1, : HPLC 11 (hydroxycitric Acid, HCA) [1], HCA ( ),,, [2], HCA [3, 4] ; HCA,, [5], [6], HCA, GB ( ), HCA,,, HCA, [7] : [8] 1,, [9] 5, [10],

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1 3 1 Vol. 3 No Journal of Food Safety and Quality Feb., 2012 HPLC 李兰英, 许丽, 丁敏, 张波, 张敏, 任淑贞, * 陈静文, 孙荣荣, 刘刚 (, ) 摘要 : 目的 (HPLC) (hydroxycitric acid, HCA), (rimonabant) (sibutramine) 方法, 99 % 0.1 % 1 %, 210 nm HCA ; 100 %, 223 nm 结果 HCA 0.001~1.0 mg/ml, %~ % 0.01~20 g/ml, 1 ng/ml 3 ng/ml 结论,,, 关键词 : (HPLC); ; ; ; HPLC analysis of the active component and two illegally added compounds in hydroxycitric acid functional food LI Lan-Ying, XU Li, DING Min, ZHANG Bo, ZHANG Min, REN Shu-Zhen, CHEN Jing-Wen, SUN Rong-Rong, LIU Gang * (Shanghai Institute of Measurement and Testing Technology, Shanghai, , China) ABSTRACT: Objective To established an HPLC method for the analysis of hydroxycitric acid (HCA) and the simultaneous determination of illegally added rimonabant and sibutramine in functional food. Methods After ultrasonic extraction of the sample, HCA was analyzed at 210 nm wave length using 99% phosphoric acid (0.1%) 1% methanol as the mobile phase; rimonabant and sibutramine was detected at 223 nm wave length using 100% methanol as the mobile phase. Results HCA showed a good liner range between 0.001~1.0 mg/ml with an average spiked recovery of 90.49% ~103.90%; Rimonabant and sibutramine detection reached a liner range between 0.01~20 g/ml, and the limit of detection (LOD) was 1 ng/ml and 3 ng/ml, respectively. Conclusion The analysis of HCA and detection of rimonabant and sibutramine were simply operated with high analysis speed and high accuracy which was proved to be practical when used for quality monitoring of functional foods. KEY WORDS: high performance liquid chromatography; hydroxycitric acid; functional food; rimonabant; sibutramine 基金项目 : (11R ); (2010QK294) * 通讯作者 :,,, liug@simt.com.cn

2 1, : HPLC 11 (hydroxycitric Acid, HCA) [1], HCA ( ),,, [2], HCA [3, 4] ; HCA,, [5], [6], HCA, GB ( ), HCA,,, HCA, [7] : [8] 1,, [9] 5, [10], [11] ;, [12, 13], FDA, [14],, (HPLC) HCA,,, 10 min HCA, 1 材料与方法 1.1 材料与试剂 (HCA %, ); 5 ( 1 5)( ); (, HCA 60 %); (, MERCK ); (, Sigma ) ( ); ( >98%, TIC); 4 ( ); (, MERCK ); Milli-Q (Milli-Q ) 1.2 仪器与设备 Agilent 1100 ( Agilent ) KQ-600B ( ) MS2 ( IKA ) Milli-Q ( Millipore ) 1.3 试验方法储备液的配制 HCA 10.0 mg,, 10 mg/ml, g 10 ml,,, 2 mg/ml, 标准混合工作溶液的配制, HCA mg/ml g/ml 样品预处理 mg 50 ml, 40 ml, 20 min,,,, 0.22 m, HPLC , 0.10 g, 250 ml, 200 ml, 20 min,,,, 0.22 m, HPLC ,,

3 12 3, 50 ml, 40 ml 50 %, 20 min,, 50 %,, 0.22 m, HPLC HPLC 色谱条件 : : Waters Atlantis T3- ( mm); : 20 ; : 10 L; : 99% 0.1% +1% ; : 0.6 ml/min; : 210 nm : : Waters Atlantis T3( mm); : 20 ; : 10 L; : ; : 0.6 ml/min; : 223 nm Fig. 1 1 HCA(A), (B) (C) Chemical structural formula of hydroxycitric acid (A), rimonabant (B) and sibutramine (C) 2 结果与分析 2.1 羟基柠檬酸分析色谱柱和流动相的选择 HCA, HPLC, 0.1 % HCA, ph 100 % Waters Atlantis T3( mm) 1 mg/ml HCA, 0.1%, 99% 0.1% 1% HCA, 7.3 min, HCA 标准曲线及线性关系, (A) HCA (mg/ml), HCA 0.001~1.0 mg/ml, 1 Y=95476X 106, r = 检出限测定 HCA (LOD) 0.1 g/ml- (S/N>3); HCA (LQD) 0.3 g/ml(s/n>10) 精密度实验, 1.0 mg/ml HCA 6, HCA A, RSD 0.09 %(n=6), 2 表 1 羟基柠檬酸标准品检测结果 Table 1 Analysis result of hydroxycitric acid standard solution (mg/ml) (A) 表 2 精密度实验峰面积及其相对标准偏差 (n=6) Table 2 Precision of the HPLC detection of HCA A 1 A 2 A 3 A 4 A 5 A 6 RSD %

4 1, : HPLC 加标回收率实验 HCA 1, HCA,, HPLC 3 ( 6 ) %~ %, 3 HCA HPLC 2 ( 1 ) 实际样品检测 5 HCA,, 4 表 3 羟基柠檬酸检测加标回收率试验结果 (n=6) Table 3 Spiked recovery experiment results of HCA analysis (mg/ml) RSD % 0.37 % % 0.51 % % 0.21 % % 2.51 % % 0.08 % % 0.52 % 2 HPLC A: 10 mg/ml ; B: 1 HPLC ; C: mg/ml HCA Fig. 2 HPLC chromatograph of HCA in different matrix samples A: 10 mg/ml HCA solution; B: HCA in functional food No. 1; C: functional food No.1 after mg/ml HCA was spiked 表 4 实际样品中羟基柠檬酸的含量检测 Table 4 Real sample analysis results (mg/ ) (mg/ ) N.D N.D (N.D. ) 2.2 利莫那班和西布曲明同时检测检测条件确定 nm, 200 nm, 223 nm,, 223 nm 样品处理方法的确定,,,,,, [15], min,, 20 min, 20 min 标准曲线及线性关系, (A) ( g/ml), 0.01~20 g/ml, 5 表 5 利莫那班和西布曲明的线性回归方程和相关系数 Table 5 The linear regression equation and correlation coefficient of rimonabant and sibutramine A=6116.4C r = A=3246.1C r =0.9999

5 检出限测定 (S/N) 3 (LOD), 1 ng/ml, 3 ng/ml; (S/N) 10 (LQD), 3.3 ng/ml, 10.0 ng/ml, 精密度实验, 10 g/ml, 10 L, 6, A, RSD 1.47 %, 1.25 %(n=6), 稳定性实验,, h,,,, RSD 2.62 %, 2.13 %, 24 h 加标回收实验 HCA 1 HCA 2,,,,, 8 HPLC ( HCA 1 ) , 9, mg/g 3 结论 HPLC HCA,,, 表 6 高效液相色谱检测利莫那班和西布曲明的精密度 (n=6) Table 6 Precision of the HPLC detection of rimonabant and sibutramine (n=6) A1 A2 A3 A4 A5 A6 RSD % % 表 7 利莫那班和西布曲明溶液稳定性实验结果 Table 7 Stability of rimonabant and sibutramine in solution (h) RSD ( g/ml) % ( g/ml) % 表 8 加标回收实验结果 Table 8 Results of spiked recovery experiment (mg/ ) ( g/ml) (%) RSD(%) HCA 1 HCA 2 HCA 1 HCA

6 1, : HPLC 15 3 HPLC A: ; B: HCA 1; C: HCA 1 5 g/ml 5 g/ml Fig. 3 HPLC chromatograph A: Mixed standard solution in water; B: HCA functional food No. 1; C: 5 g/ml sibutramin and 5 g/ml rimonabant spiked into HCA functional food No. 1. 表 9 实际样品测定结果 Table 9 Real sample analysis results (mg/g) (mg/g) HCA 1 N.D. N.D. HCA 2 N.D HCA 3 N.D. N.D. HCA 4 N.D. N.D. HCA 1 N.D. N.D. HCA 2 N.D. N.D. HCA 3 N.D. N.D. HCA 4 N.D. N.D. (N.D. ) HCA,,,, HCA,, 5 HCA,, 参考文献 [1] McCarty MF, Gustin JC. Pyruvate and hydroxycitrate/carnitine may synergize to promote reverse electron transport in hepatocyte mitochondria, effectively 'uncoupling' the oxidation of fatty acids [J]. Med Hypotheses, 1999, 52(5): [2], [J]., 2006, 42(2): [3] Preuss HG, Bagchi D, Bagchi M, et al. Effects of a natural extract of (-) -hydroxycitric acid (HCA-SX) and a combination of HCA-SX plus niacin-bound chromium and Gymnema sylvestre extract on weight loss [J]. Diabetes Obes Metab, 2004, 6(3): [4] Shara M, Ohia SE, Yasmin T, et al. Dose- and time-dependent effects of a novel (-)-hydroxycitric acid extract on body weight, hepatic and testicular lipid peroxidation, DNA fragmentation and histopathological data over a period of 90 days [J]. Mol Cell Biochem, 2003, 254(1 2): [5] Soni MG, Burdock GA, Preuss HG, et al. Safety assessment of (-)-hydroxycitric acid and Super CitriMax, a novel calcium/potassium salt [J]. Food Chem Toxicol, 2004, 42(9): [6] Rao GV, Karunakara AC, Babu RRS, et al. Hydroxycitric acid lactone and its salts: Preparation and appetite suppression studies [J]. Food Chem, 2010, 120(1): [7] Burch J, McKenna C, Palmer S, et al. Rimonabant for the treatment of overweight and obese people [J]. Health Technol Assess, 2009, 13(Suppl 3):

16 3 [8] Leite CE, Mocelin CA, Petersen GO, et al. Rimonabant: an antagonist drug of the endocannabinoid system for the treatment of obesity [J]. Pharmacol Rep, 2009, 61(2): 217 224.

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7 16 3 [8] Leite CE, Mocelin CA, Petersen GO, et al. Rimonabant: an antagonist drug of the endocannabinoid system for the treatment of obesity [J]. Pharmacol Rep, 2009, 61(2): [9] Grassin Delyle S, Abe E, Devillier P, et al. Determination of rimonabant in human plasma and hair by liquid chromatography- -mass spectrometry [J]. J Chromatogr B Analyt Technol Biomed Life Sci, 2008, 872(1 2): [10],,. [J]., 2008, 29(2): [11] de Mattos Viana B, Prais H A, Daker M V. Melancholic features related to rimonabant [J]. Gen Hosp Psychiatry, 2009, 31(6): [12] de Simone G, D'Addeo G. Sibutramine: balancing weight loss benefit and possible cardiovascular risk [J]. Nutr Metab Cardiovasc Dis, 2008, 18(5): [13],. [J]., 2006, 8(4): [14]. [J]., 2007, 28(10): [15],,,. [J]., 2010, 28(1): ( 责任编辑 : 孙媛媛 ) 作者简介李兰英,,, lily@simt.com.cn 刘刚,,, liug@simt.com.cn ˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇˆˇ 食品添加剂专题约稿 ,,,, 2011 OMP,,,, 2012,,,, : tougao@chinafoodj.com 食品安全质量检测学报编辑部

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.