用反相高效液相色谱法测定小鼠心肌、骨骼肌中AT P、AD P和AM P的含量

采用反相高效液相色谱法同时测定小鼠心肌、骨骼肌组织中ATP(三磷酸腺苷 )、ADP(二磷酸腺苷 )、AMP(一磷酸腺苷 )的含量。以150mmol/L磷酸二氢钾缓冲液 (pH=6.25)为流动相 ,采用BDShypersilC18 不锈钢色谱柱 ,紫外检测波长为254nm ,线性范围为1～200mg/L。ATP、ADP和AMP的平均加标回收率均在90 %以上 ,检出限分别为10ng、10ng、15ng。该法灵敏、准确 ,试剂费用低。     

反相高效液相色谱法同时测定人膀胱癌T24细胞中三磷酸腺苷及其代谢物的含量北大核心CSCD

所有生物过程都伴随着能量代谢,细胞内的能量供应主要依赖于线粒体合成的三磷酸腺苷(ATP)[1-2]。因此,建立有效的细胞内ATP及其代谢产物[二磷酸腺苷(ADP)和一磷酸腺苷(AMP)]的定量方法是非常有必要的。ATP、ADP和AMP的结构和理化性质非常相似,同时测定会有一定困难。     

高效液相色谱法测定棉籽中的ATP，ADP，AMP

测定生物组织中腺苷三磷酸(ATP)、腺苷二磷酸(ADP)和腺苷一磷酸(AMP)的含量,在研究生物的一些生理生化现象中有重要意义。利用不同方法定量分析这三种腺苷酸的报道已有很多。本文所述是用反相高效液相色谱法,对棉籽中的ATP、ADP和AMP进行测定。     

蜂王浆中磷酸腺苷的提取及超高效液相色谱分析

比较了高氯酸提取、热水提取和热硫酸镁溶液提取3种提取方式对蜂王浆中磷酸腺苷三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和单磷酸腺苷(AMP)的提取效果,发现在低温(低于4 ℃)下以5%高氯酸的提取效果最佳。采用超高效液相色谱-紫外检测法分析蜂王浆中的ATP, ADP和AMP的含量。以BEH Shield RP18柱(100 mm×2.1 mm,1.7 μm)为分析柱,以50 mmol/L的磷酸二氢铵(pH 6.5)和乙腈为流动相进行梯度洗脱,3种磷酸腺苷在4 min内实现了较好的分离。以加标王浆样品作添加回收率测定,ATP, ADP和AMP的回收率分别为84.1%～94.3%,86.2%～93.7%和91.0%～104.3%,相对标准偏差均小于10%。方法已被用于一些实际样品的分析,以了解ATP, ADP和AMP在蜂王浆样品中的分布情况。     

反相离子对高效液相色谱法测定心肌组织中的三磷酸腺苷

 采用反相离子对高效液相色谱法测定心肌组织中三磷酸腺苷 (ATP)的含量。样品经高氯酸溶液沉淀蛋白 ,上清液用KOH溶液中和后用反相离子对高效液相色谱法分离测定。色谱柱为SpherisorbODS2柱 ,流动相为甲醇 KH2 PO4缓冲液 (内含 5mmol/LIPR A离子对试剂 ) ,在 2 59nm波长处检测。方法最低检测限为 2mg/L,在 5mg/L～ 1 0 0 mg/L范围内有良好的线性关系 (r=0 9998) ,方法的回收率为 97 8%～ 1 0 4 % ,日内精密度 <4 85% ,日间精密度 <8 81 %。方法准确、灵敏、快速 ,适用于动物和人心肌组织中ATP含量的测定。     

对氯苯酚提取-液相色谱法分析烟草中的磷酸腺苷

建立了新鲜烟叶中三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和单磷酸腺苷(AMP)3种磷酸腺苷同时测定的方法。烟草样品经过对氯苯酚提取、纯化后,采用三乙胺缓冲液作为流动相洗脱,经C18色谱柱分离,在259 nm波长对样品进行检测。与高氯酸(PCA)法,以及DNA常用的苯酚提取法进行了比较,结果均表明:低温下以对氯苯酚提取的效果较佳,使用三乙胺缓冲液流动相具有分离效率高、检出灵敏的优点。在线性范围(5~200μg/mL)内,磷酸腺苷浓度与峰面积具有很好的相关系数(r>0.9999),检出限达到纳克级水平,氯苯酚法对ATP,ADP和AMP的平均回收率分别为96.8%,97.2%和93.3%,RSD均小于5%。     

离子对反相高效液相色谱法同时测定大鼠血浆和红细胞中外源性磷酸肌酸及其代谢产物和相关三磷酸腺苷

建立了离子对反相高效液相色谱(IP-RPHPLC)同时测定大鼠血浆和红细胞(RBC)中磷酸肌酸(PCr)及其代谢产物肌酸(Cr)以及相关三磷酸腺苷(ATP)浓度的方法.采用Kromasil-C18色谱柱(250 mm×4.6 mm,5 μm),流动相A:0.2%KH2PO4+0.08%四丁基硫酸氢铵混合溶液(pH 3....     

高效液相色谱-质谱法测定人体滑液中腺苷的含量

腺苷是三磷酸腺苷的代谢产物,它对包括免疫细胞在内的许多细胞具有广泛的生理活性,特别是对淋巴细胞、嗜中性白细胞、单核细胞和巨噬细胞有免疫抑制作用。因此,测定人体滑液中腺苷的含量具有重要意义。测定生物样品中的腺苷的方法主要有高效液相色谱法,电渗毛细管电泳法和流动注射法。本实验建立了高效液相色谱一电喷雾质谱联用(HPLC-ESI-MS)方法,用于人体滑液中腺苷浓度的测定。该方法简单、快速、灵敏度高,已成功应用于腺苷含量和风湿性关节炎的关系研究。     

铜绿微囊藻中磷酸腺苷的提取及分析

比较了酸提取、有机溶剂提取、MgSO4水溶液加热提取以及MgSO4水溶液加热超声波提取4种方式对磷酸腺苷(ATP、ADP和AMP)的提取效果,确定以MgSO4水溶液加热超声波提取效果最佳。采用MgSO4加热超声波提取时,2 mL提取液对ATP、ADP和AMP的提取效果较好。将ATP、ADP和AMP的混合标准溶液放于沸水浴中保温时,随着保温时间的延长,对ATP和AMP的影响较大,而对ADP的影响相对较小。实验结果证明,以MgSO4水溶液为提取液,用100℃加热10 min后,在超声波细胞粉碎机中超声破碎10 min的提取效率最高,既简单又无毒。用反相高效液相色谱等强度洗脱分离与紫外检测分析藻细胞中的ATP、ADP和AMP的含量,在较短的时间内(10 min)实现了较好的分离,分析准确而快速,是一较好的定性和定量分析方法。ATP、ADP和AMP的回收率分别为88%~97%、103%~107%和109%~115%,均在80%~120%之间,并且标准偏差和相对标准偏差均小于10%,证实了可以用加热超声波破碎提取,HPLC分析ATP、ADP和AMP的方法来提取和分析藻细胞中的ATP、ADP和AMP。     

超高效液相色谱-串联质谱结合内标法快速检测毛发中15种毒品及代谢物

建立了毛发中15种毒品及其代谢物的超高效液相色谱-串联质谱(UPLC-MS/MS)快速确证与内标定量分析方法.向洗净的毛发样品中加入甲醇研磨提取,研磨液经过滤后,以5 mmol/L NH4 Ac-0.1％甲酸水溶液及甲醇作为流动相,经Phenomenex Kinetex Biphenyl色谱柱(100 mm×3.0 m...     

Simultaneous determination of myocardial creatine phosphate and adenine nucleotides by reversed-phase HPLC

An isocratic HPLC system has been developed which allows for the rapid (single run of 20 min) measurement of creatine phosphate (PCr) and adenine nucleotides (ATP, ADP and AMP) in extracts from freeze-clamped and freeze-dried myocardial tissues. The separation was achieved at room temperature by using a RP18 column and a dual variable wavelength spectrophotometer, set at 210 and 254 nm. The solvent was 30 mM potassium dihydrogen phosphate, 15 mM tetrabutylammonium hydrogen sulfate, pH 6.7, 19% (v/v) acetonitrile. A distinct separation (confirmed with the retention time of standard sample) of these high energy compounds was achieved. Standard curves were linear. In isolated rat hearts the following values were obtained (mumol/g dry wt, mean +/- SEM): ATP 21.5 +/- 1.3, ADP 4.6 +/- 0.2, AMP 1.5 +/- 1.1 and PCr 32.5 +/- 1.3; which are consistent with previously published values for high energy compounds in this tissue.     

Validation of a Fast and Simple HPLC-UV Method for the Quantification of Adenosine Phosphates in Human Bronchial Epithelial Cells

A new HPLC method for the simultaneous quantitative analysis of adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) was developed and validated. ATP, ADP, and AMP were extracted from human bronchial epithelial cells with a rapid extraction procedure and separated with a C18 column (3 × 150 mm, 2.7 µm) using isocratic elution with a mobile phase consisting of 50 mM of potassium hydrogen phosphate (pH 6.80). The absorbance was monitored at 254 nm. The calibration curves were linear in 0.2 to 10 µM, selective, precise, and accurate. This method allowed us to quantify the nucleotides from two cell models: differentiated NHBE primary cells grown at the air–liquid interface (ALI) and BEAS-2B cell line. Our study highlighted the development of a sensitive, simple, and green analytical method that is faster and less expensive than other existing methods to measure ATP, ADP, and AMP and can be carried out on 2D and 3D cell models.     

胶束电动毛细管色谱法同时测定西洋参中人参皂苷Rg1、Re和Rb1

建立了西洋参中人参皂苷Rg1、Re及Rb1同时分离测定的胶束电动毛细管色谱新方法,以解决西洋参样品中难溶于水的3种人参皂苷的准确定量问题。以40.2 cm(有效长度30 cm)×50 μm的熔融石英毛细管柱为分离柱,分离缓冲液的组成为V(15 mmol/L Na2B4O7+30 mmol/L H3BO3 (pH 9.0)+100 mmol/L十二烷基硫酸钠(SDS)+30 g/L聚乙二醇35000):V(甲醇):V(异丙醇)=2:1:1,于214 nm下检测。详细研究了影响分离的因素。Rg1、Re及Rb1检出限(信噪比(S/N)为3)分别为30、40及30 mg/L,定量限(S/N=9)分别为90、120及90 mg/L,加标回收率为87.4%～95.2%。用该法测定了西洋参标准物质,并与高效液相色谱法的检测结果进行了比对,结果吻合。应用该方法分别测定了中国、加拿大及美国的西洋参,获得满意的结果。     

改进的高效液相色谱法测定婴幼儿配方奶粉中5种核苷酸

建立并优化了高效液相色谱检测婴幼儿配方奶粉中5种核苷酸(尿嘧啶核苷酸(UMP)、腺嘌呤核苷酸(AMP)、次黄嘌呤核苷酸(IMP)、鸟嘌呤核苷酸(GMP)、胞嘧啶核苷酸(CMP))的方法。样品用水提取后,经乙酸沉淀蛋白质和HLB固相萃取柱净化,采用Waters XBrigde Amide(150 mm×4.6 mm,3.5μm)色谱柱分离,以乙腈、10 mmol/L磷酸二氢钠溶液和0.12%(v/v)磷酸溶液为流动相进行梯度洗脱,二极管阵列检测器(波长为254nm)检测。结果表明,5种核苷酸检测的线性范围宽,相关性好,相关系数(r2)均为0.999 9;方法的加标回收率为86.9%~105.7%;定量限为5.6~8.0 mg/kg;日内和日间精密度分别为0.5%~1.7%(n=5)和0.6%~1.9%(n=9)。该法前处理简单,分离效果好,回收率高,重复性好,可作为婴幼儿配方奶粉中5种核苷酸的有效检测方法。     

哺乳动物细胞是所得多肽复合酶CAD催化反应产物的 HPLC分析

本研究报道了以150mm×4.6mmI.D.Zorbaxoqcyuosyg.10mmoI·L^-1KH~2PO~4和500mmoI·L^-1LKH~2PO~4水溶液为洗脱液,梯度程序为0～5min(φ=1,体积分数)10mmoI·L^-1KH~2PO~4溶液,5～50min由(φ=1)10mmoI·L^-1KH~2PO~4溶液转换到(φ=1)500mmol·L^-1KH~2PO~4溶液时,高效液相色谱分离了哺乳动物细胞中所得肽复合酶ACD催化反应混合物三磷酸腺苷(ATP)、二磷酸腺苷(ADP)和二氢乳清酸(DHO),确定了ATP,ADP和DHO的出峰顺序,制作了工作曲线,确定了催化反应混合物中ATP,ADP和DHO的相应浓度。     

Intracellular adenosine 5'-triphosphate, adenosine 5'-diphosphate, and adenosine 5'-monophosphate detection by short-end injection capillary electrophoresis using methylcellulose as the effective electroosmostic flow suppressor

We present a new rapid CE method to measure adenine nucleotides adenosine 5'-triphosphate (ATP), adenosine 5'-diphosphate (ADP), and adenosine 5'-monophosphate (AMP) in cells. The short-end injection mode allows a decrease in the analysis time by injecting samples at the outlet end of a silica capillary closest to the detection window, reducing the migration distance. Moreover, the use of methylcellulose (MC) as run buffer additive to suppress EOF permits to further reduce the migration times of analytes. Thus, when a capillary with an effective length of 10.2 cm was used with a 60 mmol/L sodium acetate buffer pH 3.80 in the presence of 0.01% of MC, the migration time of analytes were 1.35 min for ATP, 1.85 min for ADP, and 4.64 min for AMP. These conditions gave a good reproducibility for intra- and interassay (CV <4 and 8%, respectively) and all the procedure demonstrated an excellent analytical recovery (from 98.3 to 99 %). The method suitability was proved both on red blood cells and in spermatozoa. We compared our proposed method to a spectrophotometric assay, by measuring ATP levels in 40 spermatozoa samples. The obtained data were analyzed by the Passing and Bablok regression and Bland-Altman test.     

Determination and locational variations in the quantity of hydroxyanthraquinones and their glycosides in rhizomes of Rheum emodi using high-performance liquid chromatography

Locational variations in the quantity of five hydroxyanthraquinone derivatives (emodin glycoside (1), chrysophanol glycoside (2), emodin (3), chrysophanol (4) and physcion (5)) in the rhizomes of Rheum emodi are described. A simple and reliable method was developed for quantitation of compounds (1-5) in the methanolic extract of rhizomes of R. emodi using reverse-phase high-performance liquid chromatography (HPLC) with photo-diode array detector (PDA). The separation was carried out using a Purospher((R))-Star RP-18 e column (4.6mm i.d.x 250 mm, 5 microm) under the following conditions: acetonitrile:methanol (95:5, v/v) (solvent A) and water:acetic acid (99.9:0.1, v/v) (solvent B) as mobile phase with a linear gradient elution at a flow rate of 0.8 mL/min. The detection wavelength was set at 290 nm. Regression equation revealed a linear relationship (r(2)>0.9901) between the mass of hydroxyanthraquinone derivatives injected and the peak areas. The detection limits (S/N=3) ranged from 0.56 to 3.50 ng/mL and the recoveries ranged from 95.7 to 103.5% for five hydroxyanthraquinone derivatives. Compound 2 was found in maximum quantity (up to 2.23%) in the rhizomes from all the three locations (L(1), L(2) and L(3)) while compound 5 was found in the least quantity (up to 0.19%).     

高效液相色谱-串联质谱法同时测定血液中的15种减肥药物

建立了一种专属、灵敏的同时测定血液中咖啡因、盐酸西布曲明等15种减肥药的高效液相色谱-串联质谱(HPLC-MS/MS)分析方法。样品经乙腈沉淀后,进入HPLC-MS/MS中分析检测。以甲醇和含0.1%(v/v)冰醋酸的20 mmol/L醋酸铵溶液作为流动相,采用梯度洗脱方式,以UltimateXB-C18为色谱柱进行HPLC分析;质谱分析采用电喷雾离子源,正负离子快速切换扫描,选择反应监测模式检测。15种减肥药的定量限在0.001～0.05 mg/L内,各种药物的灵敏度较高,各成分的线性相关系数均大于0.99,精密度均小于12.3%,回收率范围为77.3%～110.8%。研究了这15种药物的质谱特征。该方法灵敏、简便、快捷、专属性强,可用于动物实验样品中减肥药物的含量测定,并且对其他药品、食品中目标减肥药物的测定具有借鉴意义。