Figure 32. The APCI-MS spectrum of trilinolein (LLL) from brown rice
20111230-BR-20%CHCl3-180min #26051-26128 RT:120.61-120.97 AV:39 NL:2.72E4 F:ITMS + c APCI corona Full ms [50.00-2000.00]
500 550 600 650 700 750 800 850 900 950 1000
m/z
二、氧化態固醇 (Oxysterols) campesterol、stigmasterol 及 β-sitosterol 的氧化態，但無法知道為何種結構。固醇
分子大多為不飽和的結構，遇到空氣及熱時仍會氧化，而本實驗中98 年期作糙米
樣品可能因存放太久而氧化形成氧化態固醇，這顯示於分析固醇時應注意樣品的 保存，否則固醇氧化後會使所測定的含量減少而使結果失真，並會干擾質譜分析。
圖33. 糙米樣品中 campesterol 氧化態的二次質譜圖
Figure 33. The APCI-MS/MS spectrum of campesterol oxide (m/z 399) from brown rice sample
20120319-Tai9-normal-1 #3631 RT:14.41AV:1 NL:8.28E2 F:ITMS + c APCI corona d Full ms2 [email protected] [95.00-810.00]
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
201.28 297.31 325.44 341.61 398.38
159.35 173.00 187.34 215.39 231.31
145.25 380.39
123.24 301.39 329.43 355.04 384.61
116.28
圖34. 糙米樣品中 stigmasterol 氧化態的二次質譜圖
Figure 34. The APCI-MS/MS spectrum of stigmasterol oxide (m/z 427) from brown rice sample
圖35. 糙米樣品中 β-sitosterol 氧化態的二次質譜圖
Figure 35. The APCI-MS/MS spectrum of β-sitosterol oxide (m/z 413) from brown rice sample
20120319-Tai9-normal-1 #3377 RT:13.40AV:1 NL:2.12E2 F:ITMS + c APCI corona d Full ms2 [email protected] [105.00-865.00]
120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
m/z
261.29283.61 313.37 339.16 367.75 385.37396.66
20120319-Tai9-normal-1 #3611 RT:14.34AV:1 NL:7.28E2 F:ITMS + c APCI corona d Full ms2 [email protected] [100.00-840.00]
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
m/z
161.12 241.35 283.41297.58
259.23
119.23133.18 311.44 355.46 370.64
三、雜質 可能為 erucamide (13-Docosenamide, M.W. = 337) 的分子離子訊號，erucamide 為 塑膠中的潤滑劑，而其二次質譜顯示連續 -14 (脫去 CH2基團) 的訊號 (圖 37)，
顯示其結構具有長鏈烷類，與 erucamide 的結構符合。
塑膠中的塑化劑，鄰苯二甲酸酯類 (phthalates) 亦可在樣品中偵測到，實驗中 最常發現者為鄰苯二甲酸二異壬酯 (diisononyl phthalate, DINP)，其特徵離子為 m/z 419，而二次質譜的訊號 (圖 38) 與文獻相符，其中 m/z 149 為所有鄰苯二甲 酸酯類皆有的特徵離子，代表 phthalic anhydride 片段 (Sørensen, 2006)。
另一 m/z 663 訊號查詢文獻後推測為塑膠中的抗氧化劑 Irgafos 168 (Tris (2,4-di-tert-butylphenyl) phosphite, M.W. = 646) 的氧化物 Irgafos 168 phosphate (Tris (2,4-di-tert-butylphenyl) phosphate, M.W. = 662) (Chen and Her, 1993; Carrott, 1998)，
其分子離子為 m/z 633，文獻中的二次質譜含有 m/z 607、551、495、439、383；
本實驗的二次質譜則含有前述4 個離子片段 (圖 39)，與文獻高度符合。
圖36. 糙米樣品中塑膠添加物的萃取離子層析圖
Figure 36. Extracted ion chromatograms of plastic additives: m/z (A) 338, (B) 419, and
RT:0.00 - 25.00 SM:15B APCI corona Full ms [200.00-1000.00] MS 20120319-Tai9-normal-2
NL: 1.12E4 Base Peak m/z=
419.00-420.00 F: ITMS + c APCI corona Full ms [200.00-1000.00] MS 20120319-Tai9-normal-2
NL: 6.26E4 Base Peak m/z=
663.00-664.00 F: ITMS + c APCI corona Full ms [200.00-1000.00] MS 20120319-Tai9-normal-2
(A) m/z 338
(B) m/z 419
(C) m/z 663
圖37. 糙米樣品中 erucamide 的二次質譜圖
Figure 37. The APCI-MS/MS spectrum of erucamide (m/z 338) from brown rice sample
圖38. 糙米樣品中 DINP 的二次質譜圖
Figure 38. The APCI-MS/MS spectrum of DINP (m/z 419) from brown rice sample
20120319-Tai9-normal-2 #1772 RT:6.99AV:1 NL:8.34E2 F:ITMS + c APCI corona d Full ms2 [email protected] [80.00-690.00]
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
m/z
121.12135.23149.25 170.25 198.23205.28 226.41 247.42 268.47282.47296.34 338.65
109.25 177.52 311.22 345.40
20120319-Tai9-normal-2 #1766 RT:6.96AV:1 NL:2.45E2 F:ITMS + c APCI corona d Full ms2 [email protected] [105.00-850.00]
100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440
m/z
126.92 152.38 195.27 213.26 250.37262.42 277.29 376.44
121.22 133.29 190.48 224.17
圖39. 糙米樣品中 Irgafos 168 phosphate 的二次質譜圖
Figure 39. The APCI-MS/MS spectrum of Irgafos 168 phosphate (m/z 663) from brown rice
雖然於實驗時多採用玻璃器具 (含針筒) 已大幅降低上述塑膠添加物的含量，
但樣品經固相萃取時仍會接觸到塑膠材質的固相萃取管，使用有機溶劑流洗仍無 法避掉上述物質一併洗出，故仍能於分析樣品時偵測到。而這些塑膠添加物多為 低極性，使用 APCI 為游離法時會比 ESI 更容易偵測到這些物質的存在。另外實 驗時發現如使用塑膠 tip 吸取甲醇後，該瓶甲醇較未接觸塑膠 tip 的甲醇相比含 有更高量的塑膠抗氧化劑 (Irgafos 168)，這顯示實驗時吸取溶劑需使用氣密針或玻 璃吸量管以避免分析時的干擾及汙染溶劑。
(二) 針筒潤滑劑
正相層析分析樣品前須事先將樣品以針筒過濾器過濾，預實驗時發現以塑膠
針筒過濾器過濾樣品時於層析時間2-4 分鐘處有數個波峰產生，但以玻璃針筒過濾
時則消失，再測試以塑膠及玻璃針筒過濾空白溶劑氯仿後分析，也有相同結果，
圖40 顯示氯仿經塑膠針筒過濾後較玻璃針筒過濾者多出至少 3 個波峰，依其滯留
時間推測極性較蠟酯及酯化態固醇低。對照文獻中以正相管柱分析低極性物質的 結果 (Moreau et al., 1990; Nordbäck and Lundberg, 1999)，這些波峰可能為碳氫化合 物 (hydrocarbon)，推測為塑膠針筒中的潤滑劑成分，因此正相層析後續實驗皆使
20120319-Tai9-normal-2 #3988 RT:15.81AV:1 NL:3.33E3 F:ITMS + c APCI corona d Full ms2 [email protected] [170.00-1340.00]
250 300 350 400 450 500 550 600 650 700
m/z 0
10 20 30 40 50 60 70 80 90 100
Relative Abundance
663.66
607.55
309.01 355.08 438.82 495.55 551.69 644.52
圖40. 氯仿經塑膠及玻璃針筒過濾後的層析圖
Figure 40. HPLC–ELSD chromatogram of chloroform filtered through 0.22 µm nylon filter with (A) plastic syringe and (B) glass syringe
mV
minute
0 5 10 15
0 50 100 150 200 250 300 350 400
(A) (B)
柒、結論
本實驗建立了以逆相液相層析串聯質譜儀同步分析自由態、steryl ferulate、醣 苷態及酯化醣苷態4 種植物固醇的方法。淨化樣品使用 NH2固相萃取管取代傳統 化學水解法，較為快速且便利。固相萃取可將三酸甘油酯及酯化態固醇去除並分 離植物固醇成分，保持固醇分子完整型態，避免水解法產生的問題。以大氣壓力 化學游離法可順利將低極性的植物固醇分子游離，植物固醇於逆相液相層析分離 後利用串聯質譜儀可推測分子的結構資訊，了解樣品中的固醇組成。
另以建立以正相液相層析搭配蒸發光散射偵測器分析植物固醇的方法，樣品 可不經其他前處理直接分析，結果顯示以正相層析管柱可將不同種類植物固醇分 離成單一波峰，由於蒸發光散射偵測器對於結構相似的固醇分子有接近的感應值，
對於植物固醇的定量較為精確。相較於質譜儀分析，不同植物固醇分子的游離化 程度差異較大，亦缺乏標準品進行定量分析，故僅以自由態固醇作為定量基準會 產生極大偏差。
但是以正相或逆相液相層析皆無法達成同步分析脂肪酸酯化態固醇的目的，
為本分析方法的缺點。由於酯化態固醇會於 C18 管柱中嚴重滯留，已使用固相萃 取方式去除；而酯化態固醇於 Diol 管柱中無法和蠟酯類化合物分離開，亦無法定 量。此結果顯示以常使用的正相及逆相層析管柱並無法同步分離植物固醇，後續 研究可能需使用較少使用的材質 (如 alumina 正相管柱) 或是不同裝填顆粒 (如 monolithic ) 的管柱進行分析，才能克服現有管柱的限制，達成同步分析植物固醇 的需求。
近年來發現醣苷態固醇與自由態及酯化態固醇不同，具有特殊的生理活性，
但傳統分析方法常忽略醣苷態固醇而未分析，現有文獻亦缺乏對其分析的內容。
本實驗建立的方法可對4 種植物固醇同步分析，並得到較完整的層析及質譜資訊，
可供後續研究參考。
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