7-甲基鸟苷在玻碳电极上的伏安行为研究

研究了修饰核苷7-甲基鸟苷(7-Methylguanosine)的电化学行为及测定方法.在pH 1.98的B-R缓冲液中,用循环伏安法(CV),线性扫描伏安法(LSV)、微分脉冲伏安法(DPV)等现代电化学技术研究7-甲基鸟苷在玻碳电极(GCE)上的伏安行为.实验表明,7-甲基鸟苷在 1.036 V(vs.SCE)电位处产生一个阳极氧化峰,峰电流与7-甲基鸟苷的浓度在5.0×10-5-2.0×10-6mol/L范围内呈良好的线性关系,最低检测限(D=2σ/K)为4.1×10-7mol/L.并用恒电位库仑电解法等方法对其氧化机理进行了较为详细的探讨,得到了可能的电极反应机理:7-甲基鸟苷在玻碳电极上的电极反应是属于失1质子和2电子的不可逆的氧化反应.     

莽吉柿中几种双苯吡酮和蒽醌类成分的分离与鉴定

对莽吉柿(Pericarpium Garciniae Mangostanae)85%乙醇提取物进一步分离,得到8个双苯吡酮类化合物和1个蒽酮类化合物,经理化性质和NMR及MS谱学数据鉴定分别为α-倒捻子素-3,6-二乙酸酯（α-mangostin-3,6-diyl diacetate,Ⅰ）、1,5,8-三羟基-3-甲氧基-2,4-双-（3-甲基丁-2-烯）双苯吡酮（8-hydroxycudraxanthone G,Ⅱ）、1,3-二羟基-6,7-二甲氧基-2,8-双-（3-甲基丁-2-烯基）双苯吡酮（Cowaxanthone B,Ⅲ）、1,6-二羟基-3,5-二甲氧基-2-（3-甲基丁-2-烯基）双苯吡酮（Cowaxanthone A,Ⅳ）、1,5-二羟基-4-（3-甲基丁-2-烯基）-6′,6′-二甲基吡喃[2′,3′:3,2]双苯吡酮（trapezifolixanthone,Ⅴ）、1,6-二羟基-6′,6′-二甲基吡喃[2′,3′:7,8]-6″,6″-二甲基吡喃[2″,3″:3,2-]双苯吡酮（brasilixanthone B,Ⅵ）、1,3,5-三羟基-2-（3-甲基丁-2-烯）-4-（1,1-二甲基-2-烯丙基）双苯吡酮（Allanxanthone A,Ⅶ）、1,3,8-三羟基-5-甲氧基-2,4-双-（3-甲基丁-2-烯）双苯吡酮(2,4-di-(3-methylbut-2-enyl)-1,3,8-trihydroxy-5-methoxyxanthone,Ⅷ)、1,4,8-三羟基-6-甲基-3-甲氧基-9,10-蒽酮(1,4,8-trihydroxy-6-methyl-3-methoxy- 9,10-anthraquinone, Ⅸ）。 其中化合物Ⅰ、Ⅴ、Ⅵ、Ⅶ、Ⅸ为首次从藤黄属植物中分离得到,化合物Ⅱ、Ⅲ、Ⅳ、Ⅷ为首次从该植物中分离得到。     

肿瘤标记物1-甲基鸟苷的微分脉冲伏安法的研究

建立一种灵敏的测定修饰核苷1 甲基鸟苷(1 MG)的电化学分析方法.在pH为2.00的BR缓冲液中,用循环伏安法(CV),线性扫描伏安法(LSV),方波伏安法(SWV),微分脉冲伏安法(DPV)等现代电化学技术研究了肿瘤标记物1 甲基鸟苷在玻碳电极(GCE)上的伏安行为.实验结果表明,pH为2.00的BR缓冲液中,1 甲基鸟苷在+1.16V(vs.甘汞)电位处产生一个灵敏的微分脉冲阳极氧化峰,该氧化峰的峰电流值与1 甲基鸟苷的浓度在4.0×10-7～1.0×10-5mol/L范围内呈良好的线性关系,最低检测限达(定义为D=2σ/K)6.6×10-8mol/L.将该法应用于兔血清及尿样中1 甲基鸟苷的测定,回收率在96.0%～102.0%之间,5次测定的相对标准偏差(RSD)均小于3 5%.并初步研究了1 甲基鸟苷的电化学机理.     

基于UPLC-UV-Q-TOF MS的僵蚕嘌呤(核苷)类成分多指标定量指纹图谱研究北大核心CSCD

嘌呤(核苷)是机体细胞生命活动中的主要遗传物质,但目前缺乏对僵蚕中该类成分的整体定量研究。该文采用ACE Excel 2 AQ水性柱(2.1 mm×150 mm,2μm)进行分离,以10 mmol/L磷酸二氢钾水溶液-甲醇为流动相,分别优化了样品提取与色谱条件,建立了中药僵蚕中嘌呤(核苷)类成分的超高效液相色谱-紫外(UPLC-UV)指纹图谱方法。利用四极杆-飞行时间质谱(Q-TOF MS)对9个特征峰物质进行二级鉴定,并对其质谱裂解规律进行总结。其中,2′-甲氧基-鸟苷单磷酸、6-(β-羟基乙基氨基)-嘌呤、2′-甲氧基-腺苷单磷酸和N6-(2-羟乙基)-腺苷首次从僵蚕中鉴定。基于该方法,对其中的鸟嘌呤、尿苷、腺嘌呤、鸟苷、6-(β-羟基乙基氨基)-嘌呤、腺苷、N6-(2-羟乙基)-腺苷7个成分进行绝对定量测定。2′-甲氧基-鸟苷单磷酸和2′-甲氧基-腺苷单磷酸因缺乏对照品,采用相对定量分析。结果表明,各指标成分的精密度、重现性、稳定性、耐用性的相对标准偏差(RSD)均不大于5.0%,7种成分的线性关系良好(r^(2)≥0.9994),检出限(LOD)为0.266~0.474μg/mL,定量下限(LOQ)为0.843~1.58μg/mL,平均加标回收率为94.0%~101%,RSD不大于3.4%。采用该方法对13批不同产地僵蚕药材进行分析,发现各成分含量具有显著差异。该研究可为僵蚕的质量评价提供技术支持和科学依据。     

9-(β-D-2''-脱氧核糖基)-6-甲基嘌呤的合成

报道了9-(β-D-2'-脱氧核糖基)-6-甲基嘌呤合成的新方法.以肌苷1为原料,经酯化、氯化、氨解得6-氯嘌呤核苷(4),再经过羟基保护、6-位甲基化反应及脱保护反应得到关键中间体6-甲基嘌呤核苷7,用1,3-二氯-1,1,3,3-四异丙基二硅氧烷保护7核糖上的3,5-二羟基,2-羟基与苯氧基硫代甲酰氯反应后得到9,然后与氢化三正丁基锡[HSn(Bu-n)3]还原脱氧,最后脱保护得到目标化合物11.产物结构经MS,1HNMR,元素分析等鉴定.     

大鼠尿液中异丙嗪及其主要代谢物的GC-MS分析

采用气相色谱-质谱(GC-MS)分析了大鼠尿液中的异丙嗪及其代谢产物。尿液经β-葡萄糖醛酸酶酶解,Oasis(HLB 3 mL固相萃取柱提取后,以Restek R tx-5MS(30 m×0.25 mm×0.25μm)石英毛细管色谱柱为分析柱,经GC-MS分离检测。根据质谱图对代谢物的结构进行鉴定。结果表明:在大鼠尿液中检出了吩噻嗪、去甲基异丙嗪、异丙嗪亚砜、2-羟基去甲基异丙嗪、二羟基异丙嗪、顺-N-(1-丙烯基)吩噻嗪、反-N-(1-丙烯基)吩噻嗪、顺-N-(1-丙烯基)吩噻嗪砜、反-N-(1-丙烯基)吩噻嗪砜、顺-N-(1-丙烯基)吩噻嗪亚砜及反-N-(1-丙烯基)吩噻嗪亚砜11种代谢物。对异丙嗪在大鼠体内的氧化、还原等主要代谢途径进行了讨论。     

毛细管电泳-安培检测法用于7-甲基鸟苷与丝裂霉素C分离检测的研究

建立了一种同时分离检测7-甲基鸟苷与丝裂霉素C的毛细管电泳-安培检测方法。在950 mV电极(工作电极:0.3 mm微型石墨圆盘电极;参比电极:Ag/AgCl;辅助电极:Pt丝)电位下,于20 mmol/L的磷酸盐缓冲体系(pH 9.4)中,采用18 kV的分离电压进行分离。在最佳条件下,7-甲基鸟苷与丝裂霉素C在10 min内实现分离,7-甲基鸟苷与丝裂霉素C的线性范围均为0.50~50 mg/L,检测限分别为0.050 mg/L与0.025 mg/L。将该方法用于模拟尿样和模拟兔血清样的检测,7-甲基鸟苷与丝裂霉素C的回收率为93.0%～97.2%,结果令人满意。     

连茚四酮类有机磁性化合物的合成及性能

分别以邻苯二甲酸酐、4-甲基邻苯二甲酸酐和四氯邻苯二甲酸酐为原料, 采用Gabriel & Leupold法合成了3个连茚四酮类化合物, 即2,2′-二茚满-1,1′,3,3′-四酮(1)、5,5′-二甲基-2,2′-二茚满-1,1′,3,3′-四酮(2)和4,4′,5,5′,6,6′,7,7′-八氯-2,2′-二茚满-1,1′,3,3′-四酮(3). 元素分析、¹H NMR、FTIR 和MS测定分析表明, 此3个分子主要以烯醇式存在, 同时存在分子内氢键; 通过电子自旋共振(ESR)谱测定, 给出了各化合物的ESR谱参数值. 结果表明, 化合物1~3均有良好的ESR图谱, 是只含有C, H, O及Cl的纯有机磁性化合物, 分子内都含有稳定的自由基. 通过量子化学计算, 推测出了自由基可能形成的位置及此类化合物最可能的存在形式(烯醇式), 且证明分子内存在1个氢键.     

超高效液相色谱/飞行时间质谱用于人参皂甙Rg3作用后大鼠尿液代谢物指纹图谱分析及标记物的鉴定

超高效液相色谱技术(UPLC)采用1.7 μm的色谱柱填料,有更好的分离效率、峰容量以及灵敏度;高分辨的时间飞行质谱(TOF-MS)能够测定化合物准确的分子质量并具有MS/MS功能。 两者的联用适合于复杂体系的分离分析和未知物的结构鉴定。 因此建立了一种基于UPLC/TOF-MS测定人参皂甙Rg3给药后大鼠尿液代谢物变化的方法,对其中2种发生显著变化的代谢物分别通过准确的质量测定得到其元素组成,通过MS/MS技术得到其结构信息,并通过检索数据库最终分别鉴定为4,8-二羟喹啉甲酸和4-羟基-2-喹啉酸。     

新型桥连双卟啉化合物的合成及结构表征

通过将4,4′-二羧基-2,2′-联吡啶、2,6-二溴甲基吡啶、2,6-二羟甲基吡啶和1,8-二氨基萘分别与5-(4-羟基苯基)-10,15,20-三苯基卟啉(1a)、5-(4-甲酰苯基)-10,15,20-三苯基卟啉(1b)和5-[4-(4′-溴代丁氧基)苯基]-10,15,20-三苯基卟啉(1c)反应,合成了3类新型的双卟啉化合物2a-2e,经IR,¹HNMR,MS,UV-Vis光谱及元素分析对中间体和目标化合物的结构进行了表征.     

Analysis of urinary nucleosides. V. Identification of urinary pyrimidine nucleosides by liquid chromatography/electrospray mass spectrometry

Modified urinary nucleosides are potentially invaluable in cancer diagnosis, as they reflect altered RNA turnovers. High-performance liquid chromatography (HPLC) was combined with full-scan mass spectrometry, tandem mass spectrometry, MS(n) analysis and accurate mass measurements in order to identify pyrimidine nucleosides purified from urine. Potential nucleosides were assessed by their evident UV absorbance in the HPLC chromatogram and then further examined by the various mass spectrometric techniques. In this manner numerous pyrimidine nucleosides were identified in the urine samples from cancer patients including pseudouridine, cytidine, two methylcytidines and an acetylcytidine. Furthermore, a number of novel modified pyrimidine nucleosides were tentatively identified via critical interpretation of the combined mass spectrometric data.     

Analysis of modified nucleosides in the urine of patients with malignant cancer by liquid chromatography/electrospray ionization mass spectrometry

As modified nucleosides reflect altered tRNA turnover which seems to be impaired in the body of cancer patients, they have been evaluated as potential tumor markers. High-performance liquid chromatography/electrosprary ionization quadrupole time-of-flight mass spectrometry (HPLC/ESI-Q-TOFMS) was used to identify nucleosides purified from urine in positive ionization mode. Potential nucleosides were assessed by their evident UV absorbance in HPLC and then further examined by mass spectrometric techniques. In this manner, 21 nucleosides were detected in the urine of a patient with lymphoid cancer including three modified nucleosides 5'-dehydro-2-deoxyinosine, N1,N2,N7-trimethylguanosine and N1-methyl-N2-ethylguanosine, which had never been reported previously.     

Detection of new radiation-induced DNA lesions by liquid chromatography coupled to tandem mass spectrometry

High-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) has been used to search for the formation of as yet unidentified radiation-induced DNA lesions. For that purpose, the characteristic fragmentation of most of 2'-deoxyribonucleosides that corresponds to the loss of the 2-deoxyribose moiety (loss of 116 mass units) has been utilized to specifically detect modified nucleosides. Aerated aqueous solutions of DNA were exposed to ionizing radiation, and subsequently DNA was digested to nucleosides with a cocktail of endo- and exonucleases. HPLC/ESI-MS/MS analysis of the resulting 2'-deoxyribonucleoside mixture allowed us to detect four novel DNA modifications. In a subsequent step, the sensitivity of the tandem mass spectrometer was used to search for the formation of the newly detected lesions in the DNA of gamma-irradiated cells. Thus, one of the four newly detected lesions was found to be significantly generated in cellular DNA upon exposure to ionizing radiation. In addition, the latter lesion was also shown to be present in untreated cells, indicating that the modified nucleoside could be formed endogenously.     

Analysis of urinary nucleosides. IV. Identification of urinary purine nucleosides by liquid chromatography/electrospray mass spectrometry

Modified urinary nucleosides are potentially invaluable in cancer diagnosis. High-performance liquid chromatography (HPLC) was combined with full scan mass spectrometry (MS), tandem mass spectrometry and MSn analysis in order to identify purine nucleosides purified from urine. UV peaks evident in the chromatogram were examined by the various mass spectrometric techniques and adenosine, 1-methyladenosine, xanthosine, N1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, N2,N2,N7-trimethylguanosine, inosine, and 1-methylinosine were each identified in the urine samples from cancer patients. The benefits of the use of LC/MS compared with HPLC alone are discussed.     

Study of the mass spectrometric fragmentation of pseudouridine: comparison of fragmentation data obtained by matrix-assisted laser desorption/ionisation post-source decay, electrospray ion trap multistage mass spectrometry, and by a method utilising electrospray quadrupole time-of-flight tandem mass spectrometry and in-source fragmentation

Many nucleosides and their modified forms have been studied by mass spectrometry elaborating the detailed fragmentation pathways under MS2 and MS(n) conditions. Although the C-nucleoside pseudouridine has been fragmented and studied briefly, usually amongst many other nucleosides, it has not been investigated to the same extent as other nucleosides. In this report a number of different mass spectrometric techniques are applied to obtain a fuller picture of pseudouridine fragmentation. At the same time this study is used to compare different tandem mass spectrometric techniques, including a novel methodology utilising a quadrupole time-of-flight (Q-ToF) instrument for MS(n) analysis comparable with that available with an ion trap mass spectrometer.     

Separation and identification of purine nucleosides in the urine of patients with malignant cancer by reverse phase liquid chromatography/electrospray tandem mass spectrometry

Urinary‐modified nucleosides have a potential role as cancer biomarkers for a number of malignant diseases. High performance liquid chromatography (HPLC) was combined with full‐scan mass spectrometry, MS/MS analysis and accurate mass measurements in order to identify purine nucleosides purified from urine. Potential purine nucleosides were assessed by their evident UV absorbance in the HPLC chromatogram and then further examined by the mass spectrometric techniques. In this manner, numerous modified purine nucleosides were identified in the urine samples from cancer patients including xanthine, adenosine, N1‐methyladenosine, 5′‐deoxy‐5′‐methylthioadenosine, 2‐methyladenosine, N6‐threonylcarbamoyladenosine, inosine, N1‐methylinosine, guanosine, N1‐methylguanosine, N7‐methylguanine, N2‐methylguanosine, N2,N2‐dimethyguanosine, N2,N2,N7‐trimethylguanosine. Furthermore, a number of novel purine nucleosides were tentatively identified via critical interpretation of the combined mass spectrometric data including N3‐methyladenosine, N7‐methyladenine, 5′‐dehydro‐2′‐deoxyinosine, N3‐methylguanine, O6‐methylguanosine, N1,N2,N7‐trimethylguanosine, N1‐methyl‐N2‐ethylguanosine and N7‐methyl‐N1‐ethylguanosine. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of urinary nucleosides as helper tumor markers in hepatocellular carcinoma diagnosis

Hepatocellular carcinoma (HCC) is a common neoplasm in Taiwan, for which early diagnosis is difficult and the prognosis is usually poor. HCC is usually diagnosed by abdominal sonography and serum alpha‐fetoprotein (AFP) detection. Modified nucleosides, regarded as indicators for the whole‐body turnover of RNAs, are excreted in abnormal amounts in the urine of patients with malignancies and can serve as tumor markers. We analyzed the excretion patterns of urinary nucleosides from 25 HCC patients and 20 healthy volunteers by high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC/ESI‐MS/MS) under optimized conditions. The HPLC/ESI‐MS/MS approach with selective reaction monitoring (SRM) allowed for the sensitive determination of nucleosides in human urine samples. The mean levels of the urinary nucleosides adenosine, cytidine, and inosine were significantly higher in HCC patients than healthy volunteers (average of 1.78‐, 2.26‐, and 1.47‐fold, respectively). However, the mean levels of urinary 1‐methyladenosine, 3‐methylcytidine, uridine, and 2′‐deoxyguanosine were not significantly different. Combined with the determination of serum AFP levels, the higher levels of urinary adenosine, cytidine, and inosine may be additional diagnosis markers for HCC in Taiwanese patients. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of 5-hydroxymethyl-2′-deoxycytidine in Rice by High-performance Liquid Chromatography–Tandem Mass Spectrometry with Isotope Dilution

5-hydroxymethyl-2′-deoxycytidine (5-hmdC), which plays important roles in the epigenetic remodeling in mammals, has been recently determined in plant genomes at trace concentrations. In this study, a method for the sensitive, specific, and rapid assessment of 5-hmdC in rice is reported using high-performance liquid chromatography–tandem mass spectrometry (HPLC–tandem MS) with isotope dilution. The recovery of this method was from 75.6% to 81.3% for 5-hmdC and the intra- and inter-day precision values were within 5.81%. The quantification limit for 5-hmdC was 5.0?pM. Genomic DNA from rice was extracted and enzymatically digested for nucleotides for the HPLC–tandem MS assay. The 5-hmdC levels were 1.32?±?0.08, 1.64?±?0.12, and 1.98?±?0.07 modifications per million nucleosides in the Shengdao-18, Shengxiang-145, and Jindao-263 rice cultivars. The application of HPLC–tandem MS for 5-hmdC demonstrated its presence in the genomic DNA of rice and is useful in further studying the role of modified cytosine in the epigenetic regulation of rice.