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1 6 2 Vol. 6 No Journal of Food Safety and Quality Feb., 2015 贾子才, 徐杰 *, 宋雨, 李兆杰, 薛长湖 (, ) 摘要 : 目的 -(high performance liquid chromatography coupled with evaporative light-scattering detection, HPLC-ELSD) 方法 TSKgel CN-80Ts, -- -,, 1.0 ml/min, 50, 2.4 L/min 结果, 18 min, 2.5~250 µg/ml, 0.5 µg/ml(s/n=3), R>0.99, 5~150 µg/ml, 0.05 µg/ml(s/n=3), R>0.99, ;, 0~4.44 mg/g 0.24~2.21 mg/g, 4.44 mg/g, 2.21 mg/g, 结论, 关键词 : ; ; ; ; Simultaneous quantitative analysis of cerebrosides and ceramides from sea cucumbers by high performance liquid chromatography coupled with evaporative light-scattering detection coupled with evaporative light-scattering detection JIA Zi-Cai, XU Jie *, SONG Yu, LI Zhao-Jie, XUE Chang-Hu (College of Food Science and Engineering, Ocean University of China, Qingdao , China) ABSTRACT: Objective To establish a high performance liquid chromatography coupled with evaporative light-scattering detection (HPLC-ELSD) method for analyzing the contents of cerebrosides and ceramides simultaneously in sea cucumbers. Methods The separation was performed on a TSK gel CN-80Ts column by using n-hexane-isopropanol-dichloromethane-methanol as the mobile phase at a flow rate of 1.0 ml/min. The temperature of drift tube was kept at 50 and the flow rate of nitrogen was 2.4 L/min. Seventeen sea cucumber species were selected as experimental subjects. Results Under optimal conditions, effective separation of cerebrosides and ceramides was completed within 18 min. The linear range of the established cerebrosides calibration curve was 2.5~250 µg/ml, and the limit of detection was 0.5 µg/ml with a regression 基金项目 : ( )( ) (2012BAD33B07) Fund: Supported by the National Natural Science Foundation of China ( ), the Special Fund for Marine Scientific Research in the Public Interest ( ) and the National Science and Technology Support Plan (2012BAD33B07) * 通讯作者 :,,, xujie9@ouc.edu.cn *Corresponding author: XU Jie, Associate Professor, College of Food Science and Engineering, Ocean University of China, No. 5, Yushan Road, Qingdao , China. xujie9@ouc.edu.cn

2 2, : 603 correlation coefficient of Meanwhile, the linear range of the established ceramides calibration curve was 5~150 µg/ml, and the limit of detection was 0.05 μg/ml with a regression correlation coefficient of There were obvious difference in cerebrosides and ceramides among these sea cucumber species. The cerebrosides content of Bohadschia marmorata was the highest, which was up to 4.44 mg/g, in contrast, that of Actinopyga mauritiana could not be detected. As for ceramides, the content of Parastichopus californicus was the highest, which was up to 2.21 mg/g, in contrast, Acaudina molpadioides had the lowest content. Conclusion The established method can provide essential data for development and utilization of sea cucumbers, which is sensitive, accurate and fast. KEY WORDS: high performance liquid chromatography; evaporative light-scattering detection; ceramides; cerebrosides; sea cucumber 1 引言 (Echinodermata) (Holothuroidea),,, [1] [2-4],, N-,,, [5,6],,,, C-1 β, [7] (glucosylceramide, GlcCer) (galactocerebroside, GalCer), GlcCer GalCer,,, [8,9] [10,11] [12,13] [14] [15] [16] S-180, ; PC12 [17], (UPTLC) [19, 20] (HPLC) [21, 22],, -2,3-, [21] ; [23] - (high performance liquid chromatography coupled with evaporative light-scattering detection, HPLC- ELSD),, HPLC-ELSD, UPTLC HPLC,, 2 材料和方法 2.1 实验原料 (Pearsonothuria graeffei) (Holothuria mexicana)(bohadschia marmorata) (Isostichopus badionotus) (Bohadschia argus) (Actinopyga mauritiana) (Cucumaria frondosa) (Thelenota ananas) (Acaudina molpadioides) (Holothurim scabras) (Parastichopus californicus) (Apostichopus japonicus, : ), 2.2 试剂和材料 ( ) (

3 604 6 )( Sigma ), 1; (, Merck ); ; Milli Q ; TSKgel CN-80Ts (4.6 mm 250 mm, 5 µm)( TOSOH ) 1 Fig. 1 The structures of GalCer and ceramide reference substances 2.3 仪器与设备 1100 ( Agilent ); 2000ES ELSD ( Alltech ); Elix5+Milli-QA ( Millipore ); FZ102 ( ); AL204 ( - ); T10 basic ( IKA ); SB5200D ( ); TDL-5-A ( ); DTU-1C ( TAITEC ) 2.4 实验方法色谱条件 A: : (99:1, v:v), B: : (60:40, v:v); : TSKgel CN-80Ts(4.6 mm 250 mm, 5 µm); : 40 ; : 1 ml/min; ( 1) :, 2.4 L/min, 50 ; : 20 µl Table 1 表 1 梯度洗脱程序表 Adjustable gradient elution program (min) A(%) B(%) 混合对照品溶液的配制,, 500 µg/ml,, µg/ml 样品溶液的配制 3, (100~200 ), 0.1 g( g) 6 ml : (2:1, v:v) 2 min, 15 min,, 3, 1 ml, 2 ml, 3, 100 µl 4 mol/l 1.0 ml, 37 1 h, 0.8 ml, 2 ml, 3,,, 10 ml 2.0 ml 0.22 µm, HPLC-ELSD 2.5 方法学考察线性范围及检出限 2.5~250 µg/ml 20 µl, 2.4.1, (lnx), (lny), 精密度 25 µg/ml, 2.4.1, 6, (RSD) 稳定性 25 µg/ml, 2.4.1, 12 h 2 h, (RSD) 回收率 g( g), 6 ( μg/ml) 2 ml, 4 ml, 2.4.3, ml

4 2, : , 3 RSD 样品中脑苷脂和神经酰胺含量的测定 µl, 统计分析方法 SPSS 19.0, Duncan 3 结果与讨论 3.1 色谱条件的优化, ZORBAX SB-C18 (4.6 mm 250 mm, 5 μm) TSKgel CN-80Ts (4.6 mm 250 mm, 5 μm) ZORBAX Rx-SIL (4.6 mm 250 mm, 5 μm) 3, ZORBAX SB-C18 98%-, GalCer,, ; ZORBAX Rx-SIL, -- -,,,, TSKgel CN-80Ts 3, - - -( ) ( ) -- - ( ),, ;,,,,,,, ELSD,, ELSD, ELSD L/min 3.2 标准曲线线性范围及检出限 2.5~250 µg/ml 20 µl, 2.4.1, ELSD,,, (lnx), (lny) HPLC-ELSD (25 µg/ml) Fig. 2 HPLC-ELSD chromatogram of mixed reference solution(25 µg/ml) 2,,,, 2, 0.99,,, 0.5 µg/ml 0.05 µg/ml, Table 2 表 2 混合对照品的回归方程及检出限 Regressing equation and detection limit of mixed reference solution (µg/ml) R (µg/ml) (S/N = 3) 2.5~250 lny = 1.848lnX ~150 lny = lnX

5 精密度 25 µg/ml, 2.5.2, RSD 3.33% 5.49%, 3.4 稳定性 25 µg/ml, RSD 1.92% 2.31% 12 h, 3.5 回收率 2.5.4, 93.83% 94.58%, RSD 4.93% 3.12% 3 Table 3 表 3 方法回收率实验结果 (n=6) Recovery rate for cerebrosides and ceramides in sea cucumber(n=6) (µg/ml) (µg/ml) RSD(%) (%) 样品测定 µl, 2.4.1, 3( ), 4 3 HPLC-ELSD Fig. 3 HPLC-ELSD chromatogram of cerebrosides and ceramides from sea cucumber Bohadschia marmorata, HPLC-ELSD,,, 4, 4.44 mg/g,,, 2.21 mg/g,, [23] Sugawara [24] 48,,,,,,,,,, ;,,, ;, (P<0.001), (P<0.05), (P<0.001), (P<0.001), ;,,,,,,

6 2, : 607 Table 4 表 4 不同种类海参中脑苷脂和神经酰胺的含量 The contents of cerebrosides and ceramides from different sea cucumber species (mg/g) (mg/g) (mg/g) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± n.d. 0.46± ± ± ± ± ± ± : n.d. ;,,,, 4 结论 HPLC-ELSD,,,,,, 参考文献 [1] Zhang K, Showalter M, Revollo J, et al. Sphingolipids are essential for differentiation but not growth in Leishmania [J]. EMBO J, 2003, 22(22): [2] Vesper H, Schmelz EM, Nikolova-Karakashian MN, et al. Sphingolipids in food and the emerging importance of sphingolipids to nutrition [J]. J Nutr, 1999, 129(7): [3] Wennekes T, Van Den Berg RJ, Boot RG, et al. Glycosphingolipids nature, function, and pharmacological modulation [J]. Angew Chem Int Ed Eng, 2009, 48(47): [4] Sugawara T, Miyazawa T. Separation and determination of glycolipids from edible plant sources by high-performance liquid chromatography and evaporative light-scattering detection [J]. Lipids, 1999, 34(11): [5] Hakomori SI. Glycosphingolipids in cellular interaction, differentiation, and oncogenesis [J]. Annu Rev Biochem, 1981, 50(1): [6] Spiegel S, Merrill A. Sphingolipid metabolism and cell growth

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g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min

g ml 10% ph 色谱条件 kinetex C μm 100 A 4. 6 mm 150 mm 25 5 μl A B 10 mmol /L ml /min 25 4 2013 8 监测技术 5 1 2 1 ( 1. 江苏省疾病预防控制中心, 江苏南京 210009; 2. 南京医科大学第二附属医院, 江苏南京 210029) 5 Phenomenex kinetex C 18 2. 6 μm 4. 6 mm 150 mm 10 mmol /L ph 2. 5 + 60 40 DAD 280 nm 5 20. 0 mg /L ~ 100 mg /L 0.