兔角膜基质中核黄素含量测定的HPLC方法建立

Establishment of HPLC assay method for the riboflavin in rabbits corneal stroma

目的:建立兔角膜基质中核黄素含量测定的高效液相色谱(high performance liquid chromatograph,HPLC)分析方法,并分析其专属性、灵敏度和准确性。方法:兔角膜组织匀浆经加热孵育和10%三氯乙酸去蛋白后离心,组织上清液经C18-SPE柱固相萃取后检测。高效液相色谱柱选择ODS-BP色谱柱(5μm,250mm×4.6mmID),以甲醇-纯水(体积比为2:3)为流动相,pH=7.0,流速:0.1mL/min;荧光检测器激发波长425nm,发射波长525nm。结果:核黄素在1.0×10-6～1.0×10-4mg/mL范围与峰面积呈现良好的线性关系(r2=0.999584),精密度结果显示:RSD=0.86%(n=10),样品溶液在24h内稳定性RSD=0.51%,测得的角膜样品核黄素加标平均回收率为99.3%,RSD为3.7%。结论:本实验中建立的样品前处理方法简单,HPLC方法专属性强、灵敏度高、准确性好,可作为紫外线A/核黄素角膜胶原交联法中角膜基质内核黄素含量测定的定量方法。

AIM：To develop a high performance liquid chromatography（HPLC） method for determination of riboflavin in rabbits corneal stroma,and investigate the method＇s specificity,sensitivity and accuracy to determinate whether it can be or not used to evaluate intrastromal concentrations of riboflavin for the study of corneal cross-linking（CXL）.METHODS：The riboflavin in rabbit＇s cornea homogenate was extracted on a C18-SPE cartridge after protein precipitated by heating incubation and adding 10% trichloroacetic acid,and analyzed on HPLC system with an ODS-BP column（5μm,250mm×4.6mm I.D.） and a mobile phase consisted of 40% methanol and 60% pure water.The flow rate was 0.1mL/min.The spectrophoto-fluorometer was set at wavelength of 425nm for excitation and 525nm for emission.RESULTS：Riboflavin had a good linearity（r2=0.999 584） existed within range of 1.0×10-6 to 1.0×10-4mg/mL.The precision of this method indicated the relative standard deviation（RSD） was 0.86%（n=10）,the sample solution stability RSD was 0.51% within 24 hours,and the corneal sample adding standard of the recovery were 99.3%（n=5）,RSD=3.7%.CONCLUSION：This method is specific,accurate and sensitive,and can be used for determination of riboflavin in rabbit＇s corneal stroma.