利用親水性作用層析串聯質譜技術定量食品及血清中的糖化終產物

Abstract

糖化終產物(Advanced glycation end products, AGEs)為具有高度異質性的一群化合物，會對人體造成發炎、氧化壓力等相關毒性。本研究為用液相層析串聯質譜儀(LC-MS/MS)分析糖化終產物中常見的生物標記N ε-(羧甲基)離胺酸(Nε-(1-Carboxymethyl)-L-lysine, CML)、N ε-(羧乙基)離胺酸(Nε-(1-Carboxyethyl)-L-lysine, CEL)及戊糖素(pentosidine)，對食品及血清樣品進行定量。對於前處理是使用Oasis MCX (Mixed Cation-Exchange)固相萃取(Solid Phase Extraction, SPE)管柱進行AGEs的萃取，以達到最大的分析物回收率。層析管柱的選擇上則是使用親水性作用層析管柱HILIC silica column分離高極性的CML、CEL及pentosidine，以乙腈和水作為移動相，並添加了5 mM的甲酸銨以改進峰型，分離過程中均無離子配對試劑(ion-pairing reagent)的使用。對於本研究開發之分析方法，線性範圍(1~1000 ng/mL)之決定係數R2> 0.995，同時具有良好的精密度(RSD < 11.00%)以及準確度(82.40 -113.67%)，證實能夠準確定量不同食品及血清樣品中的AGEs。定量結果顯示經過烘烤後的Labdiet 5010食品具有較高的AGEs含量(CML, 20.78 μg/g；CEL, 21.53 μg/g)，且其含量顯著性地高於未經加熱處理之食品，並且當小鼠攝入經過烘烤後的Labdiet 5010食品時，其體內血清呈現較高的AGEs含量，並且與攝入未經加熱處理食品之小鼠相比，其含量也有顯著性地增加(CML, 3.93 μM；CEL, 9.72 μM) (以上p值皆小於0.05)。Advanced glycation end products (AGEs) are a heterogeneous group of compounds that can cause inflammation, oxidative stress, and other toxic effects in the human body. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of common AGEs biomarkers Nε-(1-Carboxymethyl)-L-lysine (CML), Nε-(1-Carboxyethyl)-L-lysine (CEL), and pentosidine. They were quantified in food and serum. Oasis MCX solid phase extraction (SPE) cartridges were used for AGEs extraction to achieve maximum analyte recovery. A hydrophilic interaction chromatography (HILIC) silica column was used to separate the highly polar CML, CEL, and pentosidine. The mobile phase consisted of acetonitrile and water with 5 mM ammonium formate. No ion-pairing reagents were used during the separation. The developed method proved to be linear in the range of 1-1000 ng/mL with coefficients of determination, R2> 0.995 for all analytes. It also exhibited good precision (RSD < 11.00%) and accuracy (82.40 -113.67%), indicating its ability to accurately quantify AGEs in various food and serum. Quantitative results showed that the AGEs content in food was significantly increased after high-temperature processing (CML: 20.78 μg/g; CEL: 21.53 μg/g), followed by a similar increase in serum AGEs in mice (CML: 3.93 μM; CEL: 9.72 μM) (All p values < 0.05).