Shim Hyun Joo, Lee Eun Joo, Jung Young Hee, Kim So Hee, Kim Soon Hoe, Yoo Moohi, Kwon Jong Won, Kim Won Bae, Lee Myung Gull
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, San 56-1, Shinlim-Dong, Kwanak-Gu, Seoul 151-742, South Korea.
J Pharm Biomed Anal. 2002 Oct 15;30(3):527-33. doi: 10.1016/s0731-7085(02)00397-7.
A high-performance liquid chromatographic (HPLC) method using liquid-liquid extraction for sample preparation was developed for the determination of a new phosphodiesterase V inhibitor, DA-8159, in rat plasma and urine using sildenafil citrate as an internal standard. A 100 microl aliquot of 0.1 M Na(2)CO(3) (containing sildenafil citrate, 3 microg/ml as free sildenafil) and a 1 ml aliquot of ether were added to a 100 microl aliquot of biological samples (urine samples were diluted 20 times with distilled water). After vortex centrifugation at 9000 x g for 3 min, the ether layer was collected and dried under nitrogen gas. The residue was reconstituted with a 150 microl aliquot of the mobile phase, centrifuged, and a 100 microl aliquot of the supernatant was injected onto a reversed-phase column. The mobile phases, 20 mM KH(2)PO(4) (pH 4.7):acetonitrile (70:30, v/v for plasma and tissue samples, and 75:25, v/v for urine samples), were run at a flow rate of 1.0 ml/min. The column effluent was monitored by an ultraviolet detector set at 292 nm. The retention times for DA-8159 and the internal standard were approximately 10.7 and 9.1 min, respectively, in plasma and tissue samples and the corresponding values in urine samples were 47 and 33 min. The detection limits for DA-8159 in rat plasma and urine were 20 and 100 ng/ml, respectively. The coefficients of variation of the assay were generally low: below 10% for plasma and 9.9% for urine. No interferences from endogenous substances were found.
建立了一种高效液相色谱（HPLC）法，采用液-液萃取进行样品制备，以枸橼酸西地那非为内标，用于测定大鼠血浆和尿液中一种新型磷酸二酯酶V抑制剂DA-8159。将100 μl 0.1 M Na₂CO₃（含枸橼酸西地那非，以游离西地那非计为3 μg/ml）和1 ml乙醚加入100 μl生物样品（尿液样品用蒸馏水稀释20倍）中。在9000×g下涡旋离心3分钟后，收集乙醚层并在氮气下干燥。残渣用150 μl流动相复溶，离心，取100 μl上清液注入反相柱。流动相为20 mM KH₂PO₄（pH 4.7）：乙腈（血浆和组织样品为体积比70:30，尿液样品为体积比75:25），流速为1.0 ml/min。柱流出物用设定在292 nm的紫外检测器监测。在血浆和组织样品中，DA-8159和内标的保留时间分别约为10.7分钟和9.1分钟，在尿液样品中的相应值分别为47分钟和33分钟。大鼠血浆和尿液中DA-8159的检测限分别为20 ng/ml和100 ng/ml。该测定法的变异系数一般较低：血浆中低于10%，尿液中为9.9%。未发现内源性物质的干扰。
