Simultaneous determination of six main nucleosides and bases in natural and cultured Cordyceps by capillary electrophoresis

A simple method is described for simultaneous determination of six main nucleosides and bases including adenine, uracil, adenosine, guanosine, uridine and inosine in Cordyceps by capillary electrophoresis (CE). Chemometric optimization based on central composite design was employed to find the optimum resolution. The optimum factor space was defined by three parameters: buffer concentration, pH and concentration of acetonitrile as organic modifier. Resolution (Rs) was employed to evaluate the response function. A running buffer composed of 500 mM boric acid, adjusted pH to 8.6 with sodium hydroxide and 12.2% acetonitrile as modifier was found to be the most appropriate for the separation. The contents of the six components were determined by using adenosine monophosphate as an internal standard. Furthermore, hierarchical clustering analysis based on characteristics of 32 peaks in CE profiles from the tested 12 samples showed that natural and cultured Cordyceps were in different clusters. Adenosine and inosine were extracted as markers for discrimination of natural Cordyceps. The result of clustering based on the two peaks characteristics was in excellent agreement with that based on 32 peaks'. Thus, adenosine and inosine could be used as markers for quality control of natural and cultured Cordyceps.     

Optimization of CEC for simultaneous determination of eleven nucleosides and nucleobases in Cordyceps using central composite design

A CEC method is described for the simultaneous determination of 11 nucleosides and nucleobases including cytosine, uracil, uridine, hypoxanthine, 2'-deoxyuridine, inosine, guanosine, thymidine, adenine, adenosine, and cordycepin in Cordyceps using 5-chlorocytosine arabinoside as internal standard (IS). Chemometric optimization based on central composite design was employed to find the optimum conditions. The factors for optimization were defined as three parameters: voltage, pH, and concentration of ACN as organic modifier. The resolution (R(s)) between inosine and guanosine, as well as the entire run time were employed to evaluate the response function. A running buffer composed of 4 mM ammonium acetate and 2 mM triethylamine (TEA) adjusted to pH 5.3 using acetic acid, and containing 3% ACN as modifier, with gradient voltage (0-4 min: 20 kV, 4-12 min: linear gradient from 20 to 30 kV; 12-16 min: 30 kV) were found to be the optimum conditions for the separation. Separation of the 11 investigated compounds and 5-chlorocytosine arabinoside was achieved within 16 min. The contents of the 11 compounds in natural and cultured Cordyceps sinensis, and cultured Cordyceps militaris were also compared. The result showed that CEC is an efficient method for analysis of nucleosides and nucleobases in Cordyceps, which is helpful to control the quality of this valued traditional Chinese medicine.     

毛细管区带电泳法测定冬虫夏草及鹿茸制品中核苷和碱基成分

应用毛细管区带电泳法测定分别以冬虫夏草菌丝体粉和鹿茸血为主要原料制品中的多种核苷和碱基成分。对实验条件进行了优化,结果表明,以20mmol/L硼砂-15mmol/Lβ-环糊精为缓冲溶液(pH=9.4),分离电压22kV,检测波长254nm,电动进样为10kV、5s时,在10min内同时分离测定了虫草素、腺嘌呤、鸟嘌呤、尿嘧啶、腺苷、次黄嘌呤、尿苷、鸟苷和肌苷。各组分在0.2～200μg/mL范围内呈线性关系,检出限的范围是0.07～1.67μg/mL。5个批次的冬虫夏草菌丝粉保健品中腺嘌呤、尿嘧啶、腺苷、鸟苷、尿苷5组分的定量结果分别在0.15～0.19mg/g、0.72～0.92mg/g、1.44～1.59mg/g、1.51～2.32mg/g和1.77～2.56mg/g范围内,加标回收率的范围是82.83%～109.21%;2个批次的鹿茸血保健品中次黄嘌呤、尿苷的定量结果在36.55～49.97μg/mL和86.08～108.97μg/mL范围内,加标回收率的范围分别是89.68%～96.79%和99.05%～102.81%。     

Simple HPLC-UV determination of nucleosides and its application to the authentication of Cordyceps and its allies

A simple HPLC-UV method combined with a simple extraction procedure of nucleosides (adenosine, cordycepin, 2'-deoxyadenosine, guanosine and uridine) was developed and applied to the authentication of Cordyceps and its allies. The separation was performed on a C(18) column by isocratic elution with acetonitrile-water, and UV detection at 260 nm. The amounts of adenosine, cordycepin, 2'-deoxyadenosine, guanosine and uridine in Cordyceps were 0.28-14.15, 0.006-6.36, 0.01-0.14, 0.68-14.79 and 0.19-20.29 mg/g, respectively. Among the nucleosides studied, cordycepin was characteristically included in Cordyceps militaris (L.) Link. (CM), which is one of key Cordyceps allies, and might be a good marker for authenticating CM. The ratio of nucleosides to adenosine contents in Cordyceps seemed to be a useful marker for authentication and quality control of Cordyceps.     

Quality evaluation of Cordyceps through simultaneous determination of eleven nucleosides and bases by RP-HPLC

A new RP-HPLC method has been developed for the simultaneous determination of 11 nucleosides and bases, including adenosine, cordycepin, cytidine, guanosine, inosine, thymidine, uridine, cytosine, guanine, thymine, and uracil in Cordyceps. Determination was achieved on a Zorbax 300SB C18 analytical column (4.6 x 250 mm id, 5 mm) using gradient elution with diode-array detection. All calibration curves showed good linearity (r2 > 0.9995) within the test ranges. The developed method was simple, rapid, and accurate, and showed good reproducibility for the quantification of 11 nucleosides and bases in natural and cultured Cordyceps with both intra- and inter-day variations of less than 1.8%. Furthermore, hierarchical clustering analysis based on the typical peaks of adenosine, cordycepin, and inosine in HPLC profiles from the 11 tested samples showed that natural and cultured Cordyceps were in different clusters, which could provide a means of discriminating between Cordyceps of different origins. Thus, adenosine, cordycepin, and inosine could be used as markers for quality control of Cordyceps.     

毛细管区带电泳法测定板蓝根注射液中四种核苷的含量

采用毛细管区带电泳法测定了板蓝根注射液中胞苷、腺苷、鸟苷和尿苷的含量。电泳条件:采用未涂层石英毛细管(32.5 cm×50 μm i.d.,有效长度23.5 cm),以60 mmol/L硼砂溶液-10%(体积分数)异丙醇-20%（体积分数）乙腈为运行缓冲液,在25 ℃下以20 kV恒压电泳分离,压力进样 (1 kPa×10 s),检测波长254 nm。对电泳条件各因素进行了讨论,如缓冲液的种类、浓度和pH值,有机改性剂的种类和浓度,分离电压和毛细管温度等。样品经0.45 μm微孔滤膜过滤后直接进样;采用外     

Simultaneous determination of nucleosides and their bases in Cordyceps sinensis and its substitutes by matrix solid‐phase dispersion extraction and HPLC

Nine nucleosides and nucleobases, including uracil, adenine, thymine, uridine, adenosine, thymidine, cytidine, guanosine, and cordycepin in natural Cordyceps sinensis, cultured Cordyceps mycelia, and Cordyceps fruiting bodies were extracted by matrix solid‐phase dispersion (MSPD) and determined by HPLC. The experimental conditions for the MSPD extraction were optimized. Florisil was used as dispersant, petroleum ether as washing solvent, and methanol as elution solvent. The Florisil‐to‐sample ratio was selected to be 4:1 and no additional clean‐up sorbent was needed. The calibration curves had good linear relationships (r > 0.9997). The LOD and LOQ were in the range of 12 ～ 79 and 41 ～ 265 ng/mL, respectively. The intra‐ and interday precision were lower than 8.3%. The recoveries were between 61.5 and 93.2%. The present method consumed less sample compared with ultrasonic extraction and heating reflux extraction (HRE). The extraction yields obtained by using the present method are much higher than those obtained by UE and comparable to those obtained by HRE.     

碳纳米管修饰电极测定冬虫夏草有效成分含量

以多壁碳纳米管修饰金电极为工作电极测定了冬虫夏草胶囊有效成分的含量。结果表明,在pH值为7.0的缓冲溶液中,测得其极化电流与腺嘌呤核苷在浓度为1.0×10-4～1.0×10-8mol/L范围内呈良好的线性关系(R2=0.9947),检出限为1.0×10-9mol/L。所测的5批样品的平均回收率为101.5%,相对标准偏差RSD为1.5%。方法灵敏度高、简单、快速、重现性好、干扰小,而且节能、环保,无污染物排放。     

Simultaneous Determination of Ten Nucleosides and Related Compounds by MEEKC with [BMIM]PF6 as Oil Phase

In the present study, four nucleobases (adenine, cytosine, uracil, thymine), four nucleosides (adenosine, cytidine, uridine, thymidine), and two nucleotides (adenosine-5′-monophosphate, and cytidine-5′-monophosphate) were simultaneously determined by MEEKC with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as oil phase. Experimental parameters including the microemulsion compositions (surfactant, co-surfactant, and oil phase), pH, and concentration of borate buffer were intensively investigated. Finally, the ten compounds were well separated within 11 min using the running buffer composed of 140 mM SDS, 1.8 M n-butanol, and 10 mM [BMIM]PF₆ in 20 mM borate buffer of pH 9.0. The developed method was successfully applied to determine the contents of investigated compounds in three different widely used traditional Chinese medicines (cultured Cordyceps sinensis, Radix Astragali, and Radix Isatidis). The results indicated that the developed MEEKC method could be used for the rapid determination of nucleobases, nucleosides, and nucleotides in herbal medicines or other complex matrices.     

Simultaneous Determination of Ten Nucleosides and Related Compounds by MEEKC with [BMIM]PF6 as Oil Phase

In the present study, four nucleobases (adenine, cytosine, uracil, thymine), four nucleosides (adenosine, cytidine, uridine, thymidine), and two nucleotides (adenosine-5′-monophosphate, and cytidine-5′-monophosphate) were simultaneously determined by MEEKC with ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM]PF₆) as oil phase. Experimental parameters including the microemulsion compositions (surfactant, co-surfactant, and oil phase), pH, and concentration of borate buffer were intensively investigated. Finally, the ten compounds were well separated within 11 min using the running buffer composed of 140 mM SDS, 1.8 M n-butanol, and 10 mM [BMIM]PF₆ in 20 mM borate buffer of pH 9.0. The developed method was successfully applied to determine the contents of investigated compounds in three different widely used traditional Chinese medicines (cultured Cordyceps sinensis, Radix Astragali, and Radix Isatidis). The results indicated that the developed MEEKC method could be used for the rapid determination of nucleobases, nucleosides, and nucleotides in herbal medicines or other complex matrices.

     

NMR, CD and MCD studies of vanadate-nucleoside complexes

Complex formation between tetraoxovanadate(V) and each of the nucleosides adenosine, guanosine, cytidine and uridine has been studied in a constant salt medium at pH 7. 13C- and 51V NMR studies show that only complexes with the formula V2L2 (V = vanadate, L = nucleoside) are formed, and their formation constants have been determined. They have 51V NMR resonances around -523 ppm relative to VOCl3 and they exhibit no CD in the spectral region of the charge-transfer transitions. MCD spectra were also measured, and all experiments are in accord with a molecular structure composed by two edge-sharing VO6 octahedra forming an O4V(mu-O)2VO4 skeleton with each of the nucleoside ligands bridging the two vanadium centres through the ribose 2',3'-oxygens, which are the oxygens outside the V2O6 plane. Admixture of imidazole-HCl buffer at pH 7 gives rise to additional complexes of 1:1 stoichiometry. They have been characterized by 51V NMR and CD, and their formation constants are reported. Vanadate(V) and the deoxynucleosides deoxyadenosine, deoxyguanosine, deoxycytidine and thymidine form very weak complexes which cannot be detected by 51V NMR or CD under conditions for which vanadate and the nucleosides form complexes.     

N-Phenoxyacetylated guanosine and adenosine phosphoramidites in the solid phase synthesis of oligoribonucleotides: Synthesis of a ribozyme sequence

Phosphoramidites for guanosine and adenosine nucleosides with phenoxyacetyl as N-acyl protecting group were prepared. These nucleoside phosphoramidites, together with previously reported uridine and N-benzoyl cytidine phosphoramidites have been applied to the efficient solid phase synthesis of a trideca and a nonadecaribonucleotide. The later molecule has the sequence corresponding to a ribozyme.     

Separation of nucleosides using capillary electrochromatography

The analysis of nucleosides and nucleotides have in most cases been performed by HPLC using either reversed-phase HPLC with gradient elution or using reversed-phase ion-pair chromatography. In this paper we have explored the possibility of using capillary electrochromatography (CEC) in order to avoid the use of gradients or ion-pairing reagents. CEC is in many ways comparable to HPLC, but CEC is theoretically able to provide better separations due to the higher efficiency caused by the flowfront being more plug-like as also is the case in CE, which is to be compared to the more parabolic flow observed in HPLC. The separation of six nucleosides (adenosine, cytidine, guanosine, inosine, thymidine and uridine) was investigated with respect to concentration of buffers, pH, amount of acetonitrile, temperature and voltage in order to optimise the separation. Baseline separation was achieved for the six nucleosides in less than 13 min using a background electrolyte consisting of (5 mM acetic acid, 3 mM triethylamine, pH 5.0)-acetonitrile (92:8, v/v).     

同时测定核苷酸口服液中二磷酸核苷的毛细管区带电泳法

建立了一种毛细管区带电泳法，用于同时测定5种核苷酸－鸟苷二磷酸（GDP）、尿苷二磷酸（UDP）、腺苷二磷酸（ADP）、胞苷二磷酸（CDP）、次黄嘌呤核苷酸（IDP）；研究了缓冲液的pH值及磷酸盐的浓度对分离5种核苷酸的影响，5种核苷酸在75mmol/L三羟甲基氨基甲烷（Tris)－50mmol/L磷酸二氢钠（NaH2PO4）、pH为8．00的缓冲液可以基线分离；另外还研究了中入阳离子表面活性剂十六烷基三甲基溴化胺（CTAB）以及CTAB的浓度和进样时间对分离5种核苷酸的影响；该法成功地应用于测定核苷酸口服液中ADP、CDP、GDP、UDP的含量。     

High-throughput synthesis of HepDirect prodrugs of nucleoside monophosphates

A high-throughput phosphoramidite method for HepDirect prodrug synthesis was optimized on seven representative nucleosides, adenosine, inosine, guanosine, uridine, cytidine, AICA-riboside, and thymidine, each on a 5 mg scale. The variables optimized included (1) reaction time, (2) reaction temperature, (3) activating agent, (4) solvent, (5) purification method, and (6) stoichiometry. Preparative HPLC with mass-based fraction collection and yield determination from an ELSD standard curve enabled high-throughput. The optimized conditions for the representative nucleosides required 6 mol equiv of phosphoramidite to nucleoside and resulted in an average HPLC determined yield of 31 +/- 14% and HPLC purity of 93 +/- 3%.     

发酵虫草菌粉及产品指纹图谱建立及多指标成分分析

《中国药典》收载的发酵虫草菌粉产品的质量标准中,规定以鸟苷、腺苷、尿苷的含量作为评价相关产品质量的标准。但除此之外,还有许多其他的核苷类成分对发酵虫草菌粉质量控制的影响尚未被探讨。为探究发酵虫草菌粉及产品质控指标选择的合理性,采用超高效液相色谱-紫外检测法对19批发酵虫草菌粉及产品中9种核苷成分(尿嘧啶、胞苷、鸟嘌呤、尿苷、腺嘌呤、肌苷、鸟苷、胸苷、腺苷)进行了定量分析,建立了发酵虫草菌粉样品的指纹图谱,并结合统计学提供了一种分析指标性成分的方法。通过优化样品的提取方法,选择超声提取法制备19批发酵虫草菌粉及产品的供试液;采用Agilent Eclipse Plus C18色谱柱(150 mm×4.6 mm, 3.5 μm)进行色谱分析,以甲醇-水为流动相梯度洗脱,对方法的校正曲线、准确度、精密度、重复性和回收率进行了验证。结合对照品指认了指纹图谱中的9个核苷峰,并采用外标一点法测得了各核苷成分的含量。使用化学模式识别对指纹图谱中的共有峰进行分析,聚类分析和主成分分析得到了同样的分类结果:19批样品共分为5类,其中同一发酵虫草菌粉因工艺差异可分为2类,而心肝宝胶囊、百令胶囊、宁心宝胶囊则各单独分为1类。同时,使用主成分分析获得了各样品中的指标性成分,分别为尿苷、鸟苷、腺苷、腺嘌呤、尿嘧啶,并使用聚类分析再次进行确证,验证了指标性成分的合理性。     

Determination of Cordycepin in Cordyceps kyushuensis by Capillary Electrophoresis and its Antitumour Activity

~~Determination of Cordycepin in Cordyceps kyushuensis by Capillary Electrophoresis and its Antitumour Activity~~     

Investigation of unexpected migration behavior of adenosine in capillary zone electrophoresis

Summary The capillary zone electrophoresis of two common nucleosides, adenosine and inosine, was investigated. Both compounds were resolved in a 0.1 M sodium phosphate buffer, pH 7.5. Contrary to expectations, adenosine behaved at this pH— 5 pH units lower than the literature pK_a— as a negative ion, migrating behind mesityloxide (neutral marker) when working in normal polarity mode. To confirm the migration order, peaks were identified from absorption maxima, by high-speed scanning detector. The change in electrophoretic mobility with pH was investigated for the nucleosides, and 10 other background electrolytes were tried to match the separation capabilities of the sodium phosphate buffer. Most inorganic buffers showed comparable separation, while organic, Good-type buffers lacked selectivity.     

Counter-current chromatographic separation of nucleic acid constituents with a hydrophilic solvent system

Nucleic acid constituents such as nucleobases, nucleosides and nucleotides were separated by counter-current chromatography using type J coil planet centrifuge. The separation was performed with a hydrophilic solvent system composed of 1-propanol/800 mM potassium phosphate buffer (pH 7.4) (1:1, v/v) by eluting the lower aqueous phase at a flow-rate of 0.5 ml/min. Eight selected nucleic acid constituents (4.0 mg, 0.5 mg of each), uridine monophosphate (UMP), adenosine monophosphate (AMP), deoxyadenosine monophosphate (dAMP), uridine, urasile, deoxy uridine, adenosine and adenine were well resolved within 160 min.