PMP柱前衍生化HPLC法测定黄秋葵多糖的单糖组成
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摘要
采用1-苯基-3-甲基-5-吡唑啉酮为柱前衍生化试剂,结合反相高效液相色谱法,建立同时测定黄秋葵多糖中8种单糖组分的方法。采用InfinityLab Poroahell C18色谱柱为固定相,流动相为磷酸盐缓冲液(0.1 mol/L,pH 6.85)-乙腈(82∶18,V/V),流速为1.0 mL/min,柱温25℃,紫外检测器,波长250 nm。各单糖组分的线性范围较宽,相关系数均大于0.999,检出限为0.13～0.45 mg/kg,定量限为0.43～1.49 mg/kg,相对标准偏差小于5%,加标回收率为94.51%～106.44%。方法灵敏度高、准确性好、实用可靠,适用于黄秋葵多糖的单糖组分分析。同时测定从黄秋葵中顺序提取的热水浸提物(hot buffer soluble solution,HBSS)、螯合剂浸提物(chelating agent solublesolution,CHSS)和碱提物(dilute alkaline soluble solution,DASS)的单糖组成,确定其单糖组分的物质的量比。HBSS中,甘露糖∶鼠李糖∶葡萄糖醛酸∶半乳糖醛酸∶葡萄糖∶半乳糖∶阿拉伯糖=0.1∶7.0∶0.4∶5.8∶0.9∶14.6∶1.5;CHSS中,鼠李糖∶半乳糖醛酸∶半乳糖∶阿拉伯糖=4.7∶61.8∶9.6∶5.3;DASS中,甘露糖∶鼠李糖∶半乳糖醛酸∶葡萄糖∶半乳糖∶阿拉伯糖=9.5∶5.4∶1.3∶10.8∶2.4∶7.6。
A simple and sensitive high performance liquid chromatography(HPLC) method for simultaneous determination of eight monosaccharides in okra polysaccharides by pre-column derivatization with 1-phenyl-3-menthy-5-pyrazolone(PMP)was developed. The monosaccharides were separated on an InfinityLab Poroahell C18 column(4.6 mm × 150 mm, 4 μm),maintained at 25 ℃, using phosphate buffer(0.1 mol/L, pH 6.85): acetonitrile mixture(82:18, V/V) as a mobile phase at a flow rate of 1.0 mL/min. The wavelength of the UV detector was set at 250 nm. Under optimal conditions, the method had wide linear ranges for all monosaccharides, with correlation coefficients above 0.999. The detection limits(LODs) were in the range of 0.13–0.45 mg/kg and the limits of quantitation(LOQs) were in the range of 0.43–1.49 mg/kg. The spiked recoveries ranged from 94.51% to 106.44%, with relative standard deviations(RSDs) less than 5%. The method proved to be simple, rapid and accurate, and could meet the analysis of the monosaccharide composition of okra polysaccharides.This method was successfully applied to analyze the monosaccharide composition of three different polysaccharides from sequential solvent extraction of okra with hot sodium acetate buffer, sodium oxalate-EDTA?2 Na-sodium acetate buffer and sodium hydroxide-sodium borohydride solution. The first polysaccharide was composed of mannose, rhamnose,glucuronic acid, galacturonic acid, glucose, galactose and arabinose at a molar ratio of 0.1:7.0:0.4:5.8:0.9:14.6:1.5, the second one was composed of rhamnose, galacturonic acid, galactose and arabinose at a molar ratio of 4.7:61.8:9.6:5.3,and the third one was composed of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose at a ratio of 9.5:5.4:1.3:10.8:2.4:7.6.
A simple and sensitive high performance liquid chromatography(HPLC) method for simultaneous determination of eight monosaccharides in okra polysaccharides by pre-column derivatization with 1-phenyl-3-menthy-5-pyrazolone(PMP)was developed. The monosaccharides were separated on an InfinityLab Poroahell C18 column(4.6 mm × 150 mm, 4 μm),maintained at 25 ℃, using phosphate buffer(0.1 mol/L, pH 6.85): acetonitrile mixture(82:18, V/V) as a mobile phase at a flow rate of 1.0 mL/min. The wavelength of the UV detector was set at 250 nm. Under optimal conditions, the method had wide linear ranges for all monosaccharides, with correlation coefficients above 0.999. The detection limits(LODs) were in the range of 0.13–0.45 mg/kg and the limits of quantitation(LOQs) were in the range of 0.43–1.49 mg/kg. The spiked recoveries ranged from 94.51% to 106.44%, with relative standard deviations(RSDs) less than 5%. The method proved to be simple, rapid and accurate, and could meet the analysis of the monosaccharide composition of okra polysaccharides.This method was successfully applied to analyze the monosaccharide composition of three different polysaccharides from sequential solvent extraction of okra with hot sodium acetate buffer, sodium oxalate-EDTA?2 Na-sodium acetate buffer and sodium hydroxide-sodium borohydride solution. The first polysaccharide was composed of mannose, rhamnose,glucuronic acid, galacturonic acid, glucose, galactose and arabinose at a molar ratio of 0.1:7.0:0.4:5.8:0.9:14.6:1.5, the second one was composed of rhamnose, galacturonic acid, galactose and arabinose at a molar ratio of 4.7:61.8:9.6:5.3,and the third one was composed of mannose, rhamnose, galacturonic acid, glucose, galactose and arabinose at a ratio of 9.5:5.4:1.3:10.8:2.4:7.6.
引文
[1]KONTOGIORGOS V,MARGELOU I,GEORGIADIS N,et al.Rheological characterization of okra pectins[J].Food Hydrocolloids,2012,29(2):356-362.DOI:10.1016/j.foodhyd.2012.04.003.
[2]陈忆.黄秋葵果胶和超微粉的制备及性质研究[D].合肥:合肥工业大学,2015.
[3]刘晓霞.黄秋葵花果胶类多糖的提取工艺及其性质的研究[D].杭州:浙江大学,2014.
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[6]GEORGIADIS N,RITZOULIS C,SIOURA G,et al.Contribution of okra extracts to the stability and rheology of oil-in-water emulsions[J].Food Hydrocolloids,2011,25(5):991-999.DOI:10.1016/j.foodhyd.2010.09.014.
[7]TILMON R W,ROMANCHIK-CERPOVICZ J E.Feasibility of using okra exudate as a fat replacer in low fat chocolate dropped cookies[J].Journal of the American Dietetic Association,2001,101(9):A-23.DOI:10.1016/S0002-8223(01)80055-9.
[8]HIROSE K,ENDO K,HASEGAWA K.A convenient synthesis of lepidimoide from okra mucilage and its growth-promoting activity in hypocotyls[J].Carbohydrate Research,2004,339(1):9-19.DOI:10.1016/j.carres.2003.10.003.
[9]张丽芝,沈荣.单糖组成分析方法的研究进展[J].微生物学免疫学进展,2013,41(1):77-81.DOI:10.3969/j.issn.1005-5673.2013.01.017.
[10]SENGKHAMPARN N,VERHOEF R,SCHOLS H A,et al.Characterisation of cell wall polysaccharides from okra(Abelmoschus esculentus(L.)Moench)[J].Carbohydrate Research,2009,344(14):1824-1832.DOI:10.1016/j.carres.2008.10.012.
[11]潘媛媛,梁立娜,蔡亚岐,等.高效阴离子交换色谱-脉冲安培检测法分析啤酒和麦汁中的糖[J].色谱,2008,26(5):626-630.DOI:10.3321/j.issn:1000-8713.2008.05.020.
[12]裘雅渔,李巧玲,程和勇.高效液相体积排阻色谱法分析黄秋葵多糖的单糖组成[J].实验室研究与探索,2014,33(12):27-30.DOI:10.3969/j.issn.1006-7167.2014.12.008.
[13]STRYDOM D J.Chromatographic separation of 1-phenyl-3-methyl-5-pyrazolone-derivatized neutral,acidic and basic aldoses[J].Journal of Chromatography A,1994,678(1):17-23.DOI:10.1016/0021-9673(94)87069-1.
[14]FU D T,ONEILL R A.Monosaccharide composition analysis of oligosaccharides and glycoproteins by high-performance liquid chromatography[J].Analytical Biochemistry,1995,227(2):377-384.DOI:10.1006/abio.1995.1294.
[15]陈燕文,李玉娟,宋梦璐,等.PMP柱前衍生化-HPLC法分析玛咖多糖的单糖组成[J].当代化工,2017,46(8):1513-1516.DOI:10.3969/j.issn.1671-0460.2017.08.004.
[16]姜彩霞,刘晓兰,田英华,等.高效液相色谱法测定亚麻纤维果胶单糖衍生条件的优化[J].毛纺科技,2014,42(10):1-4.DOI:10.3969/j.issn.1003-1456.2014.10.001.
[17]戴军,朱松,汤坚,等.PMP柱前衍生高效液相色谱法分析杜氏盐藻多糖的单糖组成[J].分析测试学报,2007,26(2):206-210.DOI:10.3969/j.issn.1004-4957.2007.02.015.
[18]XUE S,WANG L,CHEN S,et al.Simultaneous analysis of saccharides between fresh and processed radix rehmanniae by HPLCand UHPLC-LTQ-Orbitrap-MS with multivariate statistical analysis[J].Molecules,2018,23(3):541-560.DOI:10.3390/molecules23030541.
[19]JIANG W X,DI S X,HUA H M,et al.Structural characterization of polysaccharide from Cyclocarya paliurus leaves and itsα-glucosidase inhibitory effect[J].Chinese Traditional&Herbal Drugs,2017,48(8):1524-1528.DOI:10.7501/j.issn.0253-2670.2017.08.008.
[20]张璐瑶,赵峡,陈欢欢.糖类化合物PMP衍生分析进展[J].分析测试学报,2016,35(3):367-372.DOI:10.3969/j.issn.1004-4957.2016.03.020.
[21]王文洁,唐炜,俞玲娜,等.蒽酮-硫酸法与苯酚-硫酸法测定凉粉草多糖的比较[J].食品科技,2017,42(9):274-279.DOI:10.13684/j.cnki.spkj.2017.09.053.
[22]付海宁,赵峡,杨海,等.柱前衍生高效液相色谱法测定多糖类兽药中单糖组分的方法研究[J].中国兽药杂志,2010,44(6):9-12.DOI:10.3969/j.issn.1002-1280.2010.06.003.
[23]朱照武,庄洋,向春林,等.单糖柱前PMP衍生HPLC测定方法探讨[J].湖北民族学院学报(自然科学版),2015,33(3):337-341.DOI:10.13501/j.cnki.42-1569/n.2015.09.027.
[24]郝蕾蕾,张典瑞,赵忠熙,等.柱前衍生化HPLC法测定黄河滩枣多糖的单糖组成[J].中国生化药物杂志,2012,33(6):740-743.DOI:10.3969/j.issn.1005-1678.2012.33.006.
[25]林钦恒,郑家概,蔡大川,等.柱前衍生化HPLC法测定酵母细胞壁中的甘露聚糖与β-葡聚糖[J].分析测试学报,2015,34(1):106-110.DOI:10.3969/j.issn.1004-4957.2015.01.017.
[26]张晓.桑叶多糖的提取分离纯化、结构鉴定及PMP柱前衍生HPLC分析单糖组成[D].上海:华东理工大学,2011.
[27]TEMENOUGA V,CHARITIDIS T,AVGIDOU M,et al.Novel emulsifiers as products from internal Maillard reactions in okra hydrocolloid mucilage[J].Food Hydrocolloids,2016,52(Suppl 1):972-981.DOI:10.1016/j.foodhyd.2015.08.026.
[28]ALBA K,LAWS A P,KONTOGIORGOS V.Isolation and characterization of acetylated LM-pectins extracted from okra pods[J].Food Hydrocolloids,2015,43:726-735.DOI:10.1016/j.foodhyd.2014.08.003.
[29]MA X L,SONG F F,ZHANG H,et al.Compositional monosaccharide analysis of Morus nigra Linn by HPLC and HPCE quantitative determination and comparison of polysaccharide from Morus nigra Linn by HPCE and HPLC[J].Current Pharmaceutical Analysis,2017,13(5):433-437.DOI:10.2174/1573412913666170330150807.
[30]COIMBRA M A,WALDRON K W,SELVENDRAN R R.Isolation and characterisation of cell wall polymers from olive pulp(Olea europaea L.)[J].Carbohydrate Research,1994,252(1):245-262.DOI:10.1016/0008-6215(94)90019-1.
[2]陈忆.黄秋葵果胶和超微粉的制备及性质研究[D].合肥:合肥工业大学,2015.
[3]刘晓霞.黄秋葵花果胶类多糖的提取工艺及其性质的研究[D].杭州:浙江大学,2014.
[4]MOHNEN D.Pectin structure and biosynthesis[J].Current Opinion in Plant Biology,2008,11(3):266-277.DOI:10.1016/j.pbi.2008.03.006.
[5]SENGKHAMPARN N,BAKX E J,VERHOEF R,et al.Okra pectin contains an unusual substitution of its rhamnosyl residues with acetyl and alpha-linked galactosyl groups[J].Carbohydrate Research,2009,344(14):1842-1851.DOI:10.1016/j.carres.2008.11.022.
[6]GEORGIADIS N,RITZOULIS C,SIOURA G,et al.Contribution of okra extracts to the stability and rheology of oil-in-water emulsions[J].Food Hydrocolloids,2011,25(5):991-999.DOI:10.1016/j.foodhyd.2010.09.014.
[7]TILMON R W,ROMANCHIK-CERPOVICZ J E.Feasibility of using okra exudate as a fat replacer in low fat chocolate dropped cookies[J].Journal of the American Dietetic Association,2001,101(9):A-23.DOI:10.1016/S0002-8223(01)80055-9.
[8]HIROSE K,ENDO K,HASEGAWA K.A convenient synthesis of lepidimoide from okra mucilage and its growth-promoting activity in hypocotyls[J].Carbohydrate Research,2004,339(1):9-19.DOI:10.1016/j.carres.2003.10.003.
[9]张丽芝,沈荣.单糖组成分析方法的研究进展[J].微生物学免疫学进展,2013,41(1):77-81.DOI:10.3969/j.issn.1005-5673.2013.01.017.
[10]SENGKHAMPARN N,VERHOEF R,SCHOLS H A,et al.Characterisation of cell wall polysaccharides from okra(Abelmoschus esculentus(L.)Moench)[J].Carbohydrate Research,2009,344(14):1824-1832.DOI:10.1016/j.carres.2008.10.012.
[11]潘媛媛,梁立娜,蔡亚岐,等.高效阴离子交换色谱-脉冲安培检测法分析啤酒和麦汁中的糖[J].色谱,2008,26(5):626-630.DOI:10.3321/j.issn:1000-8713.2008.05.020.
[12]裘雅渔,李巧玲,程和勇.高效液相体积排阻色谱法分析黄秋葵多糖的单糖组成[J].实验室研究与探索,2014,33(12):27-30.DOI:10.3969/j.issn.1006-7167.2014.12.008.
[13]STRYDOM D J.Chromatographic separation of 1-phenyl-3-methyl-5-pyrazolone-derivatized neutral,acidic and basic aldoses[J].Journal of Chromatography A,1994,678(1):17-23.DOI:10.1016/0021-9673(94)87069-1.
[14]FU D T,ONEILL R A.Monosaccharide composition analysis of oligosaccharides and glycoproteins by high-performance liquid chromatography[J].Analytical Biochemistry,1995,227(2):377-384.DOI:10.1006/abio.1995.1294.
[15]陈燕文,李玉娟,宋梦璐,等.PMP柱前衍生化-HPLC法分析玛咖多糖的单糖组成[J].当代化工,2017,46(8):1513-1516.DOI:10.3969/j.issn.1671-0460.2017.08.004.
[16]姜彩霞,刘晓兰,田英华,等.高效液相色谱法测定亚麻纤维果胶单糖衍生条件的优化[J].毛纺科技,2014,42(10):1-4.DOI:10.3969/j.issn.1003-1456.2014.10.001.
[17]戴军,朱松,汤坚,等.PMP柱前衍生高效液相色谱法分析杜氏盐藻多糖的单糖组成[J].分析测试学报,2007,26(2):206-210.DOI:10.3969/j.issn.1004-4957.2007.02.015.
[18]XUE S,WANG L,CHEN S,et al.Simultaneous analysis of saccharides between fresh and processed radix rehmanniae by HPLCand UHPLC-LTQ-Orbitrap-MS with multivariate statistical analysis[J].Molecules,2018,23(3):541-560.DOI:10.3390/molecules23030541.
[19]JIANG W X,DI S X,HUA H M,et al.Structural characterization of polysaccharide from Cyclocarya paliurus leaves and itsα-glucosidase inhibitory effect[J].Chinese Traditional&Herbal Drugs,2017,48(8):1524-1528.DOI:10.7501/j.issn.0253-2670.2017.08.008.
[20]张璐瑶,赵峡,陈欢欢.糖类化合物PMP衍生分析进展[J].分析测试学报,2016,35(3):367-372.DOI:10.3969/j.issn.1004-4957.2016.03.020.
[21]王文洁,唐炜,俞玲娜,等.蒽酮-硫酸法与苯酚-硫酸法测定凉粉草多糖的比较[J].食品科技,2017,42(9):274-279.DOI:10.13684/j.cnki.spkj.2017.09.053.
[22]付海宁,赵峡,杨海,等.柱前衍生高效液相色谱法测定多糖类兽药中单糖组分的方法研究[J].中国兽药杂志,2010,44(6):9-12.DOI:10.3969/j.issn.1002-1280.2010.06.003.
[23]朱照武,庄洋,向春林,等.单糖柱前PMP衍生HPLC测定方法探讨[J].湖北民族学院学报(自然科学版),2015,33(3):337-341.DOI:10.13501/j.cnki.42-1569/n.2015.09.027.
[24]郝蕾蕾,张典瑞,赵忠熙,等.柱前衍生化HPLC法测定黄河滩枣多糖的单糖组成[J].中国生化药物杂志,2012,33(6):740-743.DOI:10.3969/j.issn.1005-1678.2012.33.006.
[25]林钦恒,郑家概,蔡大川,等.柱前衍生化HPLC法测定酵母细胞壁中的甘露聚糖与β-葡聚糖[J].分析测试学报,2015,34(1):106-110.DOI:10.3969/j.issn.1004-4957.2015.01.017.
[26]张晓.桑叶多糖的提取分离纯化、结构鉴定及PMP柱前衍生HPLC分析单糖组成[D].上海:华东理工大学,2011.
[27]TEMENOUGA V,CHARITIDIS T,AVGIDOU M,et al.Novel emulsifiers as products from internal Maillard reactions in okra hydrocolloid mucilage[J].Food Hydrocolloids,2016,52(Suppl 1):972-981.DOI:10.1016/j.foodhyd.2015.08.026.
[28]ALBA K,LAWS A P,KONTOGIORGOS V.Isolation and characterization of acetylated LM-pectins extracted from okra pods[J].Food Hydrocolloids,2015,43:726-735.DOI:10.1016/j.foodhyd.2014.08.003.
[29]MA X L,SONG F F,ZHANG H,et al.Compositional monosaccharide analysis of Morus nigra Linn by HPLC and HPCE quantitative determination and comparison of polysaccharide from Morus nigra Linn by HPCE and HPLC[J].Current Pharmaceutical Analysis,2017,13(5):433-437.DOI:10.2174/1573412913666170330150807.
[30]COIMBRA M A,WALDRON K W,SELVENDRAN R R.Isolation and characterisation of cell wall polymers from olive pulp(Olea europaea L.)[J].Carbohydrate Research,1994,252(1):245-262.DOI:10.1016/0008-6215(94)90019-1.