Chen Xijing, He Hui, Wang Guangji, Yang Bing, Ren Weichao, Ma Le, Yu Qiaoling
Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing, Jiangsu 210009, China.
Biomed Chromatogr. 2007 Mar;21(3):257-65. doi: 10.1002/bmc.747.
A simple, accurate, precise, specific and reproducible high-performance liquid chromatography (HPLC) method was developed for simultaneous determination of resveratrol isomers in rat plasma. Cis-resveratrol was made by exposure of a trans-resveratrol solution to sunlight for 5 days followed by separation by HPLC and identification by mass spectrometry (MS). The assay procedure involved simple liquid-liquid extraction of resveratrol isomers and internal standard (IS, caffeine) from a small plasma volume directly into acetonitrile. The supernatant liquid was added an equal volume of water and injected onto a Hypersil ODS(2) C(18) column (5 microm, 4.6 x 250 mm). Mobile phase consisting of methanol and distilled water was used at a flow rate of 1.0 mL/min for the effective separation of cis-, trans-resveratrol and caffeine (IS). The detection of the analyte peak was achieved by monitoring the eluate using a UV detector set at 303 nm. The ratio of peak area of analyte to IS was used for quantification of plasma samples. Nominal retention times of cis-, trans-resveratrol and IS were 3.2, 4.3 and 6.1 min, respectively. The calibration curve was linear ranging from 0.066 to 6.64 and 0.134 to 13.4 microg/mL with correlation coefficients of 0.9998 and 0.9997 for trans and cis isomers, respectively. The absolute recovery of both isomers was more than 85%. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.066, 0.664 and 6.64 microg/mL of trans-resveratrol, were in the range 2.37-6.95% relative standard deviation (RSD) and 0.77-6.97% RSD, respectively. The inter- and intra-day precisions in the measurement of quality control (QC) samples, 0.134, 1.34 and 13.4 microg/mL of cis-resveratrol, were in the range 1.93-3.72% relative standard deviation (RSD) and 1.13-6.57% RSD, respectively. Both analytes and IS were stable in the battery of stability studies and freeze-thaw cycles. Resveratrol isomers were found to be stable for a period of 30 days on storage at -20 degrees C. The application of the assay to determine the pharmacokinetic disposition after a single oral dose to rats is described.
建立了一种简单、准确、精密、特异且可重复的高效液相色谱（HPLC）法，用于同时测定大鼠血浆中的白藜芦醇异构体。顺式白藜芦醇是通过将反式白藜芦醇溶液暴露于阳光下5天，然后通过HPLC分离并经质谱（MS）鉴定制得。测定步骤包括从小体积血浆中直接进行简单的液-液萃取，将白藜芦醇异构体和内标（IS，咖啡因）萃取到乙腈中。向上清液中加入等体积的水，然后注入Hypersil ODS(2) C(18)柱（5微米，4.6×250毫米）。流动相由甲醇和蒸馏水组成，流速为1.0毫升/分钟，用于有效分离顺式、反式白藜芦醇和咖啡因（内标）。通过使用设置在303纳米的紫外检测器监测洗脱液来实现分析物峰的检测。分析物峰面积与内标的比值用于血浆样品的定量。顺式、反式白藜芦醇和内标的标称保留时间分别为3.2、4.3和6.1分钟。校准曲线在0.066至6.64以及0.134至13.4微克/毫升范围内呈线性，反式和顺式异构体的相关系数分别为0.9998和0.9997。两种异构体的绝对回收率均超过85%。质量控制（QC）样品（反式白藜芦醇浓度为0.066、0.664和6.64微克/毫升）的日间和日内精密度，相对标准偏差（RSD）分别在2.37 - 6.95%和0.77 - 6.97%范围内。质量控制（QC）样品（顺式白藜芦醇浓度为0.134、1.34和13.4微克/毫升）的日间和日内精密度，相对标准偏差（RSD）分别在1.93 - 3.72%和1.13 - 6.57%范围内。在一系列稳定性研究和冻融循环中，分析物和内标均稳定。发现白藜芦醇异构体在-20℃储存30天内稳定。描述了该测定法在大鼠单次口服给药后药代动力学处置测定中的应用。
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