以高效液相層析及紫外光偵測測定血中Sirolimus濃度之經驗與回顧   全文下載

Determination of Sirolimus in Blood by High-Performance Liquid Chromatography with Ultraviolet Detection – Experience and Review

Sirolimus（SRL）為一強效的免疫抑制劑。療劑監測（TDM）對於讓SRL達到最適切的治療非常重要。免疫分析法、高效液相層析加紫外光偵測（HPLC-UV）及高效液相層析加質譜儀偵測（HPLC-MS或HPLC/MS/MS）均曾用於SRL之分析。本研究之目的是提供對某一分析SRL之HPLC-UV方法進行確效的經驗，並回顧各種分析SRL 之HPLC-UV方法。作者使用由Wyeth-Ayerst Research 發展出來、經確效的HPLC/UV方法來決定血中SRL濃度，並做些許修改。固定相使用Alltima C18 column（5 μm, 150 × 2.1 mm），移動相為60% acetonitrile in water ，流速為0.5 mL/min 。樣品使用人類全血（0.5 mL）加入內標準品及特定SRL的量來製備。Zinc sulfate（50 g/L, 1 mL）及acetone（1 mL）用於溶血及使蛋白質變性。取上清液以0.2或0.3 mL 之100 mM NaOH 鹼化後用1-chlorobutane（2 mL）萃取。將1-chlorobutane 層吹乾，以移動相溶解，再使用n-hexane（0.5 mL）反萃取。最低定量濃度為2.5 ng/mL ，且標準曲線在濃度2.5-75 ng/mL 為直線。同日內及不同日間變異係數分別為2.1-5.2% 及2.8-5.7% 。同日內及不同日間偏差值分別為-0.03-5.3% 及0.7-3.3% 。SRL及IS 之回收率分別為78.5-92.8% 及76.9 ± 3.9% 。樣品經三次冷凍／解凍的安定性無疑，萃取液在4°C 自動取樣器24 小時安定性也被證實。本文最後回顧SRL 之化學性質及應用在血中SRL 濃度分析之不同HPLC-UV方法，揭示建立血中SRL 濃度分析之不同HPLC-UV方法之關鍵分析技巧。"

Sirolimus (SRL) is a potent immunosuppressant. Therapeutic drug monitoring (TDM) of SRL is required to optimize the therapy. Immunoassay, high-performance liquid chromatography with ultraviolet detection (HPLC-UV), and HPLC with mass-spectrometric detection (HPLC-MS or HPLC/MS/MS) have been used in the analysis of SRL. The purpose of this study was to share our experience in validating a HPLC-UV method for analyzing SRL and to have an overview of HPLC-UV methods used in SRL assay. A validated HPLC/UV method developed by Wyeth-Ayerst Research with minor modification was use to determine SRL concentration in human whole blood. An Alltima C18 column (5 μm, 150 × 2.1 mm) was used as the stationary phase. The mobile phase was 60% acetonitrile in water, and the flow rate was 0.5 mL/min. Samples were prepared by spiking human whole blood (0.5 mL) with the internal standard (IS) and designated amount of SRL, except blank. Zinc sulfate (50 g/L, 1 mL) and acetone (1 mL) were used for hemolysis and deproteinization. After alkalizing supernatant with 100 mM NaOH (0.2 or 0.3 mL), 1-chlorobutane (2 mL) was used for extraction. The 1-chlorobutane layer was dried, reconstituted with mobile phase and back extracted with 0.5 mL of n-hexane. The limitation of quantification was 2.5 ng/mL and the standard curve was linear at the concentration range of 2.5-75 ng/mL. The intraday and interday coefficients of variation were 2.1-5.2% and 2.8-5.7%, respectively. The intraday and interday relative errors were -0.03-5.3% and 0.7-3.3%, respectively. The recoveries for SRL and the IS were 78.5-92.8% and 76.9 ± 3.9 %, respectively. The samples were proven to be stable after 3 freeze/thaw cycles, and the extract was stable over 24 hr at an autosampler set to 4°C. In addition to an overview of the chemical properties of SRL, different HPLC-UV methods for the quantification of SRL were provided to identify analytic parameters that are critical for the establishment of HPLC-UV assay for SRL.