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0.1113mg/mL,相对标准偏差为2.03%,扩展不确定度为0.0027mg/mL(k=2),检出限和定量限分别为1.16×10-3mg/mL和3.86×10-3mg/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。
Abstract
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 0.1113mg/mL. The relative standard deviation was 2.03%, the expanded uncertainty was 0.0027mg/mL (k=2), and the detection limit and quantification limit were 1.16×10-3mg/mL and 3.86×10-3mg/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.
关键词
计量学 /
心型脂肪酸结合蛋白 /
定量分析 /
柱前衍生超高效液相色谱法 /
同位素稀释质谱法
Key words
metrology /
heart-type fatty acid-binding protein /
mass fraction /
pre-column derivatization ultra performance liquid chromatography /
high performance liquid chromatography-isotope dilution mass spectrometry
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
［1］Ono T. Studies of the FABP family: A retrospective［J］. Molecular and Cellular Biochemistry: An International Journal for Chemical Biology,2005,277(1/2):1-6.
［2］王羊洋,高大胜.心脏型脂肪酸结合蛋白在急性心肌梗死诊断中的应用进展［J］.安徽医药,2017,21(11):1968-1971.
Wang Y Y, Gao D S. The progress in applications of heart-type fatty acid binding protein in the diagnosis of acute myocardial infarction ［J］. Anhui Medical and Pharmaceutical Journal,2017,21(11):1968-1971.
［3］项欣,杨丽,郭宇璇,等.心型脂肪酸结合蛋白在急性心肌梗死中的诊断准确度的Meta分析［J］.新疆医学,2021,51(4):409-414.
Xiang X, Yang L, Guo Y X, et al. Diagnosis Accuracy of Heart- type Fatty Acid- Binding Protein in Acute Myocardial Infarction: A Systematic Review and Meta- Analysis［J］. Xinjiang Medical Journal,2021,51(04):409-414.
［4］Apple F S, Sandoval Y, Jaffe A S, et al. Cardiac Troponin Assays: Guide to Understanding Analytical Characteristics and Their Impact on Clinical Care［J］. Clinical Chemistry: Journal of the American Association for Clinical Chemists,2017,63(1):73-81.
［5］Mcmahon C G, Lamont J V, Curtin E, et al. Diagnostic accuracy of heart-type fatty acid-binding protein for the early diagnosis of acute myocardial infarction［J］. The American journal of emergency medicine,2012,30(2):267-274.
［6］赵艳丽, 李为民. 心脏型脂肪酸结合蛋白与心血管疾病关系的研究进展［J］. 心血管病学进展, 2018,39(4):636-639.
Zhao Y L, Li W M. Research Progress of Heart-type Fatty Acid-binding Protein in Cardiovascular Diseases ［J］. Adv Cardiovasc Dis,2018,39(4):636-639.
［7］Sandhu A T, Heidenreich P A, Bhattacharya J, et al. Cardiovascular Testing and Clinical Outcomes in Emergency Department Patients With Chest Pain［J］. JAMA internal medicine,2017,177(8):1175-1182.
［8］宋德伟,董晓杰,徐蓓,等.C-反应蛋白纯品的同位素稀释质谱法测定与国际比对［J］.质谱学报,2014,35(5):462-466.
Song D W, Dong X J, Xu B, et al. Measurement and International Comparison for C-Reactive Protein by Isotope Dilution Mass Spectrometry［J］. Journal of Chinese Mass Spectrometry Society,2014,35(5):462-466.
［9］ISO 17511:2020, In vitro diagnostic medical devices—Measurement of quantities in biological samples—Metrological traceability of values assigned to calibrators and control materials ［S］.
［10］李佳乐,武利庆,金有训,等.人血清白蛋白纯品含量的同位素稀释质谱方法研究［J］.计量学报,2016,37(3):328-332.
Li J L, Wu L Q, Jin Y X, et al. Determination of the Mass Fraction of Pure Human Serum Albumin by HPLC-IDMS［J］. Acta Metrologic Sinica,2016,37(3): 328-332.
［11］李佳乐,武利庆,刘文丽,等.人转铁蛋白含量同位素稀释质谱测定方法研究［J］.中国测试,2015,41(5):58-62.
Li J L, Wu L Q, Liu W L, et al. Determination on mass fraction of pure human transferrin through high performance liquid chromatography-isotope dilution mass spectrometry ［J］. China Measurement & Test, 2015, 41(5): 58-62.
［12］丁松,黄和,胡燚.氨基酸分析研究进展［J］.生物加工过程,2018,16(3):12-21.
Ding S, Huang H, Hu Y. Research progress on amino acids analysis ［J］. Chinese Journal of Bioprocess Engineering, 2018, 16(3):12-21.
［13］何继杰,张晓凤,钱莉莉,等.痕量氨基酸分析方法研究进展［J］.分析科学学报,2019,35(4):507-513.
He J J, Zhang X F, Qian L L, et al. Research progress in trace amino acid analysis methods ［J］. Journal of Analytical Science,2019,35(4):507-513.
［14］王萍亚,周勇,黄鹂,等.柱前衍生化高效液相色谱-荧光检测法测定水产品中氨苄青霉素和阿莫西林残留［J］.计量学报,2012,33(4):377-380.
Wang P Y, Zhou Y, Huang P, et al. Determination of Ampicillin and Amoxicillin Residues in Aquatic Product Using Reversed-phase High Performance Liquid Chromatography-fluorescence Detection with Pre-column Derivatization［J］.Acta Metrologic Sinica,2012,33(4): 377-380.
［15］江苏省计量科学研究院(江苏省能源计量数据中心),南京诺唯赞生物科技股份有限公司. 基于柱前衍生-超高效液相色谱的蛋白标准物质测定方法:CN202211050672.4［P］. 2022-09-27.
［16］JJG 1036—2022 电子天平检定规程［S］. 2022.
［17］JJG 196—2006 常用玻璃量器检定规程［S］. 2006.
［18］李兰,贾铮,周剑,等.高效液相色谱法测定鱼粉中色氨酸及不确定度评定［J］.食品安全质量检测学报,2020,11(9):2714-2721.
Li L, Jia Z, Zhou J, et al. Uncertainty evaluation of tryptophan in fishmeal by high performance liquid chromatography method ［J］. Journal of Food Safety & Quality, 2020,11(9):2714-2721.
基金
江苏省市场监督管理局科技计划(KJ185602)
PDF(15880 KB)
