RP_HPLC梯度洗脫測定葉下珠中沒食子酸的含量

1、RP-HPLC梯度洗脫測定葉下珠中沒食子酸的含量王伽伯1,2,陳雷3,王玉4,肖小河2(11解放軍第302醫(yī)院,北京100039;21軍事醫(yī)學科學院,北京100850;31錦州醫(yī)學院,遼寧錦州121001;41長春中醫(yī)學院,吉林長春130117葉下珠P hy llan thus u rina ria L1為大戟科葉下珠屬植物葉下珠的干燥全草,具有平肝清熱、利水解毒的功效?，F(xiàn)代藥理實驗證明,葉下珠具有抗乙肝病毒、保肝等作用1。沒食子酸為其有效成分之一,是可水解鞣質的單體,具有抗病毒作用,在多種中藥材中存在。然而,已報道的沒食子酸測定方法對雜質干擾耐受力不強,重現(xiàn)性較差。沒食子酸相對分子質量小,結

2、構中帶有羥基和羧基,極性很大,通常只適合用正相鍵和柱分析,但是正相柱使用成本高,不及反相柱普及率高,如何建立適合反相色譜柱分析的方法尤顯重要。在反相柱上測定沒食子酸含量,關鍵是增大流動相中水相比例,延長沒食子酸出峰時間,同時又能在較短時間內將樣品中的低極性雜質干擾去除,保證進樣分析的連續(xù)性。梯度洗脫即可實現(xiàn)這一目的。為此,筆者采用梯度洗脫技術測定沒食子酸的含量,方法簡便、快速、準確、重現(xiàn)性好。1儀器與試藥高效液相色譜儀:惠普H P1110四元梯度泵;H P1110DAD檢測器;H P Chem stati on化學工作站;甲醇為色譜純;磷酸等其他試劑均為分析純;沒食子酸對照品(08312950

3、1(供含量測定用,中國藥品生物制品檢定所;葉下珠藥材由中國醫(yī)學科學院藥用植物研究所西雙版納分所高海泉研究員提供。2方法與結果211色譜條件:K rom asil C18分析柱(250mm×416 mm,5m;檢測波長:280nm;流動相:A甲醇,B 011%磷酸水溶液;梯度洗脫條件:015m in,A2B (1585;1517m in,A2B(9010,1732 m in,A2B(9010;3235m in,A2B(1585;體積流量:015mL m in;柱溫為室溫。212供試品溶液的制備:取葉下珠藥材粗粉,精密稱取012g,置錐形瓶中,精密加入甲醇25mL,稱定質量,冷浸24h2

4、,再稱定質量,用甲醇補足減失的質量,搖勻,濾過,取續(xù)濾液作為供試品溶液。213對照品溶液的制備:精密稱取經(jīng)五氧化二磷減壓干燥36h的沒食子酸對照品適量,加甲醇制成010235m g mL的溶液,即得。214線性關系的考察:分別精密吸取沒食子酸對照品溶液2、4、8、12、16、20L,依次注入高效液相色譜儀,按上述色譜條件測定峰面積,以峰面積積分值為縱坐標,沒食子酸的量(g為橫坐標,繪制標準曲線,回歸方程為:Y=487914X-3416,r= 019999(n=6,結果表明沒食子酸在01047 01470g呈線性關系,可用外標一點法測定含量。215穩(wěn)定性試驗:取供試品溶液,分別于配制后0、2、4

5、、8、12、24h,依樣品測定法測定,其峰面積的R SD=0151%,供試品溶液在24h內基本穩(wěn)定。216精密度試驗:精密吸取沒食子酸對照品溶液10L,重復進樣6次,測定其峰面積的R SD=0137%。217重復性試驗:對同一樣品取6份,平行測定,以外標法計算質量分數(shù),平均質量分數(shù)11153m g g, R SD=0133%。218加樣回收率試驗:精密稱取已知含量的同一批樣品012g,精確加入沒食子酸對照品溶液適量,按樣品溶液的制備方法操作,測定其含量,并計算平均回收率為98105%,R SD=1105%(n=6。219樣品測定:按樣品測定方法測定3批藥材樣品,測定結果見表1。色譜圖見圖1。表

6、1樣品測定結果(n=3Table1D eter m i nation results of sam ples(n=3批號沒食子酸 (m gg-11111412111593111643討論311流動相的選擇:在C18柱上用文獻2報道的流動相甲醇2水2磷酸(4960105進行分離,沒食子酸峰形良好,但是大量極性相對較低的雜質吸附在(下轉第131頁621中草藥Ch inese T raditi onal and H erbal D rugs第36卷第1期2005年 1月收稿日期:2004204211作者簡介:王伽伯(1981,男,現(xiàn)為軍事醫(yī)學科學院2002級碩士研究生,研究方向藥劑學。E2m ail

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