新型在线净化前处理HPLC-MS/MS测定香精香料中的麦角甾醇和麦角甾酮
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摘要
建立一种新型的在线净化前处理装置结合高效液相色谱-串联质谱(high performance liquid chromatography-tandem with mass spectrometry,HPLC-MS/MS)法测定香精香料中的麦角甾醇和麦角甾酮。该前处理装置结合基质分散萃取和柱层析对样品进行萃取和净化,之后对样品进行浓缩并利用HPLC-MS/MS分析测定。HPLC-MS/MS方法以甲醇和水为流动相,0.4 mL/min流速条件下梯度洗脱,采用C_(18)色谱柱进行液相色谱分离,大气压正离子方式电离,多重反应监测模式检测,内标法进行定量分析。结果表明,该方法前处理方便且稳定性强,溶剂可回收,净化萃取浓缩为一体;目标物质麦角甾醇和麦角甾酮能在5 min内得到分离检测,在1.5~250μg/m L范围内具有较好的线性,各待测物的线性相关系数均大于0.999,检出限为0.32~0.35μg/mL,在不同添加水平条件下,平均回收率为86%~96%,相对标准偏差在3.1%~3.6%之间。本方法能较好地应用于香精香料实际样品中麦角甾醇和麦角甾酮的测定。
A method using a new online purifying pretreatment system and high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)was established for the determination of ergosterol and ergotsterone in food flavors and spices.The pretreatment method in combination with matrix solid-phase dispersion and column chromatography was used for sample extraction and purification,followed by concentration and analysis by HPLC-MS/MS.The analysis was performed on a C_(18) column with gradient elution with a mobile phase consisting of water and methanol at a flow rate of0.4 mL/min.The mass spectrometer with an atmospheric pressure chemical ionization(APCI)source in the positive ion mode was operated using multiple reaction monitoring(MRM)and an internal standard method was used for quantification.The results showed that the pretreatment method was simple and stable with solvent recycle,integrating extraction,purification and concentration.The two analytes could be separated in 5 min with good linearity in the concentration range of1.5–250μg/m L with correlation coefficients larger than 0.999.The limits of detection(LODs)were 0.32μg/mL for ergosterol and 0.35μg/mL for ergotsterone.The mean recoveries of the target compounds at three different spiked levels were 86%–96%with relative deviations(RSDs)of 3.1%–3.6%.This method is suitable for the analysis of ergosterol and ergotsterone in food flavors and spices.
A method using a new online purifying pretreatment system and high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS)was established for the determination of ergosterol and ergotsterone in food flavors and spices.The pretreatment method in combination with matrix solid-phase dispersion and column chromatography was used for sample extraction and purification,followed by concentration and analysis by HPLC-MS/MS.The analysis was performed on a C_(18) column with gradient elution with a mobile phase consisting of water and methanol at a flow rate of0.4 mL/min.The mass spectrometer with an atmospheric pressure chemical ionization(APCI)source in the positive ion mode was operated using multiple reaction monitoring(MRM)and an internal standard method was used for quantification.The results showed that the pretreatment method was simple and stable with solvent recycle,integrating extraction,purification and concentration.The two analytes could be separated in 5 min with good linearity in the concentration range of1.5–250μg/m L with correlation coefficients larger than 0.999.The limits of detection(LODs)were 0.32μg/mL for ergosterol and 0.35μg/mL for ergotsterone.The mean recoveries of the target compounds at three different spiked levels were 86%–96%with relative deviations(RSDs)of 3.1%–3.6%.This method is suitable for the analysis of ergosterol and ergotsterone in food flavors and spices.
引文
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[17]相聪坤,宿树兰,关胜江,等.HPLC法测定不同产地不同品种马勃药材中麦角甾醇[J].中草药,2016,47(8):1397-1400.DOI:10.7501/j.issn.0253-2670.2016.08.025.
[18] 张薇薇,龚韬,韩东河,等.RP-HPLC测定人工虫草和天然虫草中麦角甾醇含量[J].中国中医药信息杂志,2014,21(4):67-69.DOI:10.3969/j.issn.1005-5304.2014.04.021.
[19]易承学,童珊珊,徐希明,等.HPLC法同时测定金针菇中麦角甾醇和22,23-二氢麦角甾醇的含量[J].江苏大学学报(医学版),2014,24(2):126-128.DOI:10.13312/j.issn.1671-7783.y130281.
[20]ZHAO Y Y,QIN X Y,CHENG X L,et al.Rapid resolution liquid chromatography-mass spectrometry and high-performance liquid chromatography-fluorescence detection for metabolism and pharmacokinetic studies of ergosta-4,6,8(14),22-tetraen-3-one[J].Analytica Chimica Acta,2010,675(2):199-206.DOI:10.1016/j.aca.2010.07.029.
[21]ZHAO Y Y,ZHAO Y,ZHANG Y M,et al.Qualitative and quantitative analysis of the diuretic component ergone inPolyporus umbellatus by HPLC with fluorescence detection and HPLC-APCI-MS/MS[J]. Pharmazie, 2009, 64(6): 366-370.DOI:10.1691/ph.2009.8364.
[22]李思明,冯怡,曾星.HPLC-APCI-MS/MS法同时测定猪苓颗粒中麦角甾酮与麦角甾醇的含量[J].药物分析杂志,2014,34(4):649-653.
[23]戚大伟,林华清,沙云菲,等.气相色谱-质谱联用法测定烟草及烟草制品中的麦角甾醇[J].烟草科技,2013(5):41-45.DOI:10.3969/j.issn.1002-0861.2013.05.011.
[24]黄龙,王进元,罗诚浩,等.烟草中游离植物甾醇的G C/M S/S I M分析[J].烟草科技,2006(10):41-45.DOI:10.3969/j.issn.1002-0861.2006.10.011.
[25]LIU W H,DING B,RUAN X M,et al.Analysis of free and conjugated phytosterols in tobacco by an improved method using gas chromatography-flame ionization detection[J].Journal of Chromatography A,2007,1163(1/2):304-311.DOI:10.1016/j.chroma.2007.06.043.
[26]SHA Y F,DENG C H,LIU B Z.Development of C18-functionalized magnetic silica nanoparticles as sample preparation technique for the determination of ergosterol in cigarettes by microwave-assisted derivatization and gas chromatography/mass spectrometry[J].Journal of Chromatography A,2008,1198/1199:27-33.DOI:10.1016/j.chroma.2008.05.049.
[27]唐艳斌,段晓艳,王丽娟,等.麦角甾醇含量检测分析柑橘真菌污染程度的方法研究[J].中国酿造,2016,35(6):50-54.DOI:10.11882/j.issn.0254-5071.2016.06.011.
[28]李蓉,卢俊文,杨芳,等.基质固相分散萃取-气相色谱-串联质谱法同时测定蔬菜中195种农药残留[J].食品科学,2014,35(24):301-307.DOI:10.7506/spkx1002-6630-201424058.
[29]乌日娜,李建科.基质固相分散在食品安全分析中的应用[J].食品科学,2005,26(6):266-268.DOI:10.3321/j.issn:1002-6630.2005.06.063.
[30]罗自生,冯思敏,张炎冰,等.一种测定甘蔗中不同形式植物甾醇含量的方法:201510244497.6[P].2015-09-16.
[2]吕丽爽,陶菲.香精香料分析方法进展[J].食品科学,2005,26(8):478-482.DOI:10.3321/j.issn:1002-6630.2005.08.124.
[3]徐杨斌,王凯,刘强,等.香精香料中芳香化合物的分析方法研究进展[J].化学与生物工程,2015,32(6):6-10.DOI:10.3969/j.issn.1672-5425.2015.06.002.
[4]邓玉清,王纪,虞龙.微生物麦角甾醇的研究进展[J].微生物学杂志,2001,21(3):45-47.DOI:10.3969/j.issn.1005-7021.2001.03.017.
[5]MAERTENS J A.History of the development of azole derivatives[J].Clinical Microbiology&Infection,2004,10(1):1-10.DOI:10.1111/j.1470-9465.2004.00841.x.
[6]REESLEV M,MILLER M,NIELSEN K F.Quantifying mold biomass on gypsum board:comparison of ergosterol and beta-Nacetylhexosaminidase as mold biomass parameters[J].Applied&Environmental Microbiology,2003,69(7):3996-4003.DOI:10.1128/AEM.69.7.3996-3998.2003.
[7]WEETE J D. Fungal lipid biochemistry: distribution and metabolism[M].New York:Plenum Press,1974:160.
[8]NES W R.Role of sterols in membranes[J].Lipids,1974,9(8):596-612.DOI:10.1007/BF02532509.
[9]BARAJAS-ACEVES M,HASSAN M,TINOCO R,et al.Effect of pollutants on the ergosterol content as indicator of fungal biomass[J].Journal of Microbiological Methods,2002,50(12):227-236.DOI:10.1016/S0167-7012(02)00031-3.
[10]ABRAMSON D,HULASARE R,YORK R K,et al.Mycotoxins,ergosterol,and odor volatiles in durum wheat during granary storage at 16%and 20%moisture content[J].Journal of Stored Products Research,2005,41(1):67-76.DOI:10.1016/j.jspr.2003.11.002.
[11]国家标准化管理委员会.粮油检验粮食中麦角甾醇的测定正相高效液相色谱法:GB/T 25221—2010[S].北京:中国标准出版社,2010.
[12]MARTIN F,DELARUELLE C,HILBERT J L.An improved ergosterol assay to estimate fungal biomass in ectomycorrhizas[J].Mycological Research,1990,94(8):1059-1064.DOI:10.1016/S0953-7562(09)81333-6.
[13]DJAJAKIRANA G,JOERGENSEN R G,MEYER B.Ergosterol and microbial biomass relationship in soil[J].Biology&Fertility of Soils,1996,22(4):299-304.DOI:10.1007/BF00334573.
[14]胡月芳.毛细管电泳-电化学检测法测定淮山中三种植物甾醇含量[J].北方园艺,2016(12):162-165.DOI:10.11937/bfyy.201612040.
[15]LI S P,LAI C M,GONG Y X,et al.Simultaneous determination of ergosterol,nucleosides and their bases from natural and cultured Cordyceps by pressurised liquid extraction and high-performance liquid chromatography[J].Journal of Chromatography A,2004,1306(2):239-242.DOI:10.1016/j.chroma.2004.02.080.
[16]李迎雪,戴肖东,詹亚光.H P L C法测定猴头菌菌丝体中麦角甾醇含量[J].食用菌,2010(6):63-64.DOI:10.3969/j.issn.1000-8357.2010.06.037.
[17]相聪坤,宿树兰,关胜江,等.HPLC法测定不同产地不同品种马勃药材中麦角甾醇[J].中草药,2016,47(8):1397-1400.DOI:10.7501/j.issn.0253-2670.2016.08.025.
[18] 张薇薇,龚韬,韩东河,等.RP-HPLC测定人工虫草和天然虫草中麦角甾醇含量[J].中国中医药信息杂志,2014,21(4):67-69.DOI:10.3969/j.issn.1005-5304.2014.04.021.
[19]易承学,童珊珊,徐希明,等.HPLC法同时测定金针菇中麦角甾醇和22,23-二氢麦角甾醇的含量[J].江苏大学学报(医学版),2014,24(2):126-128.DOI:10.13312/j.issn.1671-7783.y130281.
[20]ZHAO Y Y,QIN X Y,CHENG X L,et al.Rapid resolution liquid chromatography-mass spectrometry and high-performance liquid chromatography-fluorescence detection for metabolism and pharmacokinetic studies of ergosta-4,6,8(14),22-tetraen-3-one[J].Analytica Chimica Acta,2010,675(2):199-206.DOI:10.1016/j.aca.2010.07.029.
[21]ZHAO Y Y,ZHAO Y,ZHANG Y M,et al.Qualitative and quantitative analysis of the diuretic component ergone inPolyporus umbellatus by HPLC with fluorescence detection and HPLC-APCI-MS/MS[J]. Pharmazie, 2009, 64(6): 366-370.DOI:10.1691/ph.2009.8364.
[22]李思明,冯怡,曾星.HPLC-APCI-MS/MS法同时测定猪苓颗粒中麦角甾酮与麦角甾醇的含量[J].药物分析杂志,2014,34(4):649-653.
[23]戚大伟,林华清,沙云菲,等.气相色谱-质谱联用法测定烟草及烟草制品中的麦角甾醇[J].烟草科技,2013(5):41-45.DOI:10.3969/j.issn.1002-0861.2013.05.011.
[24]黄龙,王进元,罗诚浩,等.烟草中游离植物甾醇的G C/M S/S I M分析[J].烟草科技,2006(10):41-45.DOI:10.3969/j.issn.1002-0861.2006.10.011.
[25]LIU W H,DING B,RUAN X M,et al.Analysis of free and conjugated phytosterols in tobacco by an improved method using gas chromatography-flame ionization detection[J].Journal of Chromatography A,2007,1163(1/2):304-311.DOI:10.1016/j.chroma.2007.06.043.
[26]SHA Y F,DENG C H,LIU B Z.Development of C18-functionalized magnetic silica nanoparticles as sample preparation technique for the determination of ergosterol in cigarettes by microwave-assisted derivatization and gas chromatography/mass spectrometry[J].Journal of Chromatography A,2008,1198/1199:27-33.DOI:10.1016/j.chroma.2008.05.049.
[27]唐艳斌,段晓艳,王丽娟,等.麦角甾醇含量检测分析柑橘真菌污染程度的方法研究[J].中国酿造,2016,35(6):50-54.DOI:10.11882/j.issn.0254-5071.2016.06.011.
[28]李蓉,卢俊文,杨芳,等.基质固相分散萃取-气相色谱-串联质谱法同时测定蔬菜中195种农药残留[J].食品科学,2014,35(24):301-307.DOI:10.7506/spkx1002-6630-201424058.
[29]乌日娜,李建科.基质固相分散在食品安全分析中的应用[J].食品科学,2005,26(6):266-268.DOI:10.3321/j.issn:1002-6630.2005.06.063.
[30]罗自生,冯思敏,张炎冰,等.一种测定甘蔗中不同形式植物甾醇含量的方法:201510244497.6[P].2015-09-16.