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研究生: 廖琬柔
Liao, Wan-Rou
論文名稱: 以液相層析串聯式質譜技術檢測蜂王乳中的單、雙、三磷酸腺苷化合物
Quantification of Adenosine Mono-, Di- and Triphosphate from Royal Jelly using Liquid Chromatography - Tandem Mass Spectrometry
指導教授: 陳頌方
Chen, Sung-Fang
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2018
畢業學年度: 106
語文別: 中文
論文頁數: 81
中文關鍵詞: 核苷酸化合物蜂王乳固相萃取法分散式固相萃取法液相層析-串聯式質譜儀
英文關鍵詞: Nucleotide, Royal jelly, SPE, dSPE, HPLC-MS/MS
DOI URL: http://doi.org/10.6345/THE.NTNU.DC.043.2018.B05
論文種類: 學術論文
相關次數: 點閱:144下載:7
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  • 核苷酸化合物由含氮鹼基、核糖和磷酸基團所組成,以腺嘌呤(Adenine, Ade)所組成的三磷酸腺苷(Adenosine triphosphate, ATP)、二磷酸腺苷(Adenosine diphosphate, ADP)、單磷酸腺苷(Adenosine monophosphate, AMP),在生理機能中扮演重要的角色。蜂王乳(Royal Jelly)為蜂巢中工蜂上顎腺(Mandibular gland)以及咽下腺(Hypopharyngeal gland)的分泌物,富含多種純天然成分,更有研究指出蜂王乳可提供人體所需之營養。本實驗以此為出發點,開發一種可靠且準確的方法,檢測蜂王乳中的核苷酸含量,並探討二日齡和三日齡蜂王乳的含量差異。由於蜂王乳為複雜的基質樣品,為了準確定量蜂王乳中的Ade、AMP、ADP和ATP含量,本實驗開發快速且有效的固相萃取法(Solid-Phase Extraction, SPE)以及分散式固相萃取法(Dispersive Solid-Phase Extraction, dSPE)兩種前處理方法,並優化參數以達到最佳的萃取效率。本實驗的分析方法,使用cyanopropryl (-CN)管柱搭配四元流動幫浦,藉由改變層析環境之pH值,使分析物在12分鐘內達到良好的分離,再結合串聯式質譜進行檢測。本實驗所開發的分析方法有良好的精確度(RSD% < 10.5%)與準確度(回收率81.3 - 118.4%),線性範圍在2.5 - 1000 ng/mL之間,相關係數皆在r = 0.9995以上,偵測極限(LOD)和定量極限(LOQ)最低分別可達1.0 ng / mL和2.5 ng / mL。應用分散式固相萃取法搭配液相層析串聯式質譜法,成功定量蜂王乳中的核苷酸含量(6.2 - 2126.0 mg/kg),二日齡與三日齡蜂王乳的核苷酸含量並無顯著差異。

    Nucleotides are composed of nitrogenous base, ribose and phosphate groups. Adenine (Ade), adenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosine triphosphate (ATP) all play important roles in physiological metabolism. Royal jelly is the worker bee’s secretions, it is rich in a variety of pure natural ingredients, several studies have shown that royal jelly can supply abundant nutrition to the human. In this study, a rapid and effective LC/MS method coupled with pre-processing methods were developed and validated for the accurate quantification of Ade, AMP, ADP and ATP contents in royal jelly. To achieve the best extraction efficiency, two pretreatment methods, solid-phase extraction (SPE) and dispersive solid-phase extraction (dSPE), were developed and investigated. The silica-based cyanopropyl (CN) liquid chromatography was employed using pH programming with quaternary mobile phase system. The total LC/MS run time was within 12 min with a constant flow rate of 0.25 mL/min. The linear range were 2.5-1000 ng/mL with correlation coefficient r = 0.9995. The limit of detection (LOD) and limit of quantitation (LOQ) of 1.0 ng/mL and 2.5 ng/mL were achieved, respectively. Precision (RSD% <10.5%) and accuracy (recovery 81.3-118.4%) were both satisfactory in two pre-processing methods. Eventually, nucleotides were successfully quantified from the 2-day and 3-day royal jelly samples, with concentrations within 6.2 to 2126.0 mg/kg.

    謝誌 I 目錄 II 圖目錄 V 表目錄 VII Abstract 1 中文摘要 2 第一章序論 3 第一節 核苷酸化合物與蜂王乳樣品 3 一、核苷酸化合物 3 二、蜂王乳 5 第二節 樣品前處理 6 一、固相萃取法(SPE) 7 二、分散式固相萃取法(dSPE) 9 第三節 高效能液相層析技術 11 一、高效能液相層析法(HPLC) 11 二、偵測器 13 三、層析管柱 14 第四節 質譜儀技術 16 一、三段式四極柱串聯式質譜儀(QqQ) 18 二、電噴灑游離法(ESI) 22 三、電子倍增器 24 第五節 多重反應監測(MRM)之定量分析 26 第六節 實驗目的與動機 28 第二章 實驗材料與分析方法 29 第一節 實驗試劑 29 第二節 實驗樣品 30 第三節 實驗設備 30 第四節 實驗方法 31 一、樣品前處理 31 二、高效能液相層析參數設定 36 三、質譜儀參數設定 37 四、校正曲線之繪製 38 五、方法驗證 39 第三章 結果與討論 40 第一節 優化高效能液相層析參數 40 一、層析管柱的選擇 40 二、移動相的選擇 42 三、優化梯度沖提條件 43 第二節 優化質譜儀參數 45 一、離子源條件之設定 45 二、離子對的選擇與電壓優化 47 第三節 優化樣品前處理條件 50 一、優化固液萃取法條件 50 二、優化固相萃取法(SPE)之條件 54 三、優化分散式固相萃取法(dSPE)之條件 57 第四節 方法驗證 59 一、檢量線 59 二、準確度與精密度 63 三、同日間與異日間之回收率 66 四、基質效應 67 第五節 蜂王乳中核苷與核苷酸之定量 69 一、比較SPE及dSPE兩種前處理方法 69 二、定量二日齡與三日齡蜂王乳 70 第六節 回顧與比較文獻所提出之檢測方法 72 第四章 結論與未來展望 74 第五章 參考文獻 75

    1. Langen, P. and F. Hucho, Karl Lohmann and the Discovery of ATP. Angewandte Chemie International Edition, 2008. 47(10): p. 1824-1827.
    2. Hardie, D.G. and S.A. Hawley, AMP-activated protein kinase: the energy charge hypothesis revisited. BioEssays, 2001. 23(12): p. 1112-1119.
    3. Zhang, C., R.A. Rissman, and J. Feng, Characterization of ATP alternations in an Alzheimer's disease transgenic mouse model. J Alzheimers Dis, 2015. 44(2): p. 375-8.
    4. Celardo, I., L.M. Martins, and S. Gandhi, Unravelling mitochondrial pathways to Parkinson's disease. British Journal of Pharmacology, 2014. 171(8): p. 1943-1957.
    5. Richards, J.P., et al., Low O2-induced ATP release from erythrocytes of humans with type 2 diabetes is restored by physiological ratios of C-peptide and insulin. Am J Physiol Regul Integr Comp Physiol, 2014. 307(7): p. R862-8.
    6. Kumar, N. and C.S. Dey, Development of insulin resistance and reversal by thiazolidinediones in C2C12 skeletal muscle cells. Biochemical pharmacology, 2003. 65(2): p. 249-257.
    7. Idzko, M., et al., Extracellular nucleotide and nucleoside signaling in vascular and blood disease. Blood, 2014. 124(7): p. 1029-1037.
    8. Stefano, G.B. and R.M. Kream, Cancer: Mitochondrial Origins. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 2015. 21: p. 3736-3739.
    9. Zhang, C., et al., Targeted metabolic analysis of nucleotides and identification of biomarkers associated with cancer in cultured cell models. Acta Pharmaceutica Sinica B, 2013. 3(4): p. 254-262.
    10. Cohen, S., et al., Liquid chromatographic methods for the determination of endogenous nucleotides and nucleotide analogs used in cancer therapy: a review. Journal of Chromatography B, 2010. 878(22): p. 1912-1928.
    11. Carver, J.D., Advances in nutritional modifications of infant formulas. The American Journal of Clinical Nutrition, 2003. 77(6): p. 1550S-1554S.
    12. Schlimme, E., D. Martin, and H. Meisel, Nucleosides and nucleotides: natural bioactive substances in milk and colostrum. British Journal of Nutrition, 2000. 84(S1): p. 59-68.
    13. Gill, B.D., H.E. Indyk, and M. Manley-Harris, Analysis of nucleosides and nucleotides in infant formula by liquid chromatography–tandem mass spectrometry. Analytical and Bioanalytical Chemistry, 2013. 405(15): p. 5311-5319.
    14. Verkerk, R., Nucleotides: Speculation on lifestyle-induced essentiality. NHD Clinical, 2011. 64: p. 29-32.
    15. Hu, G. and F.-Q. Yang, Biological activities of nucleosides and their analogues in dietary foods. system, 2014. 27: p. 33.
    16. Ranogajec, A., S. Beluhan, and Z. Šmit, Analysis of nucleosides and monophosphate nucleotides from mushrooms with reversed‐phase HPLC. Journal of separation science, 2010. 33(8): p. 1024-1033.
    17. Ishimaru, M., et al., Simultaneous analysis of purine and pyrimidine compounds associated with the freshness and taste of marine foods. Food Analytical Methods, 2016. 9(6): p. 1606-1615.
    18. Phat, C., B. Moon, and C. Lee, Evaluation of umami taste in mushroom extracts by chemical analysis, sensory evaluation, and an electronic tongue system. Food chemistry, 2016. 192: p. 1068-1077.
    19. Studzińska, S. and B. Buszewski, Effect of mobile phase pH on the retention of nucleotides on different stationary phases for high-performance liquid chromatography. Analytical and bioanalytical chemistry, 2013. 405(5): p. 1663-1672.
    20. Wang, Y. and B. Liu, ATP detection using a label-free DNA aptamer and a cationic tetrahedralfluorene. Analyst, 2008. 133(11): p. 1593-8.
    21. Tan, Y., et al., Label-free fluorescent assays based on aptamer-target recognition. Analyst, 2012. 137(10): p. 2309-12.
    22. Li, F., et al., Selective and sensitive turn-on detection of adenosine triphosphate and thrombin based on bifunctional fluorescent oligonucleotide probe. Biosensors and Bioelectronics, 2013. 41(Supplement C): p. 907-910.
    23. Wei, Y., et al., An exonuclease I-based label-free fluorometric aptasensor for adenosine triphosphate (ATP) detection with a wide concentration range. Biosensors and Bioelectronics, 2015. 63(Supplement C): p. 311-316.
    24. Zinellu, A., et al., Capillary electrophoresis with laser-induced fluorescence detection for ATP quantification in spermatozoa and oocytes. Analytical and Bioanalytical Chemistry, 2010. 398(5): p. 2109-2116.
    25. Zhu, P., et al., A capillary zone electrophoresis method for adenine nucleotides analysis in Saccharomyces cerevisiae. Journal of Chromatography B, 2016. 1008: p. 156-163.
    26. Liu, J.-X., et al., Analysis of endogenous nucleotides by single cell capillary electrophoresis-mass spectrometry. Analyst, 2014. 139(22): p. 5835-5842.
    27. Xue, X.F., et al., HPLC determination of adenosine in royal jelly. Food Chemistry, 2009. 115(2): p. 715-719.
    28. Contreras-Sanz, A., et al., Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC. Purinergic Signalling, 2012. 8(4): p. 741-751.
    29. Magdenoska, O., et al., Dispersive solid phase extraction combined with ion-pair ultra high-performance liquid chromatography tandem mass spectrometry for quantification of nucleotides in Lactococcus lactis. Analytical biochemistry, 2013. 440(2): p. 166-177.
    30. Jia, J., et al., An optimized ion-pair HPLC method for simultaneous analysis of nucleoside triphosphate levels in hepatoma cell line. Chromatographia, 2011. 73(7-8): p. 755-759.
    31. Xue, X., et al., Online cleanup of accelerated solvent extractions for determination of adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in royal jelly using high-performance liquid chromatography. J Agric Food Chem, 2009. 57(11): p. 4500-5.
    32. Wu, L., et al., Identification of the distribution of adenosine phosphates, nucleosides and nucleobases in royal jelly. Food Chemistry, 2015. 173(Supplement C): p. 1111-1118.
    33. Mateos-Vivas, M., et al., Determination of nucleosides and nucleotides in baby foods by hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of hydrophilic ion-pairing reagents. Food chemistry, 2016. 211: p. 827-835.
    34. Mora, L., et al., Hydrophilic interaction chromatographic determination of adenosine triphosphate and its metabolites. Food chemistry, 2010. 123(4): p. 1282-1288.
    35. Inoue, K. and D. Dowell, HILIC-MS/MS method for the quantitation of nucleotides in infant formula and adult nutritional formula: First Action 2011.21. Journal of AOAC International, 2012. 95(3): p. 603-605.
    36. Marrubini, G., et al., Hydrophilic interaction chromatography in food matrices analysis: An updated review. Food chemistry, 2018.
    37. Inoue, K., et al., Determination of nucleotides in infant formula by ion-exchange liquid chromatography. Journal of agricultural and food chemistry, 2008. 56(16): p. 6863-6867.
    38. Studzińska, S., R. Rola, and B. Buszewski, Determination of nucleotides in infant milk formulas using novel dendrimer ion-exchangers. Journal of Chromatography B, 2014. 949: p. 87-93.
    39. Nagai, T. and R. Inoue, Preparation and the functional properties of water extract and alkaline extract of royal jelly. Food chemistry, 2004. 84(2): p. 181-186.
    40. Krell, R., Value-added products from beekeeping. 1996: Food & Agriculture Org.
    41. Buttstedt, A., R.F. Moritz, and S. Erler, More than royal food-Major royal jelly protein genes in sexuals and workers of the honeybee Apis mellifera. Frontiers in zoology, 2013. 10(1): p. 72.
    42. Kamakura, M., Royalactin induces queen differentiation in honeybees. Nature, 2011. 473(7348): p. 478.
    43. Haydak, M.H., Honey bee nutrition. Annual review of entomology, 1970. 15(1): p. 143-156.
    44. Sano, O., et al., Characterization of royal jelly proteins in both Africanized and European honeybees (Apis mellifera) by two-dimensional gel electrophoresis. Journal of agricultural and food chemistry, 2004. 52(1): p. 15-20.
    45. Takenaka, T. and T. Echigo, Chemical composition of royal jelly. Bulletin of the Faculty of Agriculture, Tamagawa University, 1980(20): p. 71-78.
    46. Echigo, T., T. Takenaka, and K. Yatsunami, Comparative studies on chemical composition of honey, royal jelly and pollen loads. Bulletin of the Faculty of Agriculture-Tamagawa University (Japan), 1986.
    47. Guo, H., Y. Kouzuma, and M. Yonekura, Structures and properties of antioxidative peptides derived from royal jelly protein. Food Chemistry, 2009. 113(1): p. 238-245.
    48. Bloodworth, B.C., et al., Liquid chromatographic determination of trans-10-hydroxy-2-decenoic acid content of commercial products containing royal jelly. Journal of AOAC International, 1995. 78(4): p. 1019-1023.
    49. Han, B., et al., Novel royal jelly proteins identified by gel-based and gel-free proteomics. Journal of agricultural and food chemistry, 2011. 59(18): p. 10346-10355.
    50. Marko, P., I. PECHÁŇ, and J. VITTEK, Some phosphorus compounds in royal jelly. Nature, 1964. 202(4928): p. 188.
    51. Xue, X., et al., Online Cleanup of Accelerated Solvent Extractions for Determination of Adenosine 5′-Triphosphate (ATP), Adenosine 5′-Diphosphate (ADP), and Adenosine 5′-Monophosphate (AMP) in Royal Jelly Using High-Performance Liquid Chromatography. Journal of Agricultural and Food Chemistry, 2009. 57(11): p. 4500-4505.
    52. Zamani, Z., et al., Effect of Royal Jelly on spatial learning and memory in rat model of streptozotocin-induced sporadic Alzheimer's disease. Advanced biomedical research, 2012. 1.
    53. Honda, Y., et al., Lifespan-extending effects of royal jelly and its related substances on the nematode Caenorhabditis elegans. PloS one, 2011. 6(8): p. e23527.
    54. Kim, J., et al., Royal jelly enhances migration of human dermal fibroblasts and alters the levels of cholesterol and sphinganine in an in vitro wound healing model. Nutrition research and practice, 2010. 4(5): p. 362-368.
    55. Park, H.M., et al., Royal Jelly Protects Against Ultraviolet B–Induced Photoaging in Human Skin Fibroblasts via Enhancing Collagen Production. Journal of medicinal food, 2011. 14(9): p. 899-906.
    56. Siavash, M., et al., The efficacy of topical royal jelly on healing of diabetic foot ulcers: a double‐blind placebo‐controlled clinical trial. International wound journal, 2015. 12(2): p. 137-142.
    57. Pasupuleti, V.R., et al., Honey, propolis, and royal jelly: a comprehensive review of their biological actions and health benefits. Oxidative medicine and cellular longevity, 2017. 2017.
    58. Marconi, E., et al., Furosine: a suitable marker for assessing the freshness of royal jelly. Journal of agricultural and food chemistry, 2002. 50(10): p. 2825-2829.
    59. Hennion, M.-C., Solid-phase extraction: method development, sorbents, and coupling with liquid chromatography. Journal of chromatography A, 1999. 856(1-2): p. 3-54.
    60. Anastassiades, M., et al., Fast and easy multiresidue method employing acetonitrile extraction/partitioning and “dispersive solid-phase extraction” for the determination of pesticide residues in produce. Journal of AOAC international, 2003. 86(2): p. 412-431.
    61. Harris, D.C., Quantitative chemical analysis. 2010: Macmillan.
    62. Fenn, J.B., et al., Electrospray ionization for mass spectrometry of large biomolecules. Science, 1989. 246(4926): p. 64-71.
    63. Ikonomou, M.G., A.T. Blades, and P. Kebarle, Electrospray mass spectrometry of methanol and water solutions suppression of electric discharge with SF6 gas. Journal of the American Society for Mass Spectrometry, 1991. 2(6): p. 497-505.
    64. Plakantara, S., et al., Nucleotides and nucleosides in ovine and caprine milk during lactation. Journal of dairy science, 2010. 93(6): p. 2330-2337.
    65. Gill, B.D., H.E. Indyk, and M. Manley-Harris, Determination of total potentially available nucleosides in bovine, caprine, and ovine milk. International dairy journal, 2012. 24(1): p. 40-43.
    66. Zhou, L., et al., Fast determination of adenosine 5′-triphosphate (ATP) and its catabolites in royal jelly using ultraperformance liquid chromatography. Journal of agricultural and food chemistry, 2012. 60(36): p. 8994-8999.
    67. Pina, A., et al., Targeted profiling of hydrophilic constituents of royal jelly by hydrophilic interaction liquid chromatography–tandem mass spectrometry. Journal of Chromatography A, 2018. 1531: p. 53-63.
    68. Viñas, P., et al., Anion exchange liquid chromatography for the determination of nucleotides in baby and/or functional foods. Journal of agricultural and food chemistry, 2009. 57(16): p. 7245-7249.
    69. Zhu, B., et al., A simultaneously quantitative method to profiling twenty endogenous nucleosides and nucleotides in cancer cells using UHPLC-MS/MS. Talanta, 2018. 179: p. 615-623.

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