高效液相色谱-荧光检测法分析血浆中ALA的含量

High performance liquid chromatography-fluorescence detection method for determination of ALA in plasma

目的建立血浆中δ-氨基-γ-酮戊酸(ALA)含量的高效液相色谱-荧光检测方法。方法将血浆样品与乙酰丙酮工作液(乙酰丙酮-乙醇-水,15∶10∶75,V/V)和10%甲醛溶液在沸水浴中反应10 min后,置于冰水浴中充分冷却。经微孔滤膜过滤后以C18柱为分析柱进行高效液相色谱测定。色谱分析以甲醇-水-乙酸(1 000∶1 000∶2,V/V)为流动相,流速为0.6 ml/min;荧光检测器的激发波长为370 nm,发射波长为460 nm,柱温为40℃。结果 ALA浓度为5μg/L^100μg/L时,与色谱峰面积之间呈良好的线性关系。本方法的最低检出限为0.5μg/L,定量限为2.0μg/L,多次重复测定结果的RSD为0.35%(n=6),不同浓度的平均加标回收率为86.56%~100.09%。结论所建立的血浆中ALA含量的高效液相色谱-荧光检测法,简便、准确,可用于血浆中ALA含量的测定,为生物样品中ALA低浓度水平的检测提供了新的途径。

Objective To established a method for determination of ALA in the human plasma by high performance liquid chromatography（ HPLC）- fluorescence detection. Methods The sample was reacted with acetylacetone working liquid（ acetylacetone- ethanol- water,15∶10∶75,V / V） and 10% formaldehyde in the boiling water bath for 10 min,and cooling down with ice water bath. Then filtered through filter membrane for determination by high performance liquid chromatography with C18 column as the analytical column. The mobile phase was analyzed by using methanol water- acetic- acid（ 1 000 ∶1 000 ∶2,V / V） and flow rate was 0. 6 ml / min with the column temperature of 40 ℃. The fluorescence detector＇s excitation wavelength and emission wavelength were respectively 370 nm and 460 nm. Results When the concentration of ALA was 5 μg / L-100 μg / L,the linear relationship was good between the concentration and the chromatographic peak area; the detection limit was 0. 5 μg / L,the limit of quantization was 2. 0 μg / L; the results of RSD for multiple repeated measurements were 0. 35%（ n = 6）,the average recovery of different addition was 86. 56%- 100. 09%. Conclusion The established HPLC- fluorescence detection method for the determination of ALA in plasma is accurate,simple,can be used for the analysis of the human plasma ALA and provide a new method for detecting the low concentration of ALA in the biological specimen.