高效液相色谱法同时测定乌骨鸡肌肉中的黑色素与肌苷酸

Simultaneous Determination of Melanin and IMP in Black-bone Chicken Muscles with High Performance Liquid Chromatography

研究建立了同时测定乌骨鸡肌肉中黑色素与肌苷酸含量的高效液相色谱分析方法。鸡肉样品经酶解后,分离肌苷酸待测溶液与纯化黑色素粗品;黑色素粗品再在碱性条件下经过氧化氢氧化为2,3,5-三羧酸吡咯(PTCA)。肌苷酸待测溶液溶解PTCA成供试样品,在高效液相色谱仪中通过C18柱分离,紫外检测器检测波长270 nm,甲醇-磷酸/三乙胺缓冲液作为流动相等度洗脱。PTCA定量后以乌贼黑色素为对照品换算黑色素含量,外标法定量。结果表明:PTCA回收率为68.22%~75.21%,RSD值为6.83%~6.92%(n=6);肌苷酸回收率为79.26%~88.26%,RSD值为1.92%~2.35%(n=6)。黑色素、肌苷酸的检测限分别为10μg/g、100μg/g。该方法灵敏、可靠,少量乌骨鸡样本即可实现同时、快速检测黑色素与肌苷酸含量。

A high performance liquid chromatography(HPLC)method was developed for quantitative analysis of melanin and inosinic acid(IMP)in black-bone chicken muscles. Melanin and IMP in tissue samples were isolated by enzymatic extraction. After centrifugation,IMP was dissolved in the supernatant,and melanin in the sublayer was purified by organic reagents. Then melanin was specifically measured as the marker pyrrole-2,3,5-tricarboxylic acid(PTCA)after alkaline H2O2 oxidation. The analyte of PTCA dissolved by IMP solution was separated on a C18 column by elution with methanol-phosphoric acid/triethylamine buffer as mobile phase and the detected wavelength was 270 nm.Sepia melanin was used as a reference for melanin,and the quantitative analysis was performed by the external standard method. The results showed that the recovery rate of PTCA was 68.22% to 75.21% and RSD was 6.83% to 6.92%(n=6),while the recovery rate of IMP was 79.26% to 88.26% and RSD was 1.92% to 2.35%(n=6). The limits of detection of melanin and IMP were 10 μg/g and 100 μg/g. This HPLC method was simple,reproducible and applicable. A few amount of black-bone chicken muscles could be used for simultaneously detection of melanin and IMP content rapidly.