液态乳中胆固醇、7-脱氢胆固醇及25-羟基胆固醇的同步测定方法
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摘要
试验优化了液态乳胆固醇的皂化条件(皂化时间、温度和皂化试剂添加量),采用高效液相色谱法同时测定乳品中胆固醇、7-脱氢胆固醇和25-羟基胆固醇的含量。结果表明,1 m L液态乳最佳皂化条件:80℃下2.0mL 2 mol/L KOH皂化20 min,液态乳的胆固醇含量为10.824 mg/100g;采用Eclipse plus C18 (4.6 mm×250mm,5μm)色谱柱,以甲醇为流动相,流速0.8 mL/min,在205 nm和227 nm两个波长下分离3种胆固醇,分离效果较好。胆固醇、7-脱氢胆固醇和25-羟基胆固醇分别在0.0967～0.9959 mg/mL、0.0120～0.2011 mg/mL和0.1237～0.1998 mg/mL范围内呈现良好的线性关系(R2=0.9999,R2=0.9996,R2=0.9992);胆固醇、7-脱氢胆固醇和25-羟基胆固醇的精密度试验RSD分别为0.619%,3.562%,1.696%;稳定性试验RSD分别为0.545%,1.248%,2.798%;重现性试验的RSD为1.105%,1.446%,5.284%;回收率分别为99.5%,67.6%,47.4%。该方法操作简单,试剂用量小,耗时短,结果准确可靠,可用于液态乳中胆固醇的准确测定。此外,本试验应用该方法测定了13种液态乳,发现除了母乳样品中含有少量的7-脱氢胆固醇和25-羟基胆固醇外,其它奶制品中均未检测出。
The saponification condition of liquid milk cholesterol was optimized through saponification time, temperature and reagent dosage, cholesterol contents of dairy products and human milk were determined by HPLC. The results showed that the cholesterol of 1 mL liquid milk was 10.824 mg/100 g under the optimum saponification condition of 2.0 m L 2 mol/L KOH saponifying for 20 min at 80 ℃. An ideal chromatographic separation of cholesterol, 7-dehydrocholesterol and 25-hydroxycholesterol was achieved on Eclipse plus C18(4.6 mm×250 mm, 5 μm) column using methanol was mobile phase at a flow rate of 0.8 mL/min under the wavelength of 205 nm and 227 nm. The calibration curves of cholesterol, 7-dehydrocholesterol and 25-hydroxycholesterol established showed good linearity over the concentration range of0.0967-0.9959 mg/mL(R2=0.9999), 0.0120～0.2011 mg/mL(R2=0.9996) and 0.1237～0.1998 mg/mL(R2=0.9992), respectively. RSD of precision test were 0.619%, 3.562% and 1.696%, respectively. RSD of stability were 0.545%, 1.248%and 2.798%, respectively. RSD of reproducibility were 1.105%, 1.446% and 5.284%, respectively. Recovery rates were99.5%, 67.6% and 47.4%, respectively. It is shown that the method is simple, accurate and reliable with shorter time and less reagent compared with GB/T 22220-2008, and could be used for the synchronous determination of cholesterol,7-dehydrocholesterol and 25-hydroxycholesterol in liquid milk. In addition, cholesterol of 13 kinds of liquid milk were determined by this method, 7-dehydrocholesterol and 25-hydroxycholesterol were only detected in human milk but not found in other dairy products.
The saponification condition of liquid milk cholesterol was optimized through saponification time, temperature and reagent dosage, cholesterol contents of dairy products and human milk were determined by HPLC. The results showed that the cholesterol of 1 mL liquid milk was 10.824 mg/100 g under the optimum saponification condition of 2.0 m L 2 mol/L KOH saponifying for 20 min at 80 ℃. An ideal chromatographic separation of cholesterol, 7-dehydrocholesterol and 25-hydroxycholesterol was achieved on Eclipse plus C18(4.6 mm×250 mm, 5 μm) column using methanol was mobile phase at a flow rate of 0.8 mL/min under the wavelength of 205 nm and 227 nm. The calibration curves of cholesterol, 7-dehydrocholesterol and 25-hydroxycholesterol established showed good linearity over the concentration range of0.0967-0.9959 mg/mL(R2=0.9999), 0.0120～0.2011 mg/mL(R2=0.9996) and 0.1237～0.1998 mg/mL(R2=0.9992), respectively. RSD of precision test were 0.619%, 3.562% and 1.696%, respectively. RSD of stability were 0.545%, 1.248%and 2.798%, respectively. RSD of reproducibility were 1.105%, 1.446% and 5.284%, respectively. Recovery rates were99.5%, 67.6% and 47.4%, respectively. It is shown that the method is simple, accurate and reliable with shorter time and less reagent compared with GB/T 22220-2008, and could be used for the synchronous determination of cholesterol,7-dehydrocholesterol and 25-hydroxycholesterol in liquid milk. In addition, cholesterol of 13 kinds of liquid milk were determined by this method, 7-dehydrocholesterol and 25-hydroxycholesterol were only detected in human milk but not found in other dairy products.
引文
[1]辜英杰,吴锐,闫世平,等.高效液相色谱-蒸发光散射检测器测定食品中胆固醇含量[J].分析测试学报,2006,25(6):98-100.
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[12]肖琳琳.乳酸菌降胆固醇作用及机理研究[D].南京:南京农业大学,2003.
[13]GOLD E S,DIERCKS A H,PODOLSKY I,et al25-Hydroxycholesterol acts as an amplifier of inflammatory signaling[J].Proceedings of the National Academy of Sciences,2014,111(29):10666-10671.
[14]崔晓暄,王士雯,齐鹏.25-羟基胆固醇促进培养的家兔主动脉中膜细胞体外钙化[J].中国动脉硬化杂志,2000,8(3):199-201.
[15]蒲博,张驰翔,王周,等.乳酸菌降胆固醇作用及其机理的研究进展[J].中国酿造,2014,33(7):5-9.
[16]BAUER L C,SANTANA D A,MACEDO M S,et al.Method validation for simultaneous determination of cholesterol and cholesterol oxides in milk by RP-HPLC-DAD[J].Journal of the Brazilian Chemical Society,2014,25(1):161-168.
[17]国家质量监督检验检疫总局.食品中胆固醇的测定:第2部分高效液相色谱法:GB/T 22220-2008[S]北京:中国标准出版社,2008:1-3.
[18]SALA A魣,LLATAS G G,BARBER魣R,et al Determination of cholesterol in human milk:an alternative to chromatographic methods[J].Nutrición hospitalaria:Organo Oficial De La Sociedad Espa觡ola De Nutrición Parenteral Y Enteral,2015,32(4):1535-1540.
[19]张丽,汤佳佳,徐国兵,等.高效液相色谱法测定牛奶中的胆固醇[J].食品科学,2012,33(2):216-218.
[20]BAUER L C,SANTANA D A,MACEDO M S,et al.Method validation for simultaneous determination of cholesterol and cholesterol oxides in milk by RP-HPLC-DAD[J].Journal of the Brazilian Chemical Society,2014,25(1):161-168.
[21]KAMELSKA A M,PIETRZAK-FIECKO R,BRYLK.Variation of the cholesterol content in breast milk during 10 days collection at early stages of lactation[J].Acta Biochimica Polonica,2012,59(2):243.
[22]KAMELSKA A M,PIETRZAK-FIE C KO R,BRYLK.Determination of cholesterol concentration in human milk samples using attenuated total reflectance Fourier transform infrared spectroscopy[J].Journal of Applied Spectroscopy,2013,80(1):148-152.
[23]于志会.益生性降胆固醇植物乳杆菌的筛选,发酵特性及体内功效研究[D].长春:吉林大学,2013.
[24]关海滨,董至恒.降胆固醇乳酸菌的筛选[C].第七届内蒙古自治区自然科学学术年会.呼和浩特:科技创新与经济结构调整-第七届内蒙古自治区自然科学学术年会优秀论文集,2012:556-558.
[25]郭翔.降胆固醇益生乳酸菌筛选及其功能机理的研究[D].无锡:江南大学,2009.
[26]李昵.降胆固醇乳酸菌的筛选及其降解机理研究[D]厦门:集美大学,2012.
[27]李婷婷.降胆固醇益生乳酸菌的筛选及其在大鼠体内的应用研究[D].哈尔滨:东北农业大学,2013.
[28]SCOPESI F,ZUNIN P,MAZZELLA M,et al.7-Ketocholesterol in human and adapted milk formulas[J].Clinical Nutrition,2002,21(5):379-384.
[29]何悦铭,王杉,揭琴丰,等.手性色谱法测定L-乳酸锌[J].食品科学,2015,36(14):107-110.
[2]郭涛,杜蕾蕾,万辉,等.高效液相色谱法测胆固醇含量鉴别地沟油[J].食品科学,2009,30(22):286-289.
[3]应英,张晶,冯靓,等.气相色谱法快速检验食用油中胆固醇含量[J].中国卫生检验杂志,2013,23(6):1410-1412.
[4]刘长建,姜波,安晓雯,等.气相色谱法测定发酵液中胆固醇的含量[J].微生物学杂志,2009,29(6):97-99.
[5]张晶,汤均,谭莹,等.气相色谱-氢火焰离子化检测器测定奶茶饮料中的胆固醇[J].中国卫生检验杂志,2009,19(1):106-107,128.
[6]丁卓平,王明华,刘振华,等.食品中胆固醇含量测定方法的研究与比较[J].食品科学,2004,25(1):130-135.
[7]AHN J H,JEONG I S,KWAK B M,et al.Rapid determination of cholesterol in emulsified confectioneries by ultra‐performance liquid chromatography[J].European Journal of Lipid Science and Technology,2012,114(11):1304-1311.
[8]AYCIRIEX S,REGAZZETTI A,GAUDIN M,et al Development of a novel method for quantification of sterols and oxysterols by UPLC-ESI-HRMS:Application to a neuroinflammation rat model[J].Analytical and Bioanalytical Chemistry,2012,404(10):3049-3059.
[9]ZHOU Q,WASOWICZ E,HANDLER B,et al.An excess concentration of oxysterols in the plasma is cytotoxic to cultured endothelial cells[J].Atherosclerosis,2000,149(1):191-197.
[10]MORIN R J,HU B,PENG S K,et al.Cholesterol oxides and carcinogenesis[J].Journal of Clinical Laboratory Analysis,1991,5(3):219-225.
[11]BROWN A J,JESSUP W.Oxysterols and atherosclerosis[J].Atherosclerosis,1999,142(1):1-28.
[12]肖琳琳.乳酸菌降胆固醇作用及机理研究[D].南京:南京农业大学,2003.
[13]GOLD E S,DIERCKS A H,PODOLSKY I,et al25-Hydroxycholesterol acts as an amplifier of inflammatory signaling[J].Proceedings of the National Academy of Sciences,2014,111(29):10666-10671.
[14]崔晓暄,王士雯,齐鹏.25-羟基胆固醇促进培养的家兔主动脉中膜细胞体外钙化[J].中国动脉硬化杂志,2000,8(3):199-201.
[15]蒲博,张驰翔,王周,等.乳酸菌降胆固醇作用及其机理的研究进展[J].中国酿造,2014,33(7):5-9.
[16]BAUER L C,SANTANA D A,MACEDO M S,et al.Method validation for simultaneous determination of cholesterol and cholesterol oxides in milk by RP-HPLC-DAD[J].Journal of the Brazilian Chemical Society,2014,25(1):161-168.
[17]国家质量监督检验检疫总局.食品中胆固醇的测定:第2部分高效液相色谱法:GB/T 22220-2008[S]北京:中国标准出版社,2008:1-3.
[18]SALA A魣,LLATAS G G,BARBER魣R,et al Determination of cholesterol in human milk:an alternative to chromatographic methods[J].Nutrición hospitalaria:Organo Oficial De La Sociedad Espa觡ola De Nutrición Parenteral Y Enteral,2015,32(4):1535-1540.
[19]张丽,汤佳佳,徐国兵,等.高效液相色谱法测定牛奶中的胆固醇[J].食品科学,2012,33(2):216-218.
[20]BAUER L C,SANTANA D A,MACEDO M S,et al.Method validation for simultaneous determination of cholesterol and cholesterol oxides in milk by RP-HPLC-DAD[J].Journal of the Brazilian Chemical Society,2014,25(1):161-168.
[21]KAMELSKA A M,PIETRZAK-FIECKO R,BRYLK.Variation of the cholesterol content in breast milk during 10 days collection at early stages of lactation[J].Acta Biochimica Polonica,2012,59(2):243.
[22]KAMELSKA A M,PIETRZAK-FIE C KO R,BRYLK.Determination of cholesterol concentration in human milk samples using attenuated total reflectance Fourier transform infrared spectroscopy[J].Journal of Applied Spectroscopy,2013,80(1):148-152.
[23]于志会.益生性降胆固醇植物乳杆菌的筛选,发酵特性及体内功效研究[D].长春:吉林大学,2013.
[24]关海滨,董至恒.降胆固醇乳酸菌的筛选[C].第七届内蒙古自治区自然科学学术年会.呼和浩特:科技创新与经济结构调整-第七届内蒙古自治区自然科学学术年会优秀论文集,2012:556-558.
[25]郭翔.降胆固醇益生乳酸菌筛选及其功能机理的研究[D].无锡:江南大学,2009.
[26]李昵.降胆固醇乳酸菌的筛选及其降解机理研究[D]厦门:集美大学,2012.
[27]李婷婷.降胆固醇益生乳酸菌的筛选及其在大鼠体内的应用研究[D].哈尔滨:东北农业大学,2013.
[28]SCOPESI F,ZUNIN P,MAZZELLA M,et al.7-Ketocholesterol in human and adapted milk formulas[J].Clinical Nutrition,2002,21(5):379-384.
[29]何悦铭,王杉,揭琴丰,等.手性色谱法测定L-乳酸锌[J].食品科学,2015,36(14):107-110.