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).

(1) Song, Q.; Liu, J.; Dong, L.; Wang, X.; Zhang, X. Novel advances in inhibiting advanced glycation end product formation using natural compounds. Biomed Pharmacother 2021, 140, 111750. DOI: 10.1016/j.biopha.2021.111750
(2) Evaluation in vitro of AGE-crosslinks breaking ability of rosmarinic acid. Glycative Stress Research 2015, 2 (4), 204-207. DOI: 10.24659/gsr.2.4_204
(3) Hodge, J. E. Dehydrated Foods, Chemistry of Browning Reactions in Model Systems. Journal of Agricultural and Food Chemistry 1953, 1 (15), 928-943. DOI: 10.1021/jf60015a004
(4) Peng, H.; Gao, Y.; Zeng, C.; Hua, R.; Guo, Y.; Wang, Y.; Wang, Z. Effects of Maillard reaction and its product AGEs on aging and age-related diseases. Food Science and Human Wellness 2024, 13 (3), 1118-1134. DOI: https://doi.org/10.26599/FSHW.2022.9250094
(5) Jia, W.; Guo, A.; Zhang, R.; Shi, L. Mechanism of natural antioxidants regulating advanced glycosylation end products of Maillard reaction. Food Chemistry 2023, 404, 134541. DOI: https://doi.org/10.1016/j.foodchem.2022.134541
(6) Wei, Q.; Liu, T.; Sun, D.-W. Advanced glycation end-products (AGEs) in foods and their detecting techniques and methods: A review. Trends in Food Science & Technology 2018, 82, 32-45. DOI: 10.1016/j.tifs.2018.09.020
(7) Almenglo, C.; Rodriguez-Ruiz, E.; Alvarez, E.; Lopez-Lago, A.; Gonzalez-Juanatey, J. R.; Garcia-Allut, J. L. Minimal invasive fluorescence methods to quantify advanced glycation end products (AGEs) in skin and plasma of humans. Methods 2022, 203, 103-107. DOI: 10.1016/j.ymeth.2020.12.003
(8) Chen, C.-y.; Zhang, J.-Q.; Li, L.; Guo, M.-m.; He, Y.-f.; Dong, Y.-m.; Meng, H.; Yi, F. Advanced Glycation End Products in the Skin: Molecular Mechanisms, Methods of Measurement, and Inhibitory Pathways. Frontiers in Medicine 2022, 9, 1-17. DOI: 10.3389/fmed.2022.837222
(9) Ahmed, M. U.; Thorpe, S. R.; Baynes, J. W. Identification of N epsilon-carboxymethyllysine as a degradation product of fructoselysine in glycated protein. Journal of Biological Chemistry 1986, 261 (11), 4889-4894. DOI: 10.1016/s0021-9258(19)89188-3
(10) Akillioglu, H. G.; Lund, M. N. Quantification of advanced glycation end products and amino acid cross-links in foods by high-resolution mass spectrometry: Applicability of acid hydrolysis. Food Chem 2022, 366, 130601. DOI: 10.1016/j.foodchem.2021.130601
(11) Qin, R.; Wu, R.; Shi, H.; Jia, C.; Rong, J.; Liu, R. Formation of AGEs in fish cakes during air frying and other traditional heating methods. Food Chem 2022, 391, 133213. DOI: 10.1016/j.foodchem.2022.133213
(12) Jiao, Y.; He, J.; Li, F.; Tao, G.; Zhang, S.; Zhang, S.; Qin, F.; Zeng, M.; Chen, J. N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine in tea and the factors affecting their formation. Food Chem 2017, 232, 683-688. DOI: 10.1016/j.foodchem.2017.04.059
(13) Li, L.; Zhuang, Y.; Zou, X.; Chen, M.; Cui, B.; Jiao, Y.; Cheng, Y. Advanced Glycation End Products: A Comprehensive Review of Their Detection and Occurrence in Food. Foods 2023, 12 (11). DOI: 10.3390/foods12112103
(14) Zhu, Z.; Huang, M.; Cheng, Y.; Khan, I. A.; Huang, J. A comprehensive review of Nε-carboxymethyllysine and Nε-carboxyethyllysine in thermal processed meat products. Trends in Food Science & Technology 2020, 98, 30-40. DOI: 10.1016/j.tifs.2020.01.021
(15) Troise, A. D.; Fiore, A.; Wiltafsky, M.; Fogliano, V. Quantification of Nepsilon-(2-Furoylmethyl)-L-lysine (furosine), Nepsilon-(Carboxymethyl)-L-lysine (CML), Nepsilon-(Carboxyethyl)-L-lysine (CEL) and total lysine through stable isotope dilution assay and tandem mass spectrometry. Food Chem 2015, 188, 357-364. DOI: 10.1016/j.foodchem.2015.04.137
(16) Nomi, Y.; Annaka, H.; Sato, S.; Ueta, E.; Ohkura, T.; Yamamoto, K.; Homma, S.; Suzuki, E.; Otsuka, Y. Simultaneous Quantitation of Advanced Glycation End Products in Soy Sauce and Beer by Liquid Chromatography-Tandem Mass Spectrometry without Ion-Pair Reagents and Derivatization. J Agric Food Chem 2016, 64 (44), 8397-8405. DOI: 10.1021/acs.jafc.6b02500
(17) Requena, J. R.; Price, D. L.; Thorpe, S. R.; Baynes, J. W. Measurement of Pentosidine in Biological Samples. Methods in Molecular Medicine 2000, 38, 209-217. DOI: 10.1385/1-59259-070-5:209
(18) Miyata, T.; Ishiguro, N.; Yasuda, Y.; Ito, T.; Nangaku, M.; Iwata, H.; Kurokawa, K. Increased Pentosidine, an Advanced Glycation End Product, in Plasma and Synovial Fluid from Patients with Rheumatoid Arthritis and Its Relation with Inflammatory Markers. Biochemical and Biophysical Research Communications 1998, 244 (1), 45-49. DOI: https://doi.org/10.1006/bbrc.1998.8203
(19) Prasad, C.; Davis, K. E.; Imrhan, V.; Juma, S.; Vijayagopal, P. Advanced Glycation End Products and Risks for Chronic Diseases: Intervening Through Lifestyle Modification. Am J Lifestyle Med 2019, 13 (4), 384-404. DOI: 10.1177/1559827617708991
(20) Tamanna, N.; Mahmood, N. Food Processing and Maillard Reaction Products: Effect on Human Health and Nutrition. Int J Food Sci 2015, 2015, 526762. DOI: 10.1155/2015/526762
(21) Demirer, B.; Yardimci, H.; Erem Basmaz, S. Inflammation level in type 2 diabetes is associated with dietary advanced glycation end products, Mediterranean diet adherence and oxidative balance score: A pathway analysis. J Diabetes Complications 2023, 37 (1), 108354. DOI: 10.1016/j.jdiacomp.2022.108354
(22) Kent, R.; Uribarri, J. Processing Contaminants: Advanced Glycation End Products (AGEs). Encyclopedia of Food Safety, 2014, 2, 371-375. DOI: 10.1016/B978-0-12-378612-8.00211-0
(23) Uribarri, J.; Cai, W.; Sandu, O.; Peppa, M.; Goldberg, T.; Vlassara, H. Diet-derived advanced glycation end products are major contributors to the body's AGE pool and induce inflammation in healthy subjects. Ann N Y Acad Sci 2005, 1043, 461-466. DOI: 10.1196/annals.1333.052
(24) Shen, C. Y.; Lu, C. H.; Wu, C. H.; Li, K. J.; Kuo, Y. M.; Hsieh, S. C.; Yu, C. L. The Development of Maillard Reaction, and Advanced Glycation End Product (AGE)-Receptor for AGE (RAGE) Signaling Inhibitors as Novel Therapeutic Strategies for Patients with AGE-Related Diseases. Molecules 2020, 25 (23). DOI: 10.3390/molecules25235591
(25) Takeuchi, M. Toxic AGEs (TAGE) theory: a new concept for preventing the development of diseases related to lifestyle. Diabetol Metab Syndr 2020, 12 (1), 105. DOI: 10.1186/s13098-020-00614-3
(26) Assar, S. H.; Moloney, C.; Lima, M.; Magee, R.; Ames, J. M. Determination of Nepsilon-(carboxymethyl)lysine in food systems by ultra performance liquid chromatography-mass spectrometry. Amino Acids 2009, 36 (2), 317-326. DOI: 10.1007/s00726-008-0071-4
(27) Demirer, B.; Samur, G. Intake of dietary advanced glycation end products may be associated with depression and sleep quality in young adults. J Affect Disord 2024, 352, 26-31. DOI: 10.1016/j.jad.2024.02.040
(28) Garay-Sevilla, M. E.; Rojas, A.; Portero-Otin, M.; Uribarri, J. Dietary AGEs as Exogenous Boosters of Inflammation. Nutrients 2021, 13 (8). DOI: 10.3390/nu13082802
(29) Sharma, C.; Kaur, A.; Thind, S. S.; Singh, B.; Raina, S. Advanced glycation End-products (AGEs): an emerging concern for processed food industries. J Food Sci Technol 2015, 52 (12), 7561-7576. DOI: 10.1007/s13197-015-1851-y
(30) Lee, E. J.; Park, J. H. Receptor for Advanced Glycation Endproducts (RAGE), Its Ligands, and Soluble RAGE: Potential Biomarkers for Diagnosis and Therapeutic Targets for Human Renal Diseases. Genomics Inform 2013, 11 (4), 224-229. DOI: 10.5808/GI.2013.11.4.224
(31) Bierhaus, A.; Humpert, P. M.; Morcos, M.; Wendt, T.; Chavakis, T.; Arnold, B.; Stern, D. M.; Nawroth, P. P. Understanding RAGE, the receptor for advanced glycation end products. J Mol Med (Berl) 2005, 83 (11), 876-886. DOI: 10.1007/s00109-005-0688-7
(32) Dubey, N. K.; Wei, H. J.; Yu, S. H.; Williams, D. F.; Wang, J. R.; Deng, Y. H.; Tsai, F. C.; Wang, P. D.; Deng, W. P. Adipose-derived Stem Cells Attenuates Diabetic Osteoarthritis via Inhibition of Glycation-mediated Inflammatory Cascade. Aging Dis 2019, 10 (3), 483-496. DOI: 10.14336/AD.2018.0616
(33) Luevano-Contreras, C.; Chapman-Novakofski, K. Dietary advanced glycation end products and aging. Nutrients 2010, 2 (12), 1247-1265. DOI: 10.3390/nu2121247
(34) Nowotny, K.; Schroter, D.; Schreiner, M.; Grune, T. Dietary advanced glycation end products and their relevance for human health. Ageing Res Rev 2018, 47, 55-66. DOI: 10.1016/j.arr.2018.06.005
(35) Prasad, C.; Imrhan, V.; Marotta, F.; Juma, S.; Vijayagopal, P. Lifestyle and Advanced Glycation End Products (AGEs) Burden: Its Relevance to Healthy Aging. Aging Dis 2014, 5 (3), 212-217. DOI: 10.14336/AD.2014.0500212
(36) Rahbar, S. Novel inhibitors of glycation and AGE formation. Cell Biochemistry and Biophysics 2007, 48 (2), 147-157. DOI: 10.1007/s12013-007-0021-x
(37) Perrone, A.; Giovino, A.; Benny, J.; Martinelli, F. Advanced Glycation End Products (AGEs): Biochemistry, Signaling, Analytical Methods, and Epigenetic Effects. Oxidative Medicine and Cellular Longevity 2020, 2020 (1), 3818196. DOI: https://doi.org/10.1155/2020/3818196
(38) Matsui, T.; Joo, H. D.; Lee, J. M.; Ju, S. M.; Tao, W. H.; Higashimoto, Y.; Fukami, K.; Yamagishi, S. Development of a monoclonal antibody-based ELISA system for glyceraldehyde-derived advanced glycation end products. Immunol Lett 2015, 167 (2), 141-146. DOI: 10.1016/j.imlet.2015.08.008
(39) Fang, H.; Wang, L.; Zhang, S.; Liu, H.; Li, J. Advanced glycation end products (AGEs) formation in high-protein foods processing model system. Journal of Chinese Institute of Food Science and Technology 2014, 14 (2), 28-34.
(40) Milkovska-Stamenova, S.; Schmidt, R.; Frolov, A.; Birkemeyer, C. GC-MS Method for the Quantitation of Carbohydrate Intermediates in Glycation Systems. Journal of Agricultural and Food Chemistry 2015, 63 (25), 5911-5919. DOI: 10.1021/jf505757m
(41) Zhao, D.; Sheng, B.; Wu, Y.; Li, H.; Xu, D.; Nian, Y.; Mao, S.; Li, C.; Xu, X.; Zhou, G. Comparison of Free and Bound Advanced Glycation End Products in Food: A Review on the Possible Influence on Human Health. J Agric Food Chem 2019, 67 (51), 14007-14018. DOI: 10.1021/acs.jafc.9b05891
(42) Troise, A. D.; Wiltafsky, M.; Fogliano, V.; Vitaglione, P. The quantification of free Amadori compounds and amino acids allows to model the bound Maillard reaction products formation in soybean products. Food Chem 2018, 247, 29-38. DOI: 10.1016/j.foodchem.2017.12.019
(43) Cheng, W.; Wang, X.; Zhang, Z.; Ma, L.; Liu, G.; Wang, Q.; Chen, F.; Cheng, K. W. Development of an Isotope Dilution UHPLC-QqQ-MS/MS-Based Method for Simultaneous Determination of Typical Advanced Glycation End Products and Acrylamide in Baked and Fried Foods. J Agric Food Chem 2021, 69 (8), 2611-2618. DOI: 10.1021/acs.jafc.0c07575
(44) Badawy, M. E. I.; El-Nouby, M. A. M.; Kimani, P. K.; Lim, L. W.; Rabea, E. I. A review of the modern principles and applications of solid-phase extraction techniques in chromatographic analysis. Anal Sci 2022, 38 (12), 1457-1487. DOI: 10.1007/s44211-022-00190-8
(45) Han, W.; Qiu, P.; Zhang, L. Determination of Nε-(1-Carboxymethyl)-L-Lysine in brewing soy sauce by ultrahigh performance liquid chromatography coupled with tandem mass spectrometry. Italian Journal of Food Science 2023, 35 (4), 21-30. DOI: 10.15586/ijfs.v35i4.2393
(46) Li, C.; Zhang, L.; Gao, W.; Lai, C.; Dong, H. Robust Detection of Advanced Glycation Endproducts in Milk Powder Using Ultrahigh Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC-MS/MS). Food Analytical Methods 2021, 14 (7), 1472-1481. DOI: 10.1007/s12161-021-01986-6
(47) Alpert, A. J. Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds. Journal of Chromatography A 1990, 499, 177-196. DOI: https://doi.org/10.1016/S0021-9673(00)96972-3
(48) Kahsay, G.; Song, H.; Van Schepdael, A.; Cabooter, D.; Adams, E. Hydrophilic interaction chromatography (HILIC) in the analysis of antibiotics. Journal of Pharmaceutical and Biomedical Analysis 2014, 87, 142-154. DOI: https://doi.org/10.1016/j.jpba.2013.04.015
(49) Buszewski, B.; Noga, S. Hydrophilic interaction liquid chromatography (HILIC)--a powerful separation technique. Anal Bioanal Chem 2012, 402 (1), 231-247. DOI: 10.1007/s00216-011-5308-5
(50) Wang, J.; Guo, Z.; Shen, A.; Yu, L.; Xiao, Y.; Xue, X.; Zhang, X.; Liang, X. Hydrophilic-subtraction model for the characterization and comparison of hydrophilic interaction liquid chromatography columns. Journal of Chromatography A 2015, 1398, 29-46. DOI: https://doi.org/10.1016/j.chroma.2015.03.065
(51) Griffiths, J. A Brief History of Mass Spectrometry. Analytical Chemistry 2008, 80 (15), 5678-5683. DOI: 10.1021/ac8013065
(52) Wang, D.; Wang, J.; Liu, X.; Du, K.; Liu, H.; Yang, X.; Liu, T.; Liu, Q.; Wang, M.; Guo, J. Quantifying carboxymethyl lysine and carboxyethyl lysine in human plasma: clinical insights into aging research using liquid chromatography-tandem mass spectrometry. BMC Biotechnol 2024, 24 (1), 1-11. DOI: 10.1186/s12896-024-00838-5
(53) Zhang, G.; Huang, G.; Xiao, L.; Mitchell, A. E. Determination of advanced glycation endproducts by LC-MS/MS in raw and roasted almonds (Prunus dulcis). J Agric Food Chem 2011, 59 (22), 12037-12046. DOI: 10.1021/jf202515k
(54) Hegele, J.; Buetler, T.; Delatour, T. Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products. Anal Chim Acta 2008, 617 (1-2), 85-96. DOI: 10.1016/j.aca.2007.12.027
(55) Zhang, W.; Zhang, B.; Ye, Y.; Zhu, H. Methylglyoxal-hydroimidazolones (MG-Hs) instead of Nvarepsilon-(carboxymethyl)-l-lysine (CML) is the major advanced glycation end-product during drying process in black tea. Food Chem 2020, 333, 127499. DOI: 10.1016/j.foodchem.2020.127499
(56) Poojary, M. M.; Zhang, W.; Greco, I.; De Gobba, C.; Olsen, K.; Lund, M. N. Liquid chromatography quadrupole-Orbitrap mass spectrometry for the simultaneous analysis of advanced glycation end products and protein-derived cross-links in food and biological matrices. J Chromatogr A 2020, 1615, 460767. DOI: 10.1016/j.chroma.2019.460767
(57) N, A.; K, T.; P, J.; M, P.; Gangadhariah, M. H.; A, J. L.; H, U. H.; Bs, G. K.; As, S.; Cd, N. Carboxymethyl lysine content in traditional Indian foods. Journal of Food Composition and Analysis 2024, 129, 106087. DOI: 10.1016/j.jfca.2024.106087
(58) Scheijen, J.; Clevers, E.; Engelen, L.; Dagnelie, P. C.; Brouns, F.; Stehouwer, C. D. A.; Schalkwijk, C. G. Analysis of advanced glycation endproducts in selected food items by ultra-performance liquid chromatography tandem mass spectrometry: Presentation of a dietary AGE database. Food Chem 2016, 190, 1145-1150. DOI: 10.1016/j.foodchem.2015.06.049
(59) Chen, Q.; Li, Y.; Dong, L.; Shi, R.; Wu, Z.; Liu, L.; Zhang, J.; Wu, Z.; Pan, D. Quantitative determination of N(epsilon)-(carboxymethyl)lysine in sterilized milk by isotope dilution UPLC-MS/MS method without derivatization and ion pair reagents. Food Chem 2022, 385, 132697. DOI: 10.1016/j.foodchem.2022.132697
(60) Heaton, J. C.; Russell, J. J.; Underwood, T.; Boughtflower, R.; McCalley, D. V. Comparison of peak shape in hydrophilic interaction chromatography using acidic salt buffers and simple acid solutions. J Chromatogr A 2014, 1347, 39-48. DOI: 10.1016/j.chroma.2014.04.026
(61) Gu, H.; Liu, G.; Wang, J.; Aubry, A. F.; Arnold, M. E. Selecting the correct weighting factors for linear and quadratic calibration curves with least-squares regression algorithm in bioanalytical LC-MS/MS assays and impacts of using incorrect weighting factors on curve stability, data quality, and assay performance. Anal Chem 2014, 86 (18), 8959-8966. DOI: 10.1021/ac5018265