應用液相層析串連式質譜儀（LC/MS/MS）分析中藥製劑中馬兜鈴酸成分之方法研究   全文下載

Analyzing Aristolochic Acids in Chinese Herbal Preparations Using LC/MS/MS

多種中草藥中含馬兜鈴酸，具嚴重之腎毒性，引起世人普遍重視而開發各種檢驗方法。液相層析串聯式質譜分析法（LC/MS/MS）之應用，配合附光二極體陣列檢出器（photo diode array），非但可以比對波峰滯留時間及紫外光（UV）吸收光譜圖，同時並可比對層析圖中各波峰之質譜圖，使檢出結果更精確、迅速。液相層析條件為分離管柱：Zorbax Extend-C18, 5 μm, 2.1×150 mm；移動相溶媒為乙A：緩衝水溶液（35：65），緩衝水溶液中含0.1%甲酸及0.1%醋酸銨，流速為0.3 mL/min，並以1：1比例分流。串聯式質譜分析條件為毛細管電壓：3.0 kV；進樣圓錐口電壓：20 V；碰撞能量：馬兜鈴酸I為10 eV，馬兜鈴酸II為12 eV；離子源溫度：120℃；溶媒揮散溫度：350℃等，分別選定【M+NH4】+加銨分子離子作為源頭離子（馬兜鈴酸I為m/z 359、馬兜鈴酸II為m/z 329）進行正離子電灑法（ESI+）之子代離子分析掃描（daughter ion scan），獲得質譜圖與標準品之質譜資料圖庫比對。為瞭解檢體中馬兜鈴酸之含量，進行多重離子裂解監控（MRM, multiple reaction monitoring）定量分析。馬兜鈴酸I之監測配對離子為m/z 359及m/z 298，馬兜鈴酸II之監測配對離子為m/z 329及m/z 268，以piromidic acid作為內部標準品，配製系列馬兜鈴對照標準品溶液，製作檢量線，馬兜鈴酸I、II在濃度範圍分別為0.02~16.00及0.028~22.40 μg/mL時所得檢量線之線性關係r值分別為0.9992與0.9988；同日內、異日間測試，馬兜鈴酸I之測試結果相對標準偏差分別為0.73%及10.44%，馬兜鈴酸II為1.38%及6.10%；添加回收率試驗，馬兜鈴酸I為99.0%至106.9%，馬兜鈴酸II為92.0%至104.5%；最低檢出極限，馬兜鈴酸I為2.0 ng/mL，馬兜鈴酸II為2.8 ng/mL。抽驗自市面之12件馬兜鈴酸陽性中藥製劑檢體，依上述分析方法進行多重離子裂解監控定量分析，馬兜鈴酸I、II含量範圍分別介於11.1至3376.0 μg/g及5.4至725.4 μg/g。"

This paper introduces a newly developed method of applying liquid chromatography- tandem mass spectrometry (LC/MS/MS) to the analysis of aristolochic acids (AAs) in Chinese herbal (medicinal) preparations. Sensitive and highly accurate readings were obtained for this study using both a photodiode array (PDA) detector and tandem mass spectrometer. The following optimized conditions for LC/MS/MS were set for this study: Separation was accomplished using a reverse phase C18 column (2.1 × 150 mm, 5 μm). The mobile phase comprised 35% acetonitrile and a 65% aqueous solution containing 0.1% formic acid and 0.1% ammonium acetate at a flow rate of 0.3 mL/min with a split ratio of 1:1 into the PDA detector and the tandem mass spectrometer. MS/MS qualitative analysis was performed at 3.0 kV capillary voltage, with a collision energy level for aristolochic acid I (AA-I) of 10 eV and for aristolochic acid II (AA-II) of 12eV. The electrospray ionization source was operated in the positive mode. AA-I [M + NH4]+ ions at m/z 359 and AA-II [M + NH4]+ ions at m/z 329 were selected as precursor ions for daughter ion scanning. The characteristic daughter ion mass spectra for AA-I and AA-II were generated and studied carefully. Quantification of results was done using the multiple reaction monitoring (MRM) method. The [M + NH4]+ and [(M + NH4)- NH3-CO2]+ ions of both AA-I and AA-II were selected as precursor and daughter ions for the MRM analysis. MS/MS detection limits were defined as 2.0 ng/mL of AA-I, and 2.8 ng/mL of AA-II. The linear regression correlation coefficients of the calibration cure were 0.9992 of AA-I within the range of 0.02~16.00 μg/mL and 0.9988 of AA-II within the range of 0.028~22.40 μg/mL. Relative standard deviations of 0.73% and 10.44% for AA-I, and 1.38% and 6.10% for AA-II were determined for the intraday and interday tests. Recoveries for five differing concentrations of AA-I ranged from 99.0% to 106.9% and, for five differing concentrations of AA-II, ranged from 92.0% to 104.5%. Levels of AA-I and AA-II detected in the 12 commercial Chinese medicinal preparation samples ranged from 11.1 to 3376.0 μg/g and from 5.4 to 725.4 μg/g, respectively.