心型脂肪酸结合蛋白纯品含量的柱前衍生超高效液相色谱方法研究

Determination of the Mass Fraction of Pure Heart-type Fatty Acid-binding Protein by Pre-column Derivatization UPLC

心型脂肪酸结合蛋白(H-FABP)被酸水解后,以氨基酸混合溶液国家标准物质为标准,γ-氨基丁酸为内标,经柱前衍生剂处理,再采用超高效液相色谱法可测定其中天冬氨酸、谷氨酸、组氨酸、精氨酸4种衍生物的量,进而推算出H-FABP纯品的含量。将此方法的测定结果与同位素稀释质谱法测定H-FABP含量的结果相比较,可验证所建方法的准确性。实验显示,在最优条件下,所建立的柱前衍生超高效液相色谱(UPLC)法中H-FABP含量测定结果为0.1113 mg/mL,相对标准偏差为2.03%,扩展不确定度为0.0027 mg/mL(k=2),检出限和定量限分别为1.16×10^(-3) mg/mL和3.86×10^(-3) mg/mL。此外,该方法成本较低且溯源链清晰,可作为一种补充验证方法用于纯品H-FABP标准物质的定值。

After the heart type fatty acid binding protein(H-FABP)is hydrolyzed by acid hydrolysis,the amount of four derivatives of aspartic acid,glutamic acid,histidine and arginine can be determined,and then the content of pure H-FABP can be calculated.During the determination process,the national standard substance of amino acid mixed solution was used as the standard,γ-aminobutyric acid was used as the internal standard,and treated with pre column derivatizer.Comparing the results of this method with those of isotope dilution mass spectrometry for the determination of H-FABP content can verify the accuracy of the established method.The experiment showed that under the optimal conditions,the H-FABP content in the established pre column derivatization ultra high performance liquid chromatography(UPLC)method was determined to be 0.1113 mg/mL.The relative standard deviation was 2.03%,the expanded uncertainty was 0.0027 mg/mL(k=2),and the detection limit and quantification limit were 1.16×10^(-3) mg/mL and 3.86×10^(-3) mg/mL,respectively.In addition,this method has a low cost and clear traceability chain,which may serve as a supplementary validation method for the calibration of pure H-FABP reference materials.