 mg/m2 in 100 ml normal saline was administered intravenously (i.v.) for 30 min on days 1, 15 and 29 of each course. The regimen was set by referring to pre- vious clinical trials [4-7]. Sample collection Blood samples were drawn on days 1, 3 and 15 of the first course. The object of sampling at day 1 was to monitor the plasma PK of GEM after administration of GEM alone. Subsequently, S-1 administration on day 1 of the first course began at the evening after blood sam- plings. The object of sampling at day 3 was to monitor the plasma PK of 5-FU after administration of S-1 alone. The object of sampling at day 15 was to examine the changes in individual drug PK after other drug administration. For this purpose, S-1 was administered 2 h before administration of GEM (Figure 1), when the plasma concentration of 5-FU had increased substan- tially [8]. Each peripheral blood sample (2 ml) was col- lected into a heparinized tube that contained 20 μl of tetrahydrouridine (a cytidine deaminase competitive inhibitor) solution at a concentration of 10 mg/ml. The samples were centrifuged at 3000 rpm for 10 min. Plasma was stored at -20°C until the measurement of 5- FU and GEM concentrations. GEM assay The high-performance liquid chromatography (HPLC) system consisted of a Waters 2690 liquid chromato- graph separation module and a Waters SMH column heater (all from Waters (MA, USA). The AtlantisR dC18 column (150 × 4.6 mm; particle size, 5 μm; Waters) was used for the peak separation of GEM. The HPLC mobile phase was a solution of 5 mM phosphate buffer (pH 7.2). The ultraviolet detector was a Waters 2487 (Waters), and was used at 272 nm. Plasma samples were deproteinized with 20% TCA, and the supernatants were filtered using Ultrafree-MC (Nihon Millipore, Tokyo, Japan) with pore diameters of 0.20 μm. Aliquots of 20 μl were injected into the HPLC system. The quantitative range of this method was 50-40000 ng/ml. 5-FU assay The high-performance liquid chromatographic-mass spectrometry (LC/MS) system consisted of a Micromass ZQ-2000 mass spectrometer, a Waters 2695 liquid chro- matograph separation module and a Waters SMH col- umn heater (all from Waters). The AtlantisR dC18 column (150 × 2.1 mm; particle size, 5 μm; Waters) was used for the peak separation of 5-FU. The HPLC mobile phase was a solution mixed purified water and acetoni- trile. The mass spectrometer was used in the negative ESI mode. The detector was used in SIR mode with a selected ion recording procedure at m/z = 128.9 for 5- FU and at m/z = 130.9 for 5-FU-15N2. To plasma sam- ples, internal standard solution (including 5-FU-15N2) was added, and was then extracted with ethyl acetate. The organic layer was evaporated to dryness under a stream of nitrogen. The residue was dissolved in purified water, and after vortex mixing, the mixture was filtered using Ultrafree-MC (Nihon Millipore) with pore dia- meters of 0.20 μm. Aliquots of 20 μl were injected into the LC/MS system. The quantitative range of this method was 5-500 ng/ml. Statistical analysis The area under the curve from the drug (S-1 or GEM) administration to the infinite time (AUCinf) was