HPLC-UV法测定植物甾醇中3种甾醇单体的含量
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摘要
本文建立了一种利用紫外高效液相法(HPLC-UV)测定植物甾醇中菜油甾醇、豆甾醇和β-谷甾醇的方法。采用Waters Symmetry~? C_(18)柱(4.6 mm×250 mm,5μm),以乙腈和水为流动相等度洗脱,柱温30℃,流速1.0 mL/min,紫外检测波长为208 nm。实验结果表明:菜油甾醇、豆甾醇和β-谷甾醇在38 min内均能实现基线分离,线性范围分别为2.52～50.30、5.08～101.60、5.10～102.00μg/mL时,甾醇单体的线性关系良好,相关系数r分别为0.9971、0.9989、0.9991,检测限为2.5μg/mL;日内精密度在0.06%～3.06%范围内,日间精密度介于1.56%～6.61%;加样回收率介于92.74%～106.25%之间。本方法能够准确测定4种植物甾醇中3种甾醇单体的含量,其中大豆甾醇和木甾醇中3种甾醇单体的含量组成差异较大,大豆甾醇中菜油甾醇和豆甾醇的含量分别为163.80～251.23 mg/g和134.89～235.04 mg/g,远高于木甾醇中的菜油甾醇和豆甾醇含量,而木甾醇中β-谷甾醇含量为(685.10±7.55) mg/g,则明显高于β-谷甾醇在大豆甾醇中的含量。相对现有的高效液相方法,本方法实现了对混合甾醇中菜油甾醇、豆甾醇和β-谷甾醇3种甾醇单体的精确定量分析。
This study was aimed to establish a HPLC approach for the quantitative analysis of campesterol,stigmasterol andβ-sitosterol in phytosterol.In HPLC analysis,the chromatographic separation was achieved on a Waters Symmetry~? C_(18) column (4.6 mm × 250 mm,5 μm) with a mobile phase composed of acetonitrile and water at a flow rate of 1.0 mL/min.In addition,detection wavelength and column temperature were set at 208 nm and 30 ℃,respectively.The results showed that three sterols reached the baseline separation and the concentration ranges of campsterol,stigmasterol and β-sitosterol were 2.52～50.30,5.08～101.60 and 5.10～102.00 μg/mL respectively.It indicated a good linear relationship and the correlation coefficients of three sterols was 0.9971,0.9989,0.9991.The limit of detection was 2.5 μg/mL,the intra-day precision was between 0.06% ～3.06%and the inter-day precision was between 1.56% ～ 6.61%,the recovery of the three components was 92.74% ～ 106.25%. This method could accurately measure campesterol,stigmasterol and β-sitosterol in phytosterols from 4 kinds of production places,the content of 3 individual phytosterols from deodorizer distillate and tall oil had great differences. The content of campesterol and stigmasterol in phytosterol was 163.80～251.23 mg/g and 134.89～235.04 mg/g,much higher than content in wood sterol.The content of β-sitosterol in wood sterol was (685.10 ± 7.55) mg/g,which was higer than phytosterol. It could be concluded that this was a better method for quality assessment of campesterol,stigmasterol and β-sitosterol in phytosterol.
This study was aimed to establish a HPLC approach for the quantitative analysis of campesterol,stigmasterol andβ-sitosterol in phytosterol.In HPLC analysis,the chromatographic separation was achieved on a Waters Symmetry~? C_(18) column (4.6 mm × 250 mm,5 μm) with a mobile phase composed of acetonitrile and water at a flow rate of 1.0 mL/min.In addition,detection wavelength and column temperature were set at 208 nm and 30 ℃,respectively.The results showed that three sterols reached the baseline separation and the concentration ranges of campsterol,stigmasterol and β-sitosterol were 2.52～50.30,5.08～101.60 and 5.10～102.00 μg/mL respectively.It indicated a good linear relationship and the correlation coefficients of three sterols was 0.9971,0.9989,0.9991.The limit of detection was 2.5 μg/mL,the intra-day precision was between 0.06% ～3.06%and the inter-day precision was between 1.56% ～ 6.61%,the recovery of the three components was 92.74% ～ 106.25%. This method could accurately measure campesterol,stigmasterol and β-sitosterol in phytosterols from 4 kinds of production places,the content of 3 individual phytosterols from deodorizer distillate and tall oil had great differences. The content of campesterol and stigmasterol in phytosterol was 163.80～251.23 mg/g and 134.89～235.04 mg/g,much higher than content in wood sterol.The content of β-sitosterol in wood sterol was (685.10 ± 7.55) mg/g,which was higer than phytosterol. It could be concluded that this was a better method for quality assessment of campesterol,stigmasterol and β-sitosterol in phytosterol.
引文
[1]Moreau R A,Whitaker Hitaker B D,Hicks K B.Phytosterols,phytostanols,and their conjugates in foods:structural diversity,quantitative analysis,and health-promoting uses[J].Progress in Lipid Research,2002,41(6):457-500.
[2]Carmona M A,Jimenez C,Jimenez S C,et al.Isolation of sterols from sunflower oil deodorizer distillate[J].Food Engineering,2010,101(2):210-213.
[3]Olkkonen V M,Gylling H,Ikonen E.Plant sterols,cholesterol precursors and oxysterols:Minute concentrations-major physiological effects[J].Steroid Biochemistry&Molecular Biology,2017,169:4-9.
[4]Kumar S,Mawlong I,Singh D.Phytosterol recovery from oilseeds:Recent advances[J].Food Process Engineering,2016.
[5]Hicks K B,Moreau R A.Phytosterols and phytostanols:functional food cholesterol busters[J].Food Technol,2001,55(1):63-67.
[6]Laakso P.Analysis of sterols from various food matrices[J].Eur.J.Lipid Sci.Technol.,2005,107(6):402-410.
[7]Fernandes P,Cabral J M S.Phytosterols:applications and recovery methods[J].Bioresource Technology,2007,98(12):2335-2350.
[8]鲁海龙,史宣明,张旋,等.植物甾醇制取及应用研究进展[J].中国油脂,2017,42(10):134-137.
[9]Lerma-Garcia M J,Simo-Alfonso E F,Mendez A,et al.Classification of extra virgin olive oils according to their genetic variety using linear discriminant analysis of sterol profiles established by ultra-performance liquid chromatography with mass spectrometry detection[J].Food Research International,2011,44(1):103-108.
[10]Abidi S L.Chromatographic analysis of plant sterols in foods and vegetable oils[J].Chromatographic A,2001,935(1):173-201.
[11]赵伟,韩敬美,刘春波,等.植物甾醇的分析方法研究进展[J].分析仪器,2012(4):6-11.
[12]Feng S,Liu S,Luo Z,et al.Direct saponification preparation and analysis of free and conjugated phytosterols in sugarcane(Saccharum officinarum L.)by reversed-phase high-performance liquid chromatography[J].Food Chemistry,2015,181:9-14.
[13]Careri M,Elviri L,Mangia A.Liquid chromatography-UVdetermination and liquid chromatography-atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soybean oil[J].Chromatography A2001,935(1):249-257.
[14]石晶,顾强,乙小娟,等.高效液相色谱-质谱法测定橄榄油中β-谷甾醇的含量[J].食品科技,2015(6):352-354,359.
[15]Bendner M,Schantz M M,Sander L C,et al.Development of liquid chromatographic methods for the determination of phytosterols in standard reference materials containing saw palmetto[J].Chromatography A,2008,1192(1):74-80.
[16]佘家姮.植物甾醇的高效液相色谱测定研究[J].海峡药学,2012,24(12):47-49.
[17]Lagarda M J,Garcia-Llatas G,Farre R.Analysis of phytosterols in foods[J].Pharmaceutical and Biomedical Analysis,2006,41(5):1486-1496.
[18]Nair V D P,Kanfer I,Hoogmartens J.Determination of stigmasterol,β-sitosterol and stigmastanol in oral dosage forms using high performance liquid chromatography with evaporative light scattering detection[J].Pharmaceutical and Biomedica Analysis,2006,41(3):731-737.
[19]Zhang X,Cambrai A,Miesch M,et al.Separation ofΔ5-andΔ7-phytosterols by adsorption chromatography and semipreparative reversed phase high-performance liquid chromatography for quantitative analysis of phytosterols in foods[J].Agriculture and Food Chemistry,2006,54(4):1196-1202.
[20]Zhang X,Geoffroy P,Miesch M,et al.Gram-scale chromatographic purification ofβ-sitosterol:synthesis and characterization ofβ-sitosterol oxides[J].Steroids,2005,70(13):886-895.
[21]Sanchez-Machado D I,Lopez-Hernadez J,Paseiro-Losada P,et al.An HPLC method for the quantification of sterols in edible seaweeds[J].Biomedical Chromatography,2004,18(3):183-190.
[22]石龙凯,刘玉兰.液相色谱-串联质谱法检测食用油脂中多环芳烃[J].中国粮油学报,2015,30(12):114-119.
[23]Aro T,Fatehi P.Tall oil production from black liquor:Challenges and opportunities[J].Separation Purification Technology,2017,175:469-480.
[2]Carmona M A,Jimenez C,Jimenez S C,et al.Isolation of sterols from sunflower oil deodorizer distillate[J].Food Engineering,2010,101(2):210-213.
[3]Olkkonen V M,Gylling H,Ikonen E.Plant sterols,cholesterol precursors and oxysterols:Minute concentrations-major physiological effects[J].Steroid Biochemistry&Molecular Biology,2017,169:4-9.
[4]Kumar S,Mawlong I,Singh D.Phytosterol recovery from oilseeds:Recent advances[J].Food Process Engineering,2016.
[5]Hicks K B,Moreau R A.Phytosterols and phytostanols:functional food cholesterol busters[J].Food Technol,2001,55(1):63-67.
[6]Laakso P.Analysis of sterols from various food matrices[J].Eur.J.Lipid Sci.Technol.,2005,107(6):402-410.
[7]Fernandes P,Cabral J M S.Phytosterols:applications and recovery methods[J].Bioresource Technology,2007,98(12):2335-2350.
[8]鲁海龙,史宣明,张旋,等.植物甾醇制取及应用研究进展[J].中国油脂,2017,42(10):134-137.
[9]Lerma-Garcia M J,Simo-Alfonso E F,Mendez A,et al.Classification of extra virgin olive oils according to their genetic variety using linear discriminant analysis of sterol profiles established by ultra-performance liquid chromatography with mass spectrometry detection[J].Food Research International,2011,44(1):103-108.
[10]Abidi S L.Chromatographic analysis of plant sterols in foods and vegetable oils[J].Chromatographic A,2001,935(1):173-201.
[11]赵伟,韩敬美,刘春波,等.植物甾醇的分析方法研究进展[J].分析仪器,2012(4):6-11.
[12]Feng S,Liu S,Luo Z,et al.Direct saponification preparation and analysis of free and conjugated phytosterols in sugarcane(Saccharum officinarum L.)by reversed-phase high-performance liquid chromatography[J].Food Chemistry,2015,181:9-14.
[13]Careri M,Elviri L,Mangia A.Liquid chromatography-UVdetermination and liquid chromatography-atmospheric pressure chemical ionization mass spectrometric characterization of sitosterol and stigmasterol in soybean oil[J].Chromatography A2001,935(1):249-257.
[14]石晶,顾强,乙小娟,等.高效液相色谱-质谱法测定橄榄油中β-谷甾醇的含量[J].食品科技,2015(6):352-354,359.
[15]Bendner M,Schantz M M,Sander L C,et al.Development of liquid chromatographic methods for the determination of phytosterols in standard reference materials containing saw palmetto[J].Chromatography A,2008,1192(1):74-80.
[16]佘家姮.植物甾醇的高效液相色谱测定研究[J].海峡药学,2012,24(12):47-49.
[17]Lagarda M J,Garcia-Llatas G,Farre R.Analysis of phytosterols in foods[J].Pharmaceutical and Biomedical Analysis,2006,41(5):1486-1496.
[18]Nair V D P,Kanfer I,Hoogmartens J.Determination of stigmasterol,β-sitosterol and stigmastanol in oral dosage forms using high performance liquid chromatography with evaporative light scattering detection[J].Pharmaceutical and Biomedica Analysis,2006,41(3):731-737.
[19]Zhang X,Cambrai A,Miesch M,et al.Separation ofΔ5-andΔ7-phytosterols by adsorption chromatography and semipreparative reversed phase high-performance liquid chromatography for quantitative analysis of phytosterols in foods[J].Agriculture and Food Chemistry,2006,54(4):1196-1202.
[20]Zhang X,Geoffroy P,Miesch M,et al.Gram-scale chromatographic purification ofβ-sitosterol:synthesis and characterization ofβ-sitosterol oxides[J].Steroids,2005,70(13):886-895.
[21]Sanchez-Machado D I,Lopez-Hernadez J,Paseiro-Losada P,et al.An HPLC method for the quantification of sterols in edible seaweeds[J].Biomedical Chromatography,2004,18(3):183-190.
[22]石龙凯,刘玉兰.液相色谱-串联质谱法检测食用油脂中多环芳烃[J].中国粮油学报,2015,30(12):114-119.
[23]Aro T,Fatehi P.Tall oil production from black liquor:Challenges and opportunities[J].Separation Purification Technology,2017,175:469-480.