微量果聚糖的两种高效凝胶色谱-柱后荧光衍生检测方法

Two high-performance gel permeation chromatographic methods with postcolumn fluorescence derivatization for traces of fructosan

目的:建立高专属性和高灵敏度的果聚糖测定方法。方法:以麦冬多糖(PRO)为模型多糖,利用高效凝胶过滤色谱(HPGPC)-柱后荧光衍生的方法来测定该多糖。通过对柱前和柱后因素的全面考察,提高方法的灵敏度。结果:PRO柱后先经水解再荧光衍生(方法1)或进样前用过碘酸氧化PRO(方法2)均可使方法灵敏度较直接柱后衍生提高约40倍。方法1的最佳测定条件:水解反应温度和pH值分别为150℃和1.3;衍生反应温度和pH值分别为90℃和12.2。方法2的最佳测定条件:过碘酸氧化时间≥24h;衍生反应温度和pH值分别为80℃和12.4。在2.5～160μg/mL浓度范围内两种方法的线性关系均较好(r=0.9995～0.9996,n=7),检测限均为25ng,低、中、高3种浓度的日内RSD分别≤3.2%和≤4.0%,日间RSD分别≤4.3%和≤4.6%。结论:本文所述的两种方法均可用于微量果聚糖的测定。

AIM： To establish a highly selective and sensitive method for determining fructosan. METHODS： Polysaccharide of Radix Ophiopogonis （PRO） was used as the model fructosan and determined by high-performance gel permeation chromatography （HPGPC） with postcolumn fluorescence derivatization. Improvement in sensitivity was to be done in combination with precolumn and postcolumn factors. RESULTS： The analytical sensitivity was estimated to increase 40 times employing postcolumn hydrolysis then fluorescence derivatization （method 1） or precolumn periodate oxidation then postcolumn fluorescence derivatization （method 2） than direct postcolumn fluorescence derivation. The optimum temperature and pH of hydrolysis for method 1 were 150 ℃ and 1. 3,respectively. The optimum temperature and pH of fluorescence derivatization for method 1 were 90 ℃ and 12. 2,respectively. For method 2,the periodate oxidation time of PRO should not be less than 24 h,and the optimum temperature and pH of fluorescence derivatization were 80 ℃ and 12. 4,respectively. The calibration curves for the two methods were linear over the range of 2. 5 ～ 160 μg/mL（r = 0. 999 5 ～ 0. 999 6,n = 7）,and both of the detection limits were 25 ng. The intra-day RSD of low,medium and high concentrations of PRO for the two methods were ≤3. 2% and ≤4. 0% ,respectively; the inter-day RSD were ≤4. 3% and ≤4. 6% ,respectively. CONCLUSION： The two methods described in the present paper can be used to detect traces of fructosan.