糙米及豆類中不同型態植物固醇同步分析方法之建立

Abstract

植物固醇可分為自由態、steryl ferulate、酯化態、醣苷態及酯化醣苷態5種。傳統分析植物固醇的方法具有許多缺陷，如酸水解造成的固醇降解或異構化。本實驗目的即為建立以液相層析/大氣壓力化學游離法串聯質譜 (liquid chromato- graphy/atmospheric pressure chemical ionization-mass spectrometry, LC/APCI-MS) 及液相層析-蒸發光散射偵測器 (liquid chromatography-evaporative light scattering detector, LC-ELSD) 同步分析不同型態植物固醇的方法。以 NH2固相萃取管淨化樣品可去除三酸甘油酯及酯化態固醇，並分離出植物固醇，以 LC/APCI-MS 分析自由態植物固醇的添加回收率，介於83.69-106.89 %；以固相萃取法取代傳統水解法可避免固醇分子產生異構化並保持完整的植物固醇型態。使用逆相液相層析串聯質譜可同步分析自由態、steryl ferulate、醣苷態及酯化醣苷態固醇的個別分子，植物固醇的最強特徵離子皆為其固醇基團脫去一分子水的訊號，自由態固醇偵測極限介於5-50 ng/mL。另以正相液相層析-蒸發光散射偵測器分析，樣品可不經前處理直接分析，正相層析管柱可將植物固醇依結構上不同結合態分離，並定量該型態固醇的總量。但由於酯化態固醇與蠟酯無法分離，仍無法對其定量；不同植物固醇偵測極限介於0.5-5 μg/mL。分析糙米及豆類樣品結果顯示 LC-MS 及 LC- ELSD 的結果具有差異，由於結合態固醇缺乏單一標準品而無法以 LC-MS 準確定量，而 ELSD 對於結構相似的固醇分子感應值較相同，因此定量的數值應較 MS 的結果準確。樣品中的固醇組成以avenasterol、campesterol、stigmasterol 及 β-sitosterol 為主，糙米中主要含有自由態固醇及 steryl ferulate (γ-oryzanol)，醣苷態及酯化醣苷態的含量較低；豆類樣品中含有自由態固醇及高量的酯化醣苷態固醇，但皆不含有 steryl ferulate。樣品中黃豆及黑豆的醣苷態固醇含量較高。

There are five common forms of phytosterols: free sterols (FS), steryl ferulates (SF), steryl glycosides (SG), acylated steryl glycosides (ASG) and steryl esters (SE). Traditional phytosterol analysis methods have many drawbacks, for instance, acid hydrolysis may cause sterol degradation or isomerization. The aim of this study is to develop methods to determine free and conjugated phytosterols simultaneously by LC/APCI-MS (liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry) and LC-ELSD (liquid chromatography- evaporative light scattering detector). Phytosterols could be separated by NH2 SPE cartridge while triglycerides and steryl esters were removed from sample during sample clean-up. Recoveries of added free sterols analyzed by LC/APCI-MS were 83.69 to 106.89 %. Replacing hydrolysis methods by solid phase extraction could avoid sterol isomerization and retain intact sterol conjugates. Single compounds of FS, SF, SG and ASG were separated by reverse phase C18 column and detected by APCI-MS simultaneously. All phytosterols give an intense characteristic ion corresponding to the loss of a water molecule from the sterol moiety. The limit of detection (LOD) for free sterols was 5-50 ng/mL. NPLC-ELSD could separate and detect lipid (phytosterol) classes synchronously without further sample pretreatment. Sterol esters still could not be quantified by NPLC-ELSD because of the coelution of wax esters and steryl esters. The limit of detection (LOD) for phytosterols was 0.5-5 μg/mL. Results showed significant difference in the phytosterol contents of samples between measurement by LC-MS and LC-ELSD. Since the lack of single pure standard of phytosterol conjugates, it is hard to quantify accurately by LC-MS. ELSD provides a more uniform response to structurally similar analytes, so here we speculated that the results measured by LC-ELSD were much accurate than LC-MS. Avenasterol, campesterol, stigmasterol and β-sitosterol are predominate among sample phytosterols. The major phytosterols in brown rices are FS and SF (γ-oryzanol) and there is less SG and ASG in brown rices. Legumes all have high content of FS and ASG but no SF. Soybean and black soybean contain much higher amounts of SG than other samples.