高效液相色谱-串联质谱联用法与高效液相色谱法同步测定白玉菇麦角固醇和VD_2方法比较
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摘要
建立高效液相色谱-串联质谱(high performance liquid chromatography-tandem mass spectrometry,HPLCMS/MS)联用法与HPLC法同步检测白玉菇样品中麦角固醇和VD2含量方法,经方法学评价确定两种检测方法的线性范围、灵敏度与精确性。通过对经醇碱皂化回流法提取样品中麦角固醇和VD2的分析验证,阐述两种检测方法的差异性,为白玉菇有效生物活性成分与相关产品的质量检测和对白玉菇中麦角固醇与VD2之间转化关系的研究提供技术支持。HPLC-MS/MS法中,采用色谱分离柱Agilent SB-C8 Rapid Res柱(2.1 mm×50 mm,3.5μm)及在优化的色谱条件下,麦角固醇与VD2之间色谱峰保留时间分别为4.303 min和4.22 min。电喷雾离子源正离子模式下采用多反应选择离子监测模式,分别选择m/z 379.3/125.3与m/z 397.3/125.3离子对麦角固醇与VD2进行定量,结果表明麦角固醇在0.15～6 mg/L、VD2在0.01～1 mg/L范围内与峰面积线性关系良好,平均加标回收率分别为93.51%、90.56%,日内和日间相对标准偏差均小于7%。HPLC法采用COSMOSIL Column 5C18-MS-II(4.6 mm×250 mm,5μm)柱分离体系,麦角固醇和VD2色谱峰保留时间分别为12.891 min和9.919 min,方法学评价结果显示,麦角固醇在15～750 mg/L、VD2在0.5～50 mg/L范围内与峰面积线性关系良好,两者的平均回收率分别为98.51%、94.05%,日内和日间相对标准偏差均小于1%。采集的样品采用HPLC-MS/MS联用法和HPLC法分别检测实验结果有一定的差异,与HPLC法相比,HPLC-MS/MS联用法具有快速检测时间短、检出限低、更灵敏的优势。
A high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) method and a high performance liquid chromatography(HPLC) method were established to simultaneously detect the contents of ergosterol and VD2 in white Hypsizygus marmoreus and methodological evaluation was performed to determine the linearity range,sensitivity and accuracy of the two detection methods under optimized detection parameters. The differences between the two detection methods in qualitative and quantitative analysis of ergosterol and VD2 extracted from samples by alcohol-alkali saponification reflux method were verified in order to provide technical support for the detection of effective bioactive components and the study of the conversion between ergosterol and VD2 in white H. marmoreus. In the HPLC-MS/MS method, the chromatographic peak retention times of ergosterol and VD2 were 4.303 min and 4.22 min, respectively, under optimized chromatographic conditions using Agilent SB-C8 Rapid Res column(2.1 mm × 50 mm, 3.5 μm) as the separation column. Ergosterol and VD2 were quantified by selecting m/z 379.3/125.3 and 397.3/125.3 as ion pairs under the multi-reaction monitoring(MRM) mode using an electrospray ionization(ESI) source operating in the positive ion mode. The results showed that the concentration and peak area presented a good linear relationship for ergosterol and VD2 in the range of0.15–6 mg/L and 0.01–1 mg/L, respectively. The average recoveries of ergosterol and VD2 from spiked samples were 93.51%and 90.56%, respectively, and the relative standard deviations(RSDs) of intra-day and inter-day differences were both less than 7%. In the HPLC method, the peak retention times of ergosterol and VD2 were 12.891 min and 9.919 min, respectively,under optimized separation conditions on a COSMOSIL 5 C18-MS-II column(4.6 mm × 250 mm, 5 μm). The methodology evaluation results of HPLC showed a good linear relationship between concentration and peak area for ergosterol and VD2 in the range of 15–750 and 0.5–50 mg/L, respectively. The average recoveries of ergosterol and VD2 from spiked samples were98.51% and 94.05%, respectively, and the RSDs of intraday and inter-day differences were lower than 1%. Some differences existed between the two methods. Compared with HPLC, the HPLC-MS/MS method established in this research exhibited the advantages of short detection time, low detection limit and high sensitivity.
A high performance liquid chromatography-tandem mass spectrometry(HPLC-MS/MS) method and a high performance liquid chromatography(HPLC) method were established to simultaneously detect the contents of ergosterol and VD2 in white Hypsizygus marmoreus and methodological evaluation was performed to determine the linearity range,sensitivity and accuracy of the two detection methods under optimized detection parameters. The differences between the two detection methods in qualitative and quantitative analysis of ergosterol and VD2 extracted from samples by alcohol-alkali saponification reflux method were verified in order to provide technical support for the detection of effective bioactive components and the study of the conversion between ergosterol and VD2 in white H. marmoreus. In the HPLC-MS/MS method, the chromatographic peak retention times of ergosterol and VD2 were 4.303 min and 4.22 min, respectively, under optimized chromatographic conditions using Agilent SB-C8 Rapid Res column(2.1 mm × 50 mm, 3.5 μm) as the separation column. Ergosterol and VD2 were quantified by selecting m/z 379.3/125.3 and 397.3/125.3 as ion pairs under the multi-reaction monitoring(MRM) mode using an electrospray ionization(ESI) source operating in the positive ion mode. The results showed that the concentration and peak area presented a good linear relationship for ergosterol and VD2 in the range of0.15–6 mg/L and 0.01–1 mg/L, respectively. The average recoveries of ergosterol and VD2 from spiked samples were 93.51%and 90.56%, respectively, and the relative standard deviations(RSDs) of intra-day and inter-day differences were both less than 7%. In the HPLC method, the peak retention times of ergosterol and VD2 were 12.891 min and 9.919 min, respectively,under optimized separation conditions on a COSMOSIL 5 C18-MS-II column(4.6 mm × 250 mm, 5 μm). The methodology evaluation results of HPLC showed a good linear relationship between concentration and peak area for ergosterol and VD2 in the range of 15–750 and 0.5–50 mg/L, respectively. The average recoveries of ergosterol and VD2 from spiked samples were98.51% and 94.05%, respectively, and the RSDs of intraday and inter-day differences were lower than 1%. Some differences existed between the two methods. Compared with HPLC, the HPLC-MS/MS method established in this research exhibited the advantages of short detection time, low detection limit and high sensitivity.
引文
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[9]DELUCA H F.History of the discovery of vitamin D and its active metabolites[J].BoneKEY Reports,2014,3:479.DOI:10.1038/bonekey.2013.213.
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[11]GULSETH H L,GJELSTAD I M,BIRKELAND K I,et al.Vitamin Dand the metabolic syndrome[J].Current Vascular Pharmacology,2013,11(6):968-984.DOI:10.2174/15701611113119990169.
[12]LEYSSENS C,VERLINDEN L,VERSTUYF A.The future of vitamin D analogs[J].Frontiers in Physiology,2014,5:122.DOI:10.3389/fphys.2014.00122.
[13]GIRGIS C M,CLIFTON-BLIGH R J,MOKBEL N,et al.Vitamin Dsignaling regulates proliferation,differentiation,and myotube size in C2C12 skeletal muscle cells[J].Endocrinology,2014,155(2):347-357.DOI:10.1210/en.2013-1205.
[14]ARNEZEDER C H,KOLIANDER W,HAMPEL W A.Rapid determination of ergosterol in yeast cells[J].Analytica Chimica Acta,1989,225:129-136.DOI:10.1016/S0003-2670(00)84601-2.
[15]许旭萍,李惠珍,佘晨兴,等.麦角固醇产生菌Torullopsis famata优化发酵条件的研究[J].生物学杂志,2002,18(2):15-17.DOI:10.3969/j.issn.2095-1736.2002.02.006.
[16]姚广印,张双凤,谢宗良.酵母细胞麦角固醇的提取条件[J].河北大学学报(自然科学版),2007,27(2):195-198.DOI:10.3969/j.issn.1000-1565.2007.02.022.
[17]LARSEN T,AXELSEN J,RAVN H W.Simplified and rapid method for extraction of ergosterol from natural samples and detection with quantitative and semi-quantitative methods using thin-layer chromatography[J].Journal of Chromatography A,2004,1026(1/2):301-304.DOI:10.1016/j.chroma.2003.10.128.
[18]曹永兵,姜远英,殷明,等.薄层色谱扫描法检测氟康唑对真菌麦角固醇生物合成的影响[J].第二军医大学学报,1999,20(5):312-315.DOI:10.3321/j.issn:0258-879X.1999.05.014.
[19]张萍,肖新月,黄玮,等.RP-HPLC-UV法测定5种发酵虫草制剂中麦角甾醇的含量[J].药物分析杂志,2011,31(2):258-260.DOI:10.16155/j.0254-1793.2011.02.013.
[20]赵英永,赵晔,林瑞超,等.RP-HPLC法测定猪苓中麦角甾醇的含量[J].药物分析杂志,2009,29(6):898-900.DOI:10.16155/j.0254-1793.2009.06.009.
[21]汪财生,汪乐意,高有领,等.响应面法优化根霉菌产麦角固醇的液体发酵工艺[J].食品科学,2012,33(5):229-233.
[22]PHILLIPS K M,RUGGIO D M,HORST R L,et al.Vitamin D and sterol composition of 10 types of mushrooms from retail suppliers in the United States[J].Journal of Agricultural and Food Chemistry,2011,59(14):7841-7853.DOI:10.1021/jf104246z.
[23]WANG Q,CHENG J X,WANG L X,et al.Valorization of spent shiitake substrate for recovery of antitumor fungal sterols by ultrasound-assisted extraction[J].Journal of Food Biochemistry,2018,42(5):e12602.DOI:10.1111/jfbc.12602.
[24]陈光.HPLC法测定脂溶性维生素注射液中维生素D2及维生素E的含量[J].中国医药科学,2017,7(15):46-50.DOI:10.3969/j.issn.2095-0616.2017.15.013.
[25]王珵,张济敏,林玲.RP-HPLC法同时测定维生素AD小丸中维生素A棕榈酸酯和维生素D2的含量[J].中国药品标准,2017,18(4):308-311.
[26]曹少谦,陈麒宇,程亮.脉冲强光处理对香菇中维生素D2含量的影响[J].食品工业科技,2018,39(19):80-83;301.DOI:10.13386/j.issn1002-0306.2018.19.014.
[27]李丽,蒋景龙.紫外光影响食用菌中VD2含量的研究进展[J].食品科学,2015,36(1):273-277.DOI:10.7506/spkx1002-6630-201501052.
[28]MATTILA P H,LAMPI A M,RONKAINEN R,et al.Sterol and vitamin D2 contents in some wild and cultivated mushrooms[J].Food Chemistry,2002,76(3):293-298.DOI:10.1016/S0308-8146(01)00275-8.
[29]URBAIN P,JAKOBSEN J.Dose-response effect of sunlight on vitamin D2 production in Agaricus bisporus mushrooms[J].Journal of Agricultural and Food Chemistry,2015,63(37):8156-8161.DOI:10.1021/acs.jafc.5b02945.
[30]余玲玲,李兴,陈燕丽,等.液质联用技术用于复杂混合物体系中小分子化合物的分析[J].中国科学:化学,2017,47(12):1379-1391.DOI:10.1360/N032017-00118.
[31]TAOFIQ O,FERNANDES A,BARROS L,et al.UV-irradiated mushrooms as a source of vitamin D2:a review[J].Trends in Food Science&Technology,2017,70:82-94.DOI:10.1016/j.tifs.2017.10.008.
[32]张养军,王京兰,李蕾,等.用反相液相色谱分离多肽时峰容量与色谱柱长度关系的研究[J].色谱,2004,22(1):24-26.DOI:10.3321/j.issn:1000-8713.2004.01.006.
[2]曹龙辉,李晓珺,赵文红,等.麦角甾醇的研究进展[J].中国酿造,2014,33(4):9-12.DOI:10.3969/j.issn.0254-5071.2014.04.003.
[3]YASUKAWA K,AOKI T,TAKIDO M.Inhibitory effects of ergosterol isolated from the edible mushroom Hypsizigus marmoreus on TPA-induced inflammatory ear oedema and tumour promotion in mice[J].Phytotherapy Research,1994,8(1):10-13.DOI:10.1002/ptr.2650080103.
[4]SUBBIAH M T,ABPLANALP W.Ergosterol(major sterol of baker’s and brewer’s yeast extracts)inhibits the growth of human breast cancer cells in vitro and the potential role of its oxidation products[J].International Journal for Vitamin and Nutrition Research,2003,73(1):19-23.DOI:10.1024/0300-9831.73.1.19.
[5]高虹,史德芳,杨德,等.巴西菇麦角甾醇抗肿瘤活性及作用机理初探[J].中国食用菌,2011,30(6):35-39.DOI:10.3969/j.issn.1003-8310.2011.06.012.
[6]WON D J,KIM S Y,JANG C H,et al.Optimization of UV irradiation conditions for the vitamin D2-fortified shiitake mushroom(Lentinula edodes)using response surface methodology[J].Food Science and Biotechnology,2018,27(2):417-424.DOI:10.1007/s10068-017-0266-0.
[7]SIMON R R,PHILLIPS K M,HORST R L,et al.Vitamin Dmushrooms:comparison of the composition of button mushrooms(Agaricus bisporus)treated postharvest with UVB light or sunlight[J].Journal of Agricultural and Food Chemistry,2011,59(16):8724-8732.DOI:10.1021/jf201255b.
[8]JASINGHE V J,PERERA C O.Ultraviolet irradiation:the generator of vitamin D2 in edible mushrooms[J].Food Chemistry,2006,95(4):638-643.DOI:10.1016/j.foodchem.2005.01.046.
[9]DELUCA H F.History of the discovery of vitamin D and its active metabolites[J].BoneKEY Reports,2014,3:479.DOI:10.1038/bonekey.2013.213.
[10]GALLAGHER J C.Vitamin D and aging[J].Endocrinology and Metabolism Clinics of North America,2013,42(2):319-332.DOI:10.1016/j.ecl.2013.02.004.
[11]GULSETH H L,GJELSTAD I M,BIRKELAND K I,et al.Vitamin Dand the metabolic syndrome[J].Current Vascular Pharmacology,2013,11(6):968-984.DOI:10.2174/15701611113119990169.
[12]LEYSSENS C,VERLINDEN L,VERSTUYF A.The future of vitamin D analogs[J].Frontiers in Physiology,2014,5:122.DOI:10.3389/fphys.2014.00122.
[13]GIRGIS C M,CLIFTON-BLIGH R J,MOKBEL N,et al.Vitamin Dsignaling regulates proliferation,differentiation,and myotube size in C2C12 skeletal muscle cells[J].Endocrinology,2014,155(2):347-357.DOI:10.1210/en.2013-1205.
[14]ARNEZEDER C H,KOLIANDER W,HAMPEL W A.Rapid determination of ergosterol in yeast cells[J].Analytica Chimica Acta,1989,225:129-136.DOI:10.1016/S0003-2670(00)84601-2.
[15]许旭萍,李惠珍,佘晨兴,等.麦角固醇产生菌Torullopsis famata优化发酵条件的研究[J].生物学杂志,2002,18(2):15-17.DOI:10.3969/j.issn.2095-1736.2002.02.006.
[16]姚广印,张双凤,谢宗良.酵母细胞麦角固醇的提取条件[J].河北大学学报(自然科学版),2007,27(2):195-198.DOI:10.3969/j.issn.1000-1565.2007.02.022.
[17]LARSEN T,AXELSEN J,RAVN H W.Simplified and rapid method for extraction of ergosterol from natural samples and detection with quantitative and semi-quantitative methods using thin-layer chromatography[J].Journal of Chromatography A,2004,1026(1/2):301-304.DOI:10.1016/j.chroma.2003.10.128.
[18]曹永兵,姜远英,殷明,等.薄层色谱扫描法检测氟康唑对真菌麦角固醇生物合成的影响[J].第二军医大学学报,1999,20(5):312-315.DOI:10.3321/j.issn:0258-879X.1999.05.014.
[19]张萍,肖新月,黄玮,等.RP-HPLC-UV法测定5种发酵虫草制剂中麦角甾醇的含量[J].药物分析杂志,2011,31(2):258-260.DOI:10.16155/j.0254-1793.2011.02.013.
[20]赵英永,赵晔,林瑞超,等.RP-HPLC法测定猪苓中麦角甾醇的含量[J].药物分析杂志,2009,29(6):898-900.DOI:10.16155/j.0254-1793.2009.06.009.
[21]汪财生,汪乐意,高有领,等.响应面法优化根霉菌产麦角固醇的液体发酵工艺[J].食品科学,2012,33(5):229-233.
[22]PHILLIPS K M,RUGGIO D M,HORST R L,et al.Vitamin D and sterol composition of 10 types of mushrooms from retail suppliers in the United States[J].Journal of Agricultural and Food Chemistry,2011,59(14):7841-7853.DOI:10.1021/jf104246z.
[23]WANG Q,CHENG J X,WANG L X,et al.Valorization of spent shiitake substrate for recovery of antitumor fungal sterols by ultrasound-assisted extraction[J].Journal of Food Biochemistry,2018,42(5):e12602.DOI:10.1111/jfbc.12602.
[24]陈光.HPLC法测定脂溶性维生素注射液中维生素D2及维生素E的含量[J].中国医药科学,2017,7(15):46-50.DOI:10.3969/j.issn.2095-0616.2017.15.013.
[25]王珵,张济敏,林玲.RP-HPLC法同时测定维生素AD小丸中维生素A棕榈酸酯和维生素D2的含量[J].中国药品标准,2017,18(4):308-311.
[26]曹少谦,陈麒宇,程亮.脉冲强光处理对香菇中维生素D2含量的影响[J].食品工业科技,2018,39(19):80-83;301.DOI:10.13386/j.issn1002-0306.2018.19.014.
[27]李丽,蒋景龙.紫外光影响食用菌中VD2含量的研究进展[J].食品科学,2015,36(1):273-277.DOI:10.7506/spkx1002-6630-201501052.
[28]MATTILA P H,LAMPI A M,RONKAINEN R,et al.Sterol and vitamin D2 contents in some wild and cultivated mushrooms[J].Food Chemistry,2002,76(3):293-298.DOI:10.1016/S0308-8146(01)00275-8.
[29]URBAIN P,JAKOBSEN J.Dose-response effect of sunlight on vitamin D2 production in Agaricus bisporus mushrooms[J].Journal of Agricultural and Food Chemistry,2015,63(37):8156-8161.DOI:10.1021/acs.jafc.5b02945.
[30]余玲玲,李兴,陈燕丽,等.液质联用技术用于复杂混合物体系中小分子化合物的分析[J].中国科学:化学,2017,47(12):1379-1391.DOI:10.1360/N032017-00118.
[31]TAOFIQ O,FERNANDES A,BARROS L,et al.UV-irradiated mushrooms as a source of vitamin D2:a review[J].Trends in Food Science&Technology,2017,70:82-94.DOI:10.1016/j.tifs.2017.10.008.
[32]张养军,王京兰,李蕾,等.用反相液相色谱分离多肽时峰容量与色谱柱长度关系的研究[J].色谱,2004,22(1):24-26.DOI:10.3321/j.issn:1000-8713.2004.01.006.