Di Paolo Antonello, Danesi Romano, Ciofi Laura, Vannozzi Francesca, Bocci Guido, Lastella Marianna, Amatori Federica, Martelloni Bianca Maria, Ibrahim Toni, Amadori Dino, Falcone Alfredo, Del Tacca Mario
Division of Pharmacology and Chemotherapy, Department of Oncology, Transplants and Advanced Technologies in Medicine, University of Pisa, 56126 Pisa, Italy.
Ther Drug Monit. 2005 Jun;27(3):362-8. doi: 10.1097/01.ftd.0000162016.11148.1b.
Administration of 5-fluorouracil (5-FU) may be associated with severe toxicities in patients who are deficient of dihydropyrimidine dehydrogenase (DPD) activity. For this reason, a sensitive HPLC method for the analysis of 5-FU and 5-fluoro-5,6-dihydrouracil (5-FDHU) was developed in the present study for the determination of DPD activity in nucleated cells of peripheral blood and pharmacokinetic analysis of 5-FU and 5-FDHU in humans. 5-FU and 5-FDHU were extracted from biologic matrices by adding sodium acetate, sodium sulfate, and diethyl ether/propanol. Dried samples were reconstituted in a mobile phase (KH2PO4 35 mmol/L, pH 4.0), isocratically eluted with a Hypersil C18 stationary phase (25 cm x 4.6 mm, 10 microm), and detected by a diode array detector (measurement and reference wavelengths, 215 and 360 nm, respectively). 5-Fluorocytosine (internal standard), 5-FDHU, and 5-FU were eluted within 13 minutes of the injection without interferences. Recoveries ranged between 81% to 85% for all compounds, and the method proved to be linear, with a coefficient of linearity of 0.999. The limits of detection and quantification were 3.2 and 16 ng/mL, respectively, and the within-day and between-day CV were less than 10% for both 5-FU and 5-FDHU. The present assay proved to be sufficiently sensitive and specific to evaluate cellular DPD activity and measure 5-FU and 5-FDHU plasma concentrations in cancer patients, thus allowing therapeutic 5-FU monitoring in patients and identification of DPD-deficient subjects at major risk of severe toxicities.
对于二氢嘧啶脱氢酶（DPD）活性缺乏的患者，给予5-氟尿嘧啶（5-FU）可能会导致严重毒性。因此，本研究开发了一种灵敏的高效液相色谱法，用于分析5-FU和5-氟-5,6-二氢尿嘧啶（5-FDHU），以测定外周血有核细胞中的DPD活性，并对人体中的5-FU和5-FDHU进行药代动力学分析。通过加入醋酸钠、硫酸钠和乙醚/丙醇，从生物基质中提取5-FU和5-FDHU。干燥后的样品用流动相（35 mmol/L KH2PO4，pH 4.0）复溶，采用Hypersil C18固定相（25 cm×4.6 mm，10 µm）等度洗脱，并用二极管阵列检测器检测（测量波长和参比波长分别为215和360 nm）。5-氟胞嘧啶（内标）、5-FDHU和5-FU在进样后13分钟内洗脱，无干扰。所有化合物的回收率在81%至85%之间，该方法被证明具有线性，线性系数为0.999。检测限和定量限分别为3.2和16 ng/mL，5-FU和5-FDHU的日内和日间变异系数均小于10%。本测定方法被证明具有足够的灵敏度和特异性，可用于评估细胞DPD活性并测量癌症患者血浆中5-FU和5-FDHU的浓度，从而实现对患者5-FU治疗的监测，并识别出有严重毒性重大风险的DPD缺乏受试者。
