高效液相色谱法测定骨骼肌ATP、ADP、AMP、NAD+、NADH含量

高效液相色谱法测定强化奶及食品中维生素D含量

Τηε ∆ ετερ ινατιον οφ ς ιτα ιν ∆ ιν Φορτιφιεδ Μ ιλκ ανδ Οτηερ Φοοδ σ βψ Η ιγ η Περφορ ανχε Λιθυιδ Χηρο ατογ ρα ηψ Η ΠΛΧ
Ι νστιτυτε οφ Η ψγ ιενε ανδ Ε νϖιρον ενταλ Μ εδ ιχινε Α χαδ ε ψ οφ Μ ιλιταρψ Μ εδ ιχαλ Σ χιενχεσ Τ ιανϕιν
各异含量亦相差大分析时应考虑取样量和前
处理程序∀如肉松的脂类含量多用牛奶样品皂化条
件不能将脂类完全皂化但温度升至 ε 并延长
皂化时间可取得较好的分析结果∀
表 Τ αβλε 样品名称
几种食品中维生素 ∆ 的测定结果
Χοντεντ οφ ϖιτα ιν ∆ ιν σεϖεραλ φοοδσ 含量
作为标准定量分析样品中的的∀ 测得值可
能是单一的或
亦可能是两者混合物∀ 样
品分离图谱中保留时间为
的峰与标准品
的保留时间一致图 ∀ 样品加标进样
Ξ 本文收稿日期
修回日期

色

峰值升高未见新峰出现由此确认

峰为 ∀ 另外还分析了未强化的鲜
牛奶峰消失进一步证实
峰是 ∀
烷溶解移入试管氮气吹干加入正相色谱流动相
Λ 溶解备用∀ 样品净化色谱条件
结果与讨论
样品净化
食品中含量远低于干扰物定量前需净化
富集∀ 本法用正相
净化样品以方法中简述
的色谱条件将标液进样测得的保留时间为

∀注入试样 Λ 收集保留时间为 ∗

高效液相色谱法测定人血浆中连翘苷含量

高效液相色谱法测定人血浆中连翘苷含量【摘要】目的建立高效液相色谱法，用于测定人血浆中连翘苷的质量浓度。

方法采用内标法，以ThermoC18柱（4.6mm×250mm，5μm）为固定相，以乙腈-水-冰醋酸(17：83：0.4 ) 为流动相，流速为1mL／min，检测波长为277nm。

结果血浆中连翘苷质量浓度在0.10～4.00μg／ml范围内与峰面积比值线性关系良好(R2= 0.999) ，最低检测浓度为0.05μg／ml，回收率达90％以上。

结论该方法具有较高的准确度，线性范围宽，方法灵敏，专一性好，操作简便，适用于连翘苷血药浓度的测定及临床药代动力学研究。

【关键词】高效液相色谱法；连翘苷；血药浓度HPLC determination Of Phillyrin in Human PlasmaQIN Ying-pu1，MO Wu-ming21.Guangxi Wuzhou People’s Hospital, Wuzhou 543000, China;2.Guangxi Wuzhou Sanjian Pharmaceutical Co., Ltd，Wuzhou 543001, China;【Abstract】Objective To determine the concentration of Phillyrin in human plasma by HPLC with ultraviolet detetion．Methods The internal standard method was used．The evaluation of Phillyrin was car ried out by ThermoC18 colum (4.6mm×250mm，5μm)w ith the mobile phase ofAcetonitrile - water - acetic acid (17：83：0.4 ) and the detection wavelength was 277nm．The flowrate was 1 mL／min．Results The calibration curve for plasma Phillyrin in range of 0.10～4.00μg／ml (R2= 0.999)．The detection limit of Phillyr in was 0.05μg／ml．The recovery was more than 90%．Conclusion This method is accurate，sensitive，specific and convenient，which could be used in the determination of plasma Phillyrin and its pharmacokinetic study in clinic．【Key words】HPLC；Phillyrin；blood drug concentration连翘，又名旱莲子(《药性论》)、大翘子(《新修本草》) ，为木犀科植物连翘Forsythia suspense(ThunbVah1)的干燥果实。

超高效液相色谱-质谱法测定细胞中嘌呤核苷酸的方法探究

超高效液相色谱-质谱法测定细胞中嘌呤核苷酸的方法探究姜丹丹;李伟;周怀彬;张婷;许芳;武佳;吕建新【摘要】建立了检测细胞中嘌呤核苷酸(ATP、ADP、AMP、NAD+和NADP+)的超高效液相色谱-质谱(UP-LC-MS)分析方法.采用KinetexTM HILIC柱(50 mm ×4.6 mm,2.1 μm)进行分离,对细胞样品的萃取方法、流动相组成及质谱参数进行优化.方法学确证表明:各待测物在1.0 ～ 100 μmol/L范围内线性关系良好,相关系数(r)均大于0.99,平均回收率为95.7％～101.1％,相对标准偏差为1.2％～6.1％.本方法的灵敏度高、简便快速、重复性好,可用于细胞中能量代谢的研究.【期刊名称】《分析测试学报》【年(卷),期】2013(032)010【总页数】5页(P1202-1206)【关键词】超高效液相色谱-质谱法;细胞;ATP;ADP;AMP;NAD+;NADP+【作者】姜丹丹;李伟;周怀彬;张婷;许芳;武佳;吕建新【作者单位】温州医学院检验医学院&生命科学学院检验医学教育部重点实验室,浙江温州325035;温州医学院检验医学院&生命科学学院检验医学教育部重点实验室,浙江温州325035;温州医学院检验医学院&生命科学学院检验医学教育部重点实验室,浙江温州325035;温州医学院检验医学院&生命科学学院检验医学教育部重点实验室,浙江温州325035;温州医学院检验医学院&生命科学学院检验医学教育部重点实验室,浙江温州325035;温州医学院检验医学院&生命科学学院检验医学教育部重点实验室,浙江温州325035;温州医学院检验医学院&生命科学学院检验医学教育部重点实验室,浙江温州325035【正文语种】中文【中图分类】O657.63;Q524嘌呤核苷酸是机体重要的能量物质。

其中三磷酸腺苷酸(ATP)作为细胞内能量传递的“分子通货”，是机体能量的直接来源，二磷酸腺苷酸(ADP)和一磷酸腺苷酸(AMP)可通过氧化磷酸化等转化为ATP供能，生物体中ATP－ADP－AMP系统的能量状态可通过能荷(EC)大小说明。

高效液相色谱法测定5′-三磷酸腺苷含量

高效液相色谱法测定5′-三磷酸腺苷含量张爱雷;王岚【摘要】采用高效液相色谱法测定5′-三磷酸腺苷含量.色谱条件为:XTerraTMRP18(3.9 mm×150 mm,5 μm) 色谱柱,流动相为0.05 mol·L-1 pH 值7.0 的NH4H2PO4缓冲溶液,检测波长259 nm,流速1.0 mL·min-1,柱温(25±2)℃,进样量20 μL.在10～80 mg·L-1范围内,ATP浓度与其259 nm处吸光度值呈良好线性关系,R＝0.9994;检测重复性较好,平均回收率为98.87%,整个色谱过程可在7 min内完成.该方法也可用于ADP和AMP的分离检测.【期刊名称】《化学与生物工程》【年(卷),期】2010(027)010【总页数】3页(P89-91)【关键词】三磷酸腺苷;一磷酸腺苷;二磷酸腺苷;含量;检测;高效液相色谱【作者】张爱雷;王岚【作者单位】江苏省产品质量监督检验研究院,江苏,南京,210007;常州大学化学化工学院,江苏,常州,213164【正文语种】中文【中图分类】O657.75′-三磷酸腺苷 (Adenosine triphosphate,ATP)又名腺三磷、腺嘌呤配糖三磷酸，临床应用广泛，是用于治疗进行性肌萎缩、脑溢血后遗症、心机能不全、心肌疾患及肝炎等疾病的辅酶类药物，被称为人体内的“能量货币”[1]。

在ATP的制备、存储过程中，易混入或产生一磷酸腺苷(AMP)、二磷酸腺苷(ADP)等杂质。

ATP、ADP及AMP的结构相似、理化性质相近，因此检测较为困难[2]。

我国部标和地方标准均采用纸电泳法[3]或纸层析法[4]测定ATP含量，存在操作时间长、误差较大、操作繁琐的缺点。

也有报道采用生物发光法测定ATP含量，但该方法不能对杂质进行分析[5]。

作者采用高效液相色谱法测定ATP含量，结果令人满意。

1 实验1.1 试剂和仪器对照品：ATP二钠(Sigma)，ADP (中国医药上海化学试剂公司)，AMP(中科院上海生化所东风生化技术公司)；样品：ATP二钠(批号：200209070,上海太平洋制药厂)；NH3·H2O、磷酸二氢铵，分析纯。

高效液相色谱法测定细胞内三磷酸腺苷及其代谢物的含量

高效液相色谱法测定细胞内三磷酸腺苷及其代谢物的含量2017年1月January 2017Chinese Journal of Chromatography Special issue for commemorating Professor Z O U Hanfa ( H )? A rticle Vol.35 No.154?58 D O I: 10.3724/SP.J. 1123.2016.08031Quantification of intracellular adenosine 5'-triphosphateand its metabolites by high performance liquidchromatography analysisZHU Huiyu, WU Danni, WANG Hailin**(S tate K ey Laboratory o f E n viro n m en ta l C hem istry and E cotoxicology, Research Center fo rE co-E nvironm ental S c ie n ce s, C hinese A ca d em y o f S c ie n ce s, B e ijin g, 100085, C hina)Abstract：This study was aimed to provide insight regarding the intracellular metabolites of adenosine 5'- triphosphate (A TP) and whether 2-tert-butyl- 1,4-benzoquinone (TBBQ) affects cell metabolites. A rapid high performance liquid chromatography (H PLC) protocol was developed for the separation and quantitation of A T P and its metabolites (adenosine diphosphate (A D P) and adenosine monophosphate (AMP)) in cells. Chromato-graphic separation was performed using a Shim adzu H PL C system equipped with an Agela Venusil M P C18 column; isocratic elution was adopted. The mobile phase comprised solvent A (50 mmol/L disodium hydrogen phosphate and 15 mmol/L trimethylamine (TEA); pH adjusted to 7. 88 using acetic acid (HAc)) and solvent B (methanol). The correlation coefficients of the three analytes were very high (R2^0. 999 6), and the contents of the three metabolites in the M R C-5 cells were within the linear ranges (0.1-100 pmol/L). The lim its of detection for the detected three compounds were low. Sam ples were extracted from cells (after exposure and non-exposure to quinones) using 80%(v/v) methanol aqueous solution. The method developed in this study was successfully applied to detect ATP, A D P and A M P in M R C-5 cells, and the results demonstrated that A TP, ADP, A M P levels in cells were affected by TBBQ, but the relations between the concentration of T B B Q and the level of ATP, A D P and A M P were com plex.Key words:high performance liquid chromatography (HPLC); adenosine 5’-triphosphate (ATP); quinones;metabolites; intracellularCLC number：O658 Document code：A Article IC：1000-8713(2017)01-0054-05高效液相色谱法测定细胞内三磷酸腺苷及其代谢物的含量朱会宇，吴丹妮，汪海林*(中国科学院生态环境研究中心，环境化学与生态毒理学国家重点实验室，北京100085)摘要:研究了三磷酸腺苷(ATP)及其代谢物在细胞内的含量以及2-叔丁基-1,4-苯醌（TBBQ)对ATP及其代谢产物在细胞内含量的影响。

高效液相色谱法测定龙眼果实中ATP、ADP、AMP的含量

高效液相色谱法测定龙眼果实中ATP、ADP、AMP的含量杨子琴;王惠聪;付欣雨;李建国;黄旭明【摘要】摸索了一种基于高效液相色谱仪快速测定龙眼果实腺苷三磷酸(ATP)、腺苷二磷酸(ADP)、腺苷一磷酸(AMP)含量的方法.该方法利用磷酸盐缓冲液(pH7.0)作为单一流动相,流速设定为1 mL/min,停止时间13 min,检测波长254 nm,无参比波长.ATP、ADP和AMP浓度在5～50 mg/L范围内与相应的色谱峰面积呈完美的直线关系(决定系数R2均为1.000),回收率95.2％～97.5％,保留时间分别为8.651、9.844和11.457 min.该方法测定结果表明,果实腺苷含量水平分别为30.0、6.5和2.0μg/g,能荷值为0.86.%A rapid method using high performance liquid chromatography was explored to determine the levels of adenosine triphosphate,adenosine diphosphate,adenosine monophosphate in longan fruit.The method used 0.1 mol/L KH2PO4 (pH7.0) as the single mobile phase with a flow rate of 1 mL/min,stop time 13 min,the detection wavelength 254 nm,without reference wavelength.The retention time for ATP,ADP and AMP was 8.651,9.844 and 11.457 min,respectively.Within 0～50 mg/L,ATP,ADP and AMP concentrations all displayed perfect linear correlation (R2=1.000) with their peak areas.The recovery rate reached95.2％～97.5％.This method was applied to determine levels of adenosine phosphates in young longan fruit,and the results showed that the average contents of ATP,ADP and AMP were 30.0,6.5 and 2.0 μg/g,respectively,with an energy charge of 0.86 in young fruit.【期刊名称】《热带作物学报》【年(卷),期】2013(034)007【总页数】3页(P1381-1383)【关键词】高效液相色谱;龙眼;腺苷三磷酸;腺苷二磷酸;腺苷一磷酸【作者】杨子琴;王惠聪;付欣雨;李建国;黄旭明【作者单位】华南农业大学园艺学院,广东广州 510642;中国热带农业科学院热带作物品种资源研究所,海南儋州 571737;华南农业大学园艺学院,广东广州 510642;华南农业大学园艺学院,广东广州 510642;华南农业大学园艺学院,广东广州510642;华南农业大学园艺学院,广东广州 510642【正文语种】中文【中图分类】S667.2细胞能量代谢是维持植物组织中生理生化活动的基础，细胞的生物合成、物质转运、跨膜电位维持、膜脂质的降解及恢复等均依赖于能量代谢[1]。

高效液相色谱法测定保健品中维生素D 含量

分析与检测
高效液相色谱法测定保健品中维生素 D 含量
□ 罗骁吴帅龙李彩玲广电计量检测（合肥）有限公司
摘要：建立了一种使用液相色谱 - 紫外检测器，以维生素 D2、D3 互为内标，测定保健食品中维生素 D 的方法。样品经淀粉酶水解，无水乙醇 - 氢氧化钾皂化，皂化液用石油醚萃取、水洗、干燥，旋转蒸发至约 2 mL，氮吹至干，甲醇溶解定容，内标法定量。维生素 D2 和维生素 D3 的分离度为 1.5，线性范围为 0.01 ～ 5.0 μg/mL，相关系数 R2 ≥ 0.999；相对标准偏差 RSD（n=6）为 2.82% ～ 5.10%；加标回收率 96.23% ～ 104.26%。当取样量为 5.000 g 时，VD2、VD3 的检出限均低于 0.2 μg/100 g。
关键词：维生素 D；保健食品；液相色谱（HPLC）；内标法
维生素 D3 与维生素 D2 为脂溶性维生素，主要功用是调节细胞生长分化和人体免疫功能，有利于新骨生成和钙化，促进骨骼生长 [1]，预防骨质疏松症。但维生素 D 摄入过多，可引起高血钙、甚至软组织异位骨化等 [2]。
Toledo），高效液相色谱仪（LC-20A， 5009.82-2016）第四法，准确称量某
维生素 D2 D3
线性范围 μg/mL 0.01 ～ 5.0 0.01 ～ 5.0
表 1 线性方程、相关系数（r2）及定量下限
线性方程
相关系数，r2
检出限 μg/100 g
C=1.0575A-0.0552
0.9999
0.1954
C=1.3888A-0.4966
0.9996
0.1723
Hale Waihona Puke 定量限 μg/100 g 0.6513 0.5743

高效液相色谱法测定药品的维生素A含量

高效液相色谱法测定药品的维生素A含量摘要：目的分析高效液相色谱法（HPLC）测定维生素AD滴剂中维生素A含量的效果。

方法 HPLC测定维生素AD滴剂中维生素A含量的色谱条件：分析柱为Kromasil 100-5C18（150mm×43mm，5µm）；流动相为甲醇：酒精（80：20）；流速为0.5mL/min；柱温30℃，检测器在270nm波长处检测维生素A。

结果维生素AD滴剂中维生素A分离良好，回归方程为A=775.98C+12.97（r=0.9997），线性范围为1.21-2.84mg/mL。

维生素A的平均回收率为99.94%，RSD为1.28%。

维生素A样品在6h内稳定性较好。

维生素A含量为（0.3969±0.05）mg/mL。

结论用HPLC法测定维生素AD滴剂中维生素A的含量方法简便、速度快、灵敏度和精确度高、重复性和稳定性好，值得推广应用。

关键词：维生素A；高效液相色谱法维生素A具有多种生理功能[1]：调节生长发育、提高免疫力、保护视力等，缺乏维生素A会导致发育迟缓、易感性提高、视力下降等疾病。

维生素AD滴剂是临床补充维生素A的常用方法，准确检测其中的维生素A含量对于把握药物的质量和疗效至关重要。

临床测定维生素常用的一种方法是高效液相色谱法（HPLC）[2]。

本研究采用HPLC法测定药品（维生素AD滴剂）中维生素A含量，效果颇佳，现将结果报道如下。

1 资料与方法1.1 仪器及试剂高效液相色谱仪（美国赛默飞公司）、紫外检测器（南京普阳科学仪器研究所）；离心机（德国奥豪斯公司）；旋涡器（美国SCI公司）；维生素A标准品（北京神州科创化工技术研究所）；乙醇（分析纯）；甲醇（色谱纯）；正己烷（分析纯）；高纯氮气。

1.2 色谱条件分析柱：Kromasil 100-5C18（150mm×43mm，5µm），柱温30℃。

检测波长270nm；流动相：甲醇：酒精（80：20）；流速0.5mL/min；进样量20µL。

肌肉中肌苷酸的高效液相色谱测定法

定。ｔａ２用离子对色谱法Ｋｉｄ等〔ａ〕测定。本文采用
反相色谱法对大白鼠和小白鼠腿肌中的肌苷酸用外
标法进行定量测定。
实验
部分
（一）仪器和试剂色谱仪：日本岛津ＬＣ４，ＳＤ－紫外分光检测器，ＣＲ２ＡＰ１－Ａ数据微处理
机。谱Ｈｐｒｌ， μ ，５× ．ｍ色柱：ｙｓＣ８５ｍ１４ｍ，ｅｉ１０６
ｄｔｍｎｔｎＣ２，ｅ＋Ｎ２，ｎ＋Ｃ２ａｄｅｒｉｉｏｕ＋Ｆ３，ｉＺ２，ｏ＋ｎｅａｏｆ＋，
Ｐ２ｂｉｃｍｔｒｈ．ｍｘｕｅｍｏｂ＋ｙｈｏａｇｐｏｎｏａｉｒｏ６ｍｌｔｆ
ｃｒａｉ＋３ｍｌｌａｉ＋０ｍ１ｔｉｉｃｔｃｄｍｏｏａｃｄ３ｍｏａｅｃｘｉｃｃｉ
取腿肌剪碎放入试管，管口用塑料布包扎紧，在水
浴上煮沸１，冷却后离心１ｍｎ上清液用氯仿－ｈ０ｉ，苯酚饱和溶液去一次蛋白，再用氯仿反复去蛋白，直
至氯仿与水的界面间无蛋白为止。
（Ｉ标准溶液的三）ＭＰ回收用外标法测定Ｉ、Ａ、Ｎ混合溶液中ＭＰＭＰＩＯＴ的ＭＰ浓度，如表１所列，回收率良好。
表１
标准ＩＰＭ溶液的回收
结果和讨论（一）分离条件的选择用４ｍｍｏＬ０ｌ磷酸盐／缓冲溶液作流动相，调节不同的ＰＨ值，在Ｃ８１柱上进行分离。在Ｐ０— ．Ｈ６７范围内，Ｉ５ＭＰ腺苷单磷
酸（ＭＰ和肌苷ＩＯ都Ａ）Ｎ）能很好分离，而在ｐＨ
ｗｈａｐｓｅ５ａｄ／ｉｄｔｔ．ｉａｌｉｏ０ｌＵＶｓｅｏｔｓｅｆｎＶｍｚｅｃｒｎｅｅｏｒｇｔ４１４ｏＬ４（－ｙｉｄｔｔｎｅｗｓ０ｍｌ－２Ｐｒｅｃｉｅｎａ × －／ａｉｌｏｅｒｎｌＲｉ３ｏＬｍｎｍｙａ）ｒｅｉ（Ａ）ｍ１ａｏｉｚｓｃｏＰｎ／ｍｕｈｄｏｉｅｌｏＬｅｃｄＴｉｍｔｏｙｒｘｄａｄｌａｔａｉ．ｈｎｍ／ｃｉｃｓｅｄｈｗｓｌｄｔａａｙｓｈｍｎｉａａｐｉｔｈｎｌｉｏｕａｈｒｐｅｏｅｓｆａ．

辅酶 NAD(H)Ⅰ含量试剂盒使用说明

辅酶NAD(H)Ⅰ含量试剂盒使用说明产品简介：NAD是糖酵解和TCA循环的主要氢受体，生成的NADH经呼吸电子链传递把电子交给氧，在合成ATP的同时，形成大量的ROS，同时NADH再生为NAD。

糖、脂、蛋白质三大代谢物质分解中的氧化反应绝大部分通过这一体系完成。

NAD(H)含量和NADH/NAD比值的高低可用于评价糖酵解和TCA循环的强弱。

较高的NAD(H)及NADH/NAD比值说明细胞呼吸耗氧量较高，处于过氧化状态，NADH/NAD 比值升高也可抑制糖酵解和TCA循环。

另外，NAD降解产物具有非常重要的调控作用。

NAD和NADH在254nm下有吸收峰，利用高效液相色谱法在相应波长下测定其含量。

试验中所需的仪器和试剂：高效液相色谱仪、低速离心机、溶剂抽滤装置、针头式过滤器（水系，50个，0.22µm）、滤膜（水系和有机系各2个，0.45µm）、C18柱（4.6×150mm）、可调式移液器、样品瓶（50个，2mL）、乙腈（120mL）、甲醇（色谱级，300mL）、蒸馏水注：若用自动进样器，需要样品瓶，测定时将不少于1mL的样品或标准品加入样品瓶中。

无自动进样器，需手动进样时，不需要样品瓶，用进样针将不少于20ul的样品或标准品打入进样阀。

产品内容：试剂一：粉剂1×1瓶，粉剂2×1瓶，4℃保存；临用前用少量蒸馏水将粉剂1和粉剂2溶解后倒入容量瓶中，用蒸馏水定容至100mL，形成NAD和NADH提取液（注：试剂瓶中的粉剂要冲洗干净），4℃保存3个月；试剂二：粉剂1×1瓶，粉剂2×1瓶，粉剂3×1瓶，4℃保存；临用前用少量蒸馏水将粉剂1、粉剂2和粉剂3溶解后倒入容量瓶中，用蒸馏水定容至1000mL，形成流动相缓冲液基质（注：试剂瓶中的粉剂要冲洗干净）,4℃保存3个月。

试剂三：NAD标准品5mg×1支，-20℃保存。

高效液相色谱法HPLC测定植物组织中ATP,ADP,AMP含量

ISSN1330-9862scientific note (FTB-1485)A Simple and Rapid Determination of ATP,ADP and AMPConcentrations in Pericarp Tissue of Litchi Fruit by HighPerformance Liquid ChromatographyHai Liu1,3,Yueming Jiang1*,Yunbo Luo2and Weibo Jiang21South China Botanic Garden,The Chinese Academy of Science,510650Guangzhou Leyiju,PR China2College of Food Science and Nutritional Engineering,China Agricultural University,Qinghuadonglu,100083Beijing,PR China3Graduate School of the Chinese Academy of Science,100039Beijing,PR ChinaReceived:February25,2005Accepted:May25,2005SummaryA simple and rapid method using high performance liquid chromatography(HPLC)was developed to determine levels of adenosine triphosphate(ATP),adenosine diphos-phate(ADP)and adenosine monophosphate(AMP)in litchi fruit pericarp tissue.This HPLCmethod used acetonitrile gradient elution and shortened the time required for determina-tions of adenosine phosphates.This analysis exhibited good repeatability(coefficients ofvariation1.28–1.80%)and recovery rate(94.7–97.1%).The correlation coefficients of ATP,ADP and AMP with their peak areas at a range of0–80ng were0.9946,0.9994and0.9974,respectively.This method was applied to determine levels of adenosine phosphates in pe-ricarp tissue of litchi fruit at harvest.There were27.4m g/g of ATP,35.4m g/g of ADP and7.9m g/g of AMP on a fresh mass basis.Key words:HPLC,ATP,ADP,AMP,determination,litchi fruit,pericarp tissueIntroductionEnergy metabolism is an important metabolic path-way which maintains biochemical and physiological ac-tivities in plant tissues(1).Energy charge can be used to show energy status in plant tissues(1,2),while synthe-sis,degradation and restoration of membrane lipids de-pend on energy supply(3–5).The energy charge of cells can be calculated as([ATP]+0.5[ADP])/([ATP]+[ADP] +[AMP]),as described by Pradet and Raymond(1).Some of key enzymes concerning glycolysis,the Krebs’cycle, the electron transport system and oxidative phosphory-lation are also regulated by the energy level of cells(6,7). Lipids are the essential components of plant cell mem-branes,and involvement of adenylate nucleotides in fatty acid biosynthesis in membrane lipid is well estab-lished(5,8).Thus,energy status of tissues of fruits and vegetables may play an important role in maintaining the integrity of cell membranes,which is related to post-harvest life(7,9).The luciferase system was mainly used to determine ATP concentration in organisms,while the analysis of ATP and ADP was performed by enzyme-linked immu-nosorbent assay(10–12).A high performance liquid chro-matography(HPLC)analysis of ATP and its degrada-tion products was also developed on reverse-phase col-umns using phosphate buffer as a mobile phase(13,14). Organic solvents,such as methanol(15,16)or acetonitrile (14),can be used to reduce the running time.In practice, these methods need a long running time and it is neces-sary to develop a simple and rapid method to analyze a large number of fruit samples(14,17,18).*Corresponding author;Phone:++862037252905;Fax:++862037252831;E-mail:ymjiang@There are no reports on the determinations of ATP, ADP and AMP contents by HPLC in plant tissue.The objective of this study was to develop a simple and ra-pid HPLC method for measurements of ATP,ADP and AMP content in pericarp tissue of litchi fruit. Materials and MethodsReagentsThe high purity ATP,ADP and AMP standards,per-chloric acid,potassium dihydrogen phosphate,potassi-um hydrogen phosphate and acetonitrile(HPLC grade) were purchased from Sigma Chemical Co.All reagents used were dissolved in deionized water,and then filte-red with the Sybron/Barnstead Millipore system and a 0.45-m m filter paper.Plant materialsFruit of litchi(Litchi chinensis Sonn.cv.Huaizhi)at the commercially mature stage was harvested from an orchard in Guangzhou.Litchi pericarp was collected for extraction and determinations of ATP,ADP and AMP. Preparations of standard stock soluti onsATP,ADP and AMP standards(1mg)were each dissolved in10mL of deionized water to obtain ATP, ADP and AMP standard stock solutions at100m g/mL. Aliquots of the stock standard solutions were made by diluting them in deionized water at0,0.2,0.5,1,2and4 m g/mL.A volume of20m L of each sample was taken for HPLC analysis.Mobile phasesMobile phase A consisted of0.06mol/L dipotassi-um hydrogen phosphate and0.04mol/L potassium di-hydrogen phosphate dissolved in deionized water and adjusted to pH=7.0with0.1mol/L potassium hydrox-ide,while mobile phase B consisted of100%acetoni-trile.Air bubbles in the two solutions were driven away using an ultrasonic instrument.Extraction of ATP,ADP and AMP from litchifruit pericarp tissueLitchi fruit pericarp tissue(2g)was rapidly frozen in liquid nitrogen and homogenized into powder.Adeno-sine phosphates were extracted from the powder with 10mL of0.6mol/L perchloric acid in the ice bath for1 min by the method of Yang et al.(12).The extraction mixture was centrifuged for10min at6000´g(Beckman J20-2)and4°C,and6mL of the supernatant was taken and quickly neutralized to pH=6.5–6.8with1mol/L KOH solution.The neutralized supernatant was then allowed to stand for30min in an ice bath to precipitate most of the potassium perchlorate,which was removed by paper filtration.The filtrate solution was filtered again through a0.45-m m filter.The final filtrate solution was made up to8mL and then stored at–30°C prior to the analysis. HPLC analysisThe HPLC(Gold125Solvent System,Beckman In-struments Inc.,USA)conditions were as follows:an Ultrasphere ODS EC250´4.60mm column(Beckman In-struments Inc.,USA)was equipped with a Beckman125 pump system.Peaks were detected and analyzed at254 nm by a Gold168diode array detector.HPLC separa-tion was achieved using continuous gradient elution. The elution program was as follows:0min100%A,0% B;2min95%A,5%B;4min80%A,20%B;5.3min 75%A,25%B and6min100%A,0%B.Finally,the program took a further1min to return to the initial con-ditions and stabilize.Flow rate of the mobile phase was 1.2mL/min,while the injection volume was20m L.The total retention time was about5min and the gradient was run for6min to ensure full separation.ATP,ADP and AMP in the samples were identified by comparison with retention time of standards,while the concentra-tions of ATP,ADP and AMP were determined using the external standard method.Data were expressed as means of six replicate determinations.Recovery trialThree standards(ATP,ADP and AMP)were added into litchi fruit pericarp tissue.The extraction was car-ried out according to the above-mentioned method.The concentration of each standard added for the trial was2 m g/mL.The whole experiment was repeated six times. Statistical analysisQuantitative data from the HPLC analysis were compared using either the coefficients of variation(CV) or analysis of variance(ANOVA).Least significant dif-ference was used to compare the means.Results and DiscussionHPLC analysis and calibration curves of ATP,ADP and AMPATP and its breakdown products exhibited a great absorbance at254nm(13).As shown in Fig.1,ATP, ADP and AMP were separated well and detected at254 nm in this study.Acetonitrile instead of methanol used as mobile phase gave a rapid and better separation of ATP,ADP and AMP(data not shown)because it has higher polarity than methanol.Ryder(13)obtained a012345670.0000.0010.0020.0030.0040.005ATPADPAMPA254nmRetention time/minFig.1.HPLC chromatogram of a standard mixture of ATP,ADP and AMPsufficient resolution of adenosine phosphates within 16min using an isocratic system.Veciana-Nogues et al.(16)reported a good separation of adenosine phosphates us-ing 30%methanol gradient conditions.In this study,the separation and determination of ATP ,ADP and AMP needed only 6min and,thus,it is very convenient to an-alyze a large number of samples.There was a good linear relationship among ATP ,ADP and AMP concentrations at a range of 0–80ng against their peak areas,with correlation coefficients being 0.9946,0.9994and 0.9974,respectively,at the 5%ing 2m g/mL standards,the CV of the levels of ATP ,ADP and AMP were 1.80,1.28and 1.30%,respectively,all below 5.00%(Table 1),which indicated excellent re-peatability for the analysis of the three compounds.Recovery trialHigh precision was important to determine the con-centrations of ATP ,ADP and AMP (14,19).The results indicated that the mean recovery rates of ATP ,ADP and AMP were 94.9,94.7and 97.1%,respectively .These were very high recovery rates bearing in mind the complexity of the analyses.The CV of ATP ,ADP and AMP were 4.3,3.5and 1.4%,respectively,all below 5.0%,exhibiting good precision.Analyses of ATP ,ADP and AMP of litchi fruit pericarp tissueThis HPLC method was applied to determine the concentrations of ATP ,ADP and AMP of pericarp tissue in litchi fruit at harvest.Fig.2shows the chromatograms of ATP ,ADP and AMP extracted from the litchi pericarp tissue.There were 27.4m g/g of ATP ,35.4m g/g of ADPand 7.9m g/g of AMP on a fresh mass (FM)basis.In this study,2g of fresh litchi fruit pericarp tissue was enough to determine the concentrations of ATP ,ADP and AMP .Furthermore,the authors used this method to analyze the concentrations of ATP ,ADP and AMP of banana and longan fruits,and found 10.9m g/g FM of ATP,12.2m g/g FM of ADP and 12.8m g/g FM of AMP in banana fruit peel and 6.6m g/g FM of ATP ,8.1m g/g FM of ADP and 7.1m g/g FM of AMP in longan fruit skin.In this study,the HPLC method could not separate the peaks eluting between 2and 3min,whose ultravio-let absorption spectrum was at 254nm (Fig.2).Özogul et al.(14)reported that the unknown compounds in her-ring tissue could be the degradation products of ATP ,such as inosine monophospate and hypoxanthine.How-ever,their identification in litchi fruit pericarp tissue needs further investigation.ConclusionIn conclusion,an HPLC analysis of ATP ,ADP and AMP concentrations in litchi fruit pericarp tissue was simple and rapid to use.We suggest that the improved method for identification and quantification of ATP,ADP and AMP should be a valuable tool in analyzing a large number of fruit samples.AcknowledgementsFinancial support by the National Natural Science Foundation of China (No.30425040and 30430490)and the International Foundation for Science (No.E2265-3)is highly appreciated.References1. A.Pradet,P .Raymond,Adenine nucleotide ratios and adenylate energy charge in energy metabolism,Annu.Rev.Plant Physiol.34(1983)199–224.2.R.J.Romani,S.Ozelkok,«Survival«of mitochondria in vi-tro :Physical and energy parameters,Plant Physiol.51(1973)702–707.3.K.C.Eastwell,P .K.Stumpf,Regulation of plant acetyl-CoA carboxylase by adenylate nucleotides,Plant Physiol.72(1983)50–55.4.J.L.Harwood,Fatty acid metabolism,Annu.Rev.Plant Physiol.Plant Mol.Biol.39(1988)101–138.5.J.Ohlrogge,J.Browse,Lipid biosynthesis,Plant Cell,7(1995)957–970.6.V .N.Luzikov,Stabilization of the enzymic systems of the inner mitochondrial membrane and related problems,Sub-cell.Biochem.2(1973)1–31.7. A.A.Saquet,J.Streif,F.Bangerth,Energy metabolism and membrane lipid alterations in relation to brown heart de-velopment in ’Conference’pears during delayed controlled atmosphere storage,Postharvest Biol.Technol.30(2003)123–132.8. D.J.Brown,H.Beevers,Fatty acid of rice coleoptiles in air and anoxia,Plant Physiol.84(1977)555–559.9. A.A.Saquet,J.Streif,F.Bangerth,On the involvement of adenine nucleotides in the development of brown heart in ’Conference’pears during delayed controlled atmosphere storage,Gartenbauwissenschaft,66(2001)140–144.10.Y.Sugawara,M.Takeuchi,A simple and rapid method fordetermining cell survival in the cryopreserved shoot apexTable parative HPLC analyses of ATP ,ADP and AMPCompoundMeans a CV/%ATP 0.6111 1.80ADP 0.7667 1.28AMP0.86631.30aData were expressed as peak areas (mm 2)(N =6)Retention time/min012345670.0000.0050.0100.0150.0200.0250.030ATPADPAMPA 254n mFig.2.HPLC chromatogram of ATP ,ADP and AMP from litchi fruit pericarp tissueusing luciferin-luciferase ATP assay,Plant Sci.130(1997) 107–112.11. B.Naslund,L.Stahle,A.Lundin,B.Anderstam,P.Arner,J.Bergstrom,Luminometric single step urea assay using ATP-hydrolyzing urease,Clin.Chem.44(1998)1964–1973.12.N.C.Yang,W.M.Ho,Y.H.Chen,M.L.Hu,A convenientone-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP,Anal.Biochem.306 (2002)323–327.13.J.M.Ryder,Determination of adenosine triphosphate andits breakdown products in fish muscle by high-performance liquid chromatography,J.Agric.Food Chem.33(1985)678–680.14. F.Özogul,K.D.A.Taylor,P.C.Quantick,Y.Özogul,A ra-pid HPLC-determination of ATP-related compounds and its application to herring stored under modified atmo-sphere,Int.J.Food Sci.Technol.35(2000)549–554.15. F.S.Anderson,R.C.Murphy,Isocratic separation of somepurine nucleotide,nucleoside and metabolites from biolo-gical extract by high-performance liquid chromatography, J.Chromatogr.121(1976)251–262.16.M.T.Veciana-Nogues,M.Izquierdo-Pulido,M.C.Vidal-Ca-rou,Determination of ATP related compounds in fish and canned tuna fish by HPLC,Food Chem.59(1997)467–472.17.G.Manfredi,L.C.Yang,C.D.Gajewski,M.Mattiazzi,Mea-surements of ATP in mammalian cells,Methods,26(2002) 317–326.18.J.Murray,A.B.Thomson,Reverse phase ion pair separa-tion of nucleotides and related products in fish muscle,J.High unn.6(1983)209–210.19.K.Samizo,R.Ishikawa,A.Nakamura,K.Kohama,A high-ly sensitive method for measurement of myosin ATPase activity by reversed-phase high-performance liquid chro-matography,Anal.Biochem.293(2001)212–215.。

高效液相色谱法测定肌肉组织中的磷酸肌酸

ＤｔｍｎｔｎＰｏｐｏｒａｎｉＭｕｃｌＴｓｅｂｅｒｉａｉｏｈｓｈｃｅｔｅｓｕａｉｕｓｅｏｆｉｎｒｓｙ
ＨｇＰｒｒａｃＬｑｉＣｒｍｔｇｐｙｉｅｆｍｎｅｕｈｏａｒｈｈｏｉｄｏａ
ＺＡＨＮＧａ－ａ，ＯＮＧｎＤＩＬ－ＹｎｗｎＬＣ，ＮＧｌｕｕｕ（ｕａＨｓｉｌＣｒｉｏｙＣｈｅＡａｅｙｄｃＳｉｃａｄＦｗｉｐｔｆｒｄｏｇ，ｉｓｃｍｏＭｅｉｌｎｓｏａｏａｌｎｅｄｆａｃｅｎｅＰｋｇｉＭｅｉｌｌｅＢｉｎ１０３，ｈｎ）ｅｉＵｏｄａＣｌｇ，ｅｉ００７ＣｉｎｎｎｃｏｅｊｇａＡｓａｔＴｅｎｉｔｎｎｒｓｒ（ｈｓｏｒｔｅＰｒｉｏｐｒｏｎｉｐｒｎｅｈｓｄｏｂｔｃ：ｈｑａｔａｏｏｅｅｙｅｐｏｈｃａｎ，Ｃ）ｓａｍｕｔｏａｃｉｔｔｙｒｕｔｉｆｇｔｓｏｐｅｉｆａｍｔｎｕｆｅｌｉ－ｓｅｍｔｏｓＴｉｉｍｒｓｉｔｅｒ，ｈｈｅｄｔａｙｔｎｏａｕｉｅｕｔｉｎｔｓｓａｌｍ．ｈｓｅｎｈａｔｗｉｄｐｎｓｖｒｇｅｅｔｔｎｎｒｐｄｖｇｉｕｅｂｉｓｏｏｈｅｃｅｏｅｒｘａｅｎｎｔｒｅａｂｍｔｏｓｔｍａｔｎｎｒａｆｎｔｎｖｉｂｉｏｄｔｏｍｙｃｒａｅｅｙｒｉｅｉｅｂｌｏｉａｉｏｍｌｃｏ．Ａａａｉｙａｎｏａｉｎｒｓｅｓｏｒｃａｉｍｎｉｔｓｕｉｌｌｆａｔｄｌｇｔｓｏ，ｔｒｏ，ｄｐｎｂｆａｅｉｔｅｏｓｆａｔｒｇｏｓｅｅｓｃａｉｈｍａｙｐｘｍａｈｅｒｉｉｅｓｌｏｓｓｎｈｒｐｎｅｈｒｔｄｕｓｔｓｓｈｓｅｉａｄｏｅｉｅｆｅｎｓａｅｓｓｇｅｒｓｓｅｏｒｓｕｓｒｃｎ．

同一流动相测定植物体内12种有机酸和维生素c的高效液相色谱法

同一流动相测定植物体内12种有机酸和维生素c的
高效液相色谱法
高效液相色谱法（HPLC）是一种在分子水平上分离和分析混合物的化学技术。

该技术已被广泛用于食品、化妆品、药物、环境和其他领域的分析。

这里介绍一种使用HPLC测定植物体内12种有机酸和维生素C的方法。

材料和仪器：
1. 色谱柱：C18反相色谱柱
2. 柱温：25℃
3. 甲醇
4. 乙酸
5. FIF水（80：15：5）
6. 洋葱、韭菜、蘑菇等植物样品
7. 液相色谱仪
步骤：
1. 准备样品：将洋葱、韭菜、蘑菇等植物样品切碎，然后将其加入1ml的冰冻Fruitrot中，并用离心机离心10min。

将上清取出，并进行滤过处理。

2. 准备标准曲线：将12种有机酸和维生素C标准物质（苹果酸、柠檬酸、琥珀酸、酒石酸、丙酮酸、葡萄糖酸、谷氨酸、谷酸、草酸、橙酸、马来酸、维生素C）分别制备成一定浓度的标准溶液，用HPLC进行分析得到标准曲线。

3. HPLC条件：色谱柱为C18反相色谱柱，流速为1.0 ml/min，柱温为25℃，流动相为甲醇-乙酸-FIF水（80：15：5），检测波长为210nm。

4. 样品分析：将经过滤过处理的样品加入色谱柱，进行HPLC分析，并测量其吸光度。

5. 数据分析：通过测量吸光度，根据标准曲线，计算出样品中12种有机酸和维生素C的含量。

结果：
使用上述方法，可以在植物样品中准确测量12种有机酸和维生素C的含量。

这种方法简单、快速、准确，可用于评估植物营养状况和检测植物的新陈代谢过程。

高效液相色谱法HPLC测定植物组织中ATP,ADP,AMP含量

ISSN1330-9862scientific note (FTB-1485)A Simple and Rapid Determination of ATP,ADP and AMPConcentrations in Pericarp Tissue of Litchi Fruit by HighPerformance Liquid ChromatographyHai Liu1,3,Yueming Jiang1*,Yunbo Luo2and Weibo Jiang21South China Botanic Garden,The Chinese Academy of Science,510650Guangzhou Leyiju,PR China2College of Food Science and Nutritional Engineering,China Agricultural University,Qinghuadonglu,100083Beijing,PR China3Graduate School of the Chinese Academy of Science,100039Beijing,PR ChinaReceived:February25,2005Accepted:May25,2005SummaryA simple and rapid method using high performance liquid chromatography(HPLC)was developed to determine levels of adenosine triphosphate(ATP),adenosine diphos-phate(ADP)and adenosine monophosphate(AMP)in litchi fruit pericarp tissue.This HPLCmethod used acetonitrile gradient elution and shortened the time required for determina-tions of adenosine phosphates.This analysis exhibited good repeatability(coefficients ofvariation1.28–1.80%)and recovery rate(94.7–97.1%).The correlation coefficients of ATP,ADP and AMP with their peak areas at a range of0–80ng were0.9946,0.9994and0.9974,respectively.This method was applied to determine levels of adenosine phosphates in pe-ricarp tissue of litchi fruit at harvest.There were27.4m g/g of ATP,35.4m g/g of ADP and7.9m g/g of AMP on a fresh mass basis.Key words:HPLC,ATP,ADP,AMP,determination,litchi fruit,pericarp tissueIntroductionEnergy metabolism is an important metabolic path-way which maintains biochemical and physiological ac-tivities in plant tissues(1).Energy charge can be used to show energy status in plant tissues(1,2),while synthe-sis,degradation and restoration of membrane lipids de-pend on energy supply(3–5).The energy charge of cells can be calculated as([ATP]+0.5[ADP])/([ATP]+[ADP] +[AMP]),as described by Pradet and Raymond(1).Some of key enzymes concerning glycolysis,the Krebs’cycle, the electron transport system and oxidative phosphory-lation are also regulated by the energy level of cells(6,7). Lipids are the essential components of plant cell mem-branes,and involvement of adenylate nucleotides in fatty acid biosynthesis in membrane lipid is well estab-lished(5,8).Thus,energy status of tissues of fruits and vegetables may play an important role in maintaining the integrity of cell membranes,which is related to post-harvest life(7,9).The luciferase system was mainly used to determine ATP concentration in organisms,while the analysis of ATP and ADP was performed by enzyme-linked immu-nosorbent assay(10–12).A high performance liquid chro-matography(HPLC)analysis of ATP and its degrada-tion products was also developed on reverse-phase col-umns using phosphate buffer as a mobile phase(13,14). Organic solvents,such as methanol(15,16)or acetonitrile (14),can be used to reduce the running time.In practice, these methods need a long running time and it is neces-sary to develop a simple and rapid method to analyze a large number of fruit samples(14,17,18).*Corresponding author;Phone:++862037252905;Fax:++862037252831;E-mail:ymjiang@There are no reports on the determinations of ATP, ADP and AMP contents by HPLC in plant tissue.The objective of this study was to develop a simple and ra-pid HPLC method for measurements of ATP,ADP and AMP content in pericarp tissue of litchi fruit. Materials and MethodsReagentsThe high purity ATP,ADP and AMP standards,per-chloric acid,potassium dihydrogen phosphate,potassi-um hydrogen phosphate and acetonitrile(HPLC grade) were purchased from Sigma Chemical Co.All reagents used were dissolved in deionized water,and then filte-red with the Sybron/Barnstead Millipore system and a 0.45-m m filter paper.Plant materialsFruit of litchi(Litchi chinensis Sonn.cv.Huaizhi)at the commercially mature stage was harvested from an orchard in Guangzhou.Litchi pericarp was collected for extraction and determinations of ATP,ADP and AMP. Preparations of standard stock soluti onsATP,ADP and AMP standards(1mg)were each dissolved in10mL of deionized water to obtain ATP, ADP and AMP standard stock solutions at100m g/mL. Aliquots of the stock standard solutions were made by diluting them in deionized water at0,0.2,0.5,1,2and4 m g/mL.A volume of20m L of each sample was taken for HPLC analysis.Mobile phasesMobile phase A consisted of0.06mol/L dipotassi-um hydrogen phosphate and0.04mol/L potassium di-hydrogen phosphate dissolved in deionized water and adjusted to pH=7.0with0.1mol/L potassium hydrox-ide,while mobile phase B consisted of100%acetoni-trile.Air bubbles in the two solutions were driven away using an ultrasonic instrument.Extraction of ATP,ADP and AMP from litchifruit pericarp tissueLitchi fruit pericarp tissue(2g)was rapidly frozen in liquid nitrogen and homogenized into powder.Adeno-sine phosphates were extracted from the powder with 10mL of0.6mol/L perchloric acid in the ice bath for1 min by the method of Yang et al.(12).The extraction mixture was centrifuged for10min at6000´g(Beckman J20-2)and4°C,and6mL of the supernatant was taken and quickly neutralized to pH=6.5–6.8with1mol/L KOH solution.The neutralized supernatant was then allowed to stand for30min in an ice bath to precipitate most of the potassium perchlorate,which was removed by paper filtration.The filtrate solution was filtered again through a0.45-m m filter.The final filtrate solution was made up to8mL and then stored at–30°C prior to the analysis. HPLC analysisThe HPLC(Gold125Solvent System,Beckman In-struments Inc.,USA)conditions were as follows:an Ultrasphere ODS EC250´4.60mm column(Beckman In-struments Inc.,USA)was equipped with a Beckman125 pump system.Peaks were detected and analyzed at254 nm by a Gold168diode array detector.HPLC separa-tion was achieved using continuous gradient elution. The elution program was as follows:0min100%A,0% B;2min95%A,5%B;4min80%A,20%B;5.3min 75%A,25%B and6min100%A,0%B.Finally,the program took a further1min to return to the initial con-ditions and stabilize.Flow rate of the mobile phase was 1.2mL/min,while the injection volume was20m L.The total retention time was about5min and the gradient was run for6min to ensure full separation.ATP,ADP and AMP in the samples were identified by comparison with retention time of standards,while the concentra-tions of ATP,ADP and AMP were determined using the external standard method.Data were expressed as means of six replicate determinations.Recovery trialThree standards(ATP,ADP and AMP)were added into litchi fruit pericarp tissue.The extraction was car-ried out according to the above-mentioned method.The concentration of each standard added for the trial was2 m g/mL.The whole experiment was repeated six times. Statistical analysisQuantitative data from the HPLC analysis were compared using either the coefficients of variation(CV) or analysis of variance(ANOVA).Least significant dif-ference was used to compare the means.Results and DiscussionHPLC analysis and calibration curves of ATP,ADP and AMPATP and its breakdown products exhibited a great absorbance at254nm(13).As shown in Fig.1,ATP, ADP and AMP were separated well and detected at254 nm in this study.Acetonitrile instead of methanol used as mobile phase gave a rapid and better separation of ATP,ADP and AMP(data not shown)because it has higher polarity than methanol.Ryder(13)obtained a012345670.0000.0010.0020.0030.0040.005ATPADPAMPA254nmRetention time/minFig.1.HPLC chromatogram of a standard mixture of ATP,ADP and AMPsufficient resolution of adenosine phosphates within 16min using an isocratic system.Veciana-Nogues et al.(16)reported a good separation of adenosine phosphates us-ing 30%methanol gradient conditions.In this study,the separation and determination of ATP ,ADP and AMP needed only 6min and,thus,it is very convenient to an-alyze a large number of samples.There was a good linear relationship among ATP ,ADP and AMP concentrations at a range of 0–80ng against their peak areas,with correlation coefficients being 0.9946,0.9994and 0.9974,respectively,at the 5%ing 2m g/mL standards,the CV of the levels of ATP ,ADP and AMP were 1.80,1.28and 1.30%,respectively,all below 5.00%(Table 1),which indicated excellent re-peatability for the analysis of the three compounds.Recovery trialHigh precision was important to determine the con-centrations of ATP ,ADP and AMP (14,19).The results indicated that the mean recovery rates of ATP ,ADP and AMP were 94.9,94.7and 97.1%,respectively .These were very high recovery rates bearing in mind the complexity of the analyses.The CV of ATP ,ADP and AMP were 4.3,3.5and 1.4%,respectively,all below 5.0%,exhibiting good precision.Analyses of ATP ,ADP and AMP of litchi fruit pericarp tissueThis HPLC method was applied to determine the concentrations of ATP ,ADP and AMP of pericarp tissue in litchi fruit at harvest.Fig.2shows the chromatograms of ATP ,ADP and AMP extracted from the litchi pericarp tissue.There were 27.4m g/g of ATP ,35.4m g/g of ADPand 7.9m g/g of AMP on a fresh mass (FM)basis.In this study,2g of fresh litchi fruit pericarp tissue was enough to determine the concentrations of ATP ,ADP and AMP .Furthermore,the authors used this method to analyze the concentrations of ATP ,ADP and AMP of banana and longan fruits,and found 10.9m g/g FM of ATP,12.2m g/g FM of ADP and 12.8m g/g FM of AMP in banana fruit peel and 6.6m g/g FM of ATP ,8.1m g/g FM of ADP and 7.1m g/g FM of AMP in longan fruit skin.In this study,the HPLC method could not separate the peaks eluting between 2and 3min,whose ultravio-let absorption spectrum was at 254nm (Fig.2).Özogul et al.(14)reported that the unknown compounds in her-ring tissue could be the degradation products of ATP ,such as inosine monophospate and hypoxanthine.How-ever,their identification in litchi fruit pericarp tissue needs further investigation.ConclusionIn conclusion,an HPLC analysis of ATP ,ADP and AMP concentrations in litchi fruit pericarp tissue was simple and rapid to use.We suggest that the improved method for identification and quantification of ATP,ADP and AMP should be a valuable tool in analyzing a large number of fruit samples.AcknowledgementsFinancial support by the National Natural Science Foundation of China (No.30425040and 30430490)and the International Foundation for Science (No.E2265-3)is highly appreciated.References1. A.Pradet,P .Raymond,Adenine nucleotide ratios and adenylate energy charge in energy metabolism,Annu.Rev.Plant Physiol.34(1983)199–224.2.R.J.Romani,S.Ozelkok,«Survival«of mitochondria in vi-tro :Physical and energy parameters,Plant Physiol.51(1973)702–707.3.K.C.Eastwell,P .K.Stumpf,Regulation of plant acetyl-CoA carboxylase by adenylate nucleotides,Plant Physiol.72(1983)50–55.4.J.L.Harwood,Fatty acid metabolism,Annu.Rev.Plant Physiol.Plant Mol.Biol.39(1988)101–138.5.J.Ohlrogge,J.Browse,Lipid biosynthesis,Plant Cell,7(1995)957–970.6.V .N.Luzikov,Stabilization of the enzymic systems of the inner mitochondrial membrane and related problems,Sub-cell.Biochem.2(1973)1–31.7. A.A.Saquet,J.Streif,F.Bangerth,Energy metabolism and membrane lipid alterations in relation to brown heart de-velopment in ’Conference’pears during delayed controlled atmosphere storage,Postharvest Biol.Technol.30(2003)123–132.8. D.J.Brown,H.Beevers,Fatty acid of rice coleoptiles in air and anoxia,Plant Physiol.84(1977)555–559.9. A.A.Saquet,J.Streif,F.Bangerth,On the involvement of adenine nucleotides in the development of brown heart in ’Conference’pears during delayed controlled atmosphere storage,Gartenbauwissenschaft,66(2001)140–144.10.Y.Sugawara,M.Takeuchi,A simple and rapid method fordetermining cell survival in the cryopreserved shoot apexTable parative HPLC analyses of ATP ,ADP and AMPCompoundMeans a CV/%ATP 0.6111 1.80ADP 0.7667 1.28AMP0.86631.30aData were expressed as peak areas (mm 2)(N =6)Retention time/min012345670.0000.0050.0100.0150.0200.0250.030ATPADPAMPA 254n mFig.2.HPLC chromatogram of ATP ,ADP and AMP from litchi fruit pericarp tissueusing luciferin-luciferase ATP assay,Plant Sci.130(1997) 107–112.11. B.Naslund,L.Stahle,A.Lundin,B.Anderstam,P.Arner,J.Bergstrom,Luminometric single step urea assay using ATP-hydrolyzing urease,Clin.Chem.44(1998)1964–1973.12.N.C.Yang,W.M.Ho,Y.H.Chen,M.L.Hu,A convenientone-step extraction of cellular ATP using boiling water for the luciferin-luciferase assay of ATP,Anal.Biochem.306 (2002)323–327.13.J.M.Ryder,Determination of adenosine triphosphate andits breakdown products in fish muscle by high-performance liquid chromatography,J.Agric.Food Chem.33(1985)678–680.14. F.Özogul,K.D.A.Taylor,P.C.Quantick,Y.Özogul,A ra-pid HPLC-determination of ATP-related compounds and its application to herring stored under modified atmo-sphere,Int.J.Food Sci.Technol.35(2000)549–554.15. F.S.Anderson,R.C.Murphy,Isocratic separation of somepurine nucleotide,nucleoside and metabolites from biolo-gical extract by high-performance liquid chromatography, J.Chromatogr.121(1976)251–262.16.M.T.Veciana-Nogues,M.Izquierdo-Pulido,M.C.Vidal-Ca-rou,Determination of ATP related compounds in fish and canned tuna fish by HPLC,Food Chem.59(1997)467–472.17.G.Manfredi,L.C.Yang,C.D.Gajewski,M.Mattiazzi,Mea-surements of ATP in mammalian cells,Methods,26(2002) 317–326.18.J.Murray,A.B.Thomson,Reverse phase ion pair separa-tion of nucleotides and related products in fish muscle,J.High unn.6(1983)209–210.19.K.Samizo,R.Ishikawa,A.Nakamura,K.Kohama,A high-ly sensitive method for measurement of myosin ATPase activity by reversed-phase high-performance liquid chro-matography,Anal.Biochem.293(2001)212–215.。

高效液相色谱法测定鸡软骨_型胶原蛋白含量

药物分析杂志 Ch in J Pharm Ana l 2010, 30( 9)
# 1769 #
表 2∀ 样品中型胶原蛋白含量测定 Tab 2∀ Analysis of collagen from ch ick en cartilage
样品序号 ( sam ple No. )
5次峰面积平均值 ( average of
溶解胶原蛋白采用溶剂为 0 1 m o l L- 1醋酸, 静置缓慢溶解, 全溶时间为 24 h。具体方法如下:
型胶原蛋白样品溶液配制: 将型胶原蛋白样品剪成细长的一段段, 称取 50 0 m g, 置于洁净干燥的 25 mL 量瓶中, 先加入 0 1 m o l L- 1醋酸约 20 mL, 使其溶胀, 室温静置过夜, 完全溶解后, 再用 0 1 m o l L- 1醋酸定容。
图 1∀ 标准品 ( A ) 及样品 ( B ) 色谱图 F ig 1∀ C hrom atogram s of standard( A ) and sam p le( B) 1. 型胶原蛋白 ( co llagen ) ∀ 2. 醋酸钠 ( sod ium acetate)
性好。见表 1( 表 1中重复性的 RSD为同一天内连续 3次进样所得 )。
Abstract∀ Ob jective: T o establish an HPLC m ethod for the determ ination of collagen . M ethods: Co llagen
from ch icken cartilage w as se lected, and it w as determ ined by ge l co lum n w ith m o lecular exclusion chrom atog ram

高效液相色谱法测定骨骼肌ATP、ADP、AMP、NAD+、NADH含量

高效液相色谱法测定骨骼肌ATP、ADP、AMP、NAD+、NADH含量万方数据万方数据万方数据万方数据高效液相色谱法测定骨骼肌ATP、ADP、AMP、NAD+、NADH含量作者：洪平，刘虎威，靳光华，黎燕，杨奎生作者单位：洪平,杨奎生(国家体育总局体育科学研究所,北京,100061)，刘虎威,黎燕(北京大学化学系)，靳光华(山东大学) 刊名：中国运动医学杂志英文刊名：CHINESE JOURNAL OF SPORTS MEDICINE年，卷(期)：2002,21(1)被引用次数：17次参考文献(7条)1.Harris RC;Hultman E;Nodesjo LO Glycogen, glycolyticintermediates, and high-energy phosphates determined inbiopsy samples of musculus quadriceps femoris of manat rest. Methods and variance of values 19742.Harris RC The effect of circulatory occlusion onisometric exercise capacity and energy metabolism of thequadriceps muscle in man 19753.Lowry OH A flexible system of enzymaticanalysis 19724.张龙翔生化实验方法和技术 19875.Weicker H Purine nucleotides and AMP deamina-tion during maximal and endurance swimming exercisein heart and skeletal muscle of rats[外文期刊] 1990(z2)6.朱月春HPLC同步测定肌酸、磷酸肌酸、腺苷酸及次黄嘌呤类物质 1992(01)7.冯炜权运动生物化学原理 1995本文读者也读过(9条)1.戴忠和.朱道立.田澍高效液相色谱法测定大鼠比目鱼肌和内侧腓肠肌内ATP·ADP及AMP含量的研究[期刊论文]-安徽农业科学2008,36(24)2.张玲.潘杰高效液相色谱法测定小鼠心肌组织中ATP、ADP、AMP的含量[期刊论文]-中国医院药学杂志2008,28(21)3.周玉娟.刘福林.崔秀彦.ZHOU Yu-juan.LIU Fu-lin.CUI Xiu-yan 反相离子对高效液相色谱法测定大鼠缺血再灌注心肌ATP的含量[期刊论文]-军医进修学院学报2006,27(2)4.寇瑛( ).何才云.刘智敏.石应康.佟莉高效液相色谱法在测定心肌组织中ATP、ADP、AMP方面的应用[期刊论文]-华西医学2000,15(3)5.吴珏珩.汤丽芬.谭炳炎.欧阳彬.李章旺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[期刊论文]-分析测试学报1999,18(4)6.高娜.杨勇.王世军.容蓉.蔺建军.王小平.GAO Na.YANG Yong.WANG Shi-jun.RONG Rong.LIN Jian-jun.WANG Xiao-ping HPLC测定附子对大鼠肝组织中腺苷酸含量及能荷的影响[期刊论文]-中国实验方剂学杂志2010,16(15)7.李建.陈可泉.黄秀梅.杨卓娜.姜岷.韦萍.LI Jian.CHEN Ke-quan.HUANG Xiu-mei.YANG Zhuo-na.JIANG Min. WEI Ping厌氧发酵有机酸体系中NAD+和NADH测定方法的建立[期刊论文]-食品科技2008,33(12)8.邢莉丽.管玉霞反相高效液相色谱对生物中ATP和ADP的检测[期刊论文]-科技信息(科学·教研)2007(34)9.田岳凤.吴富东.王道东.单秋华.TIAN Yue-feng.WU Fu-dong.WANG Dao-dong.SHAN Qiu-hua高效液相色谱法测定心肌组织腺苷酸的方法和意义[期刊论文]-中华中医药学刊2007,25(1) 引证文献(17条)1.戴忠和.朱道立.田澍高效液相色谱法测定大鼠比目鱼肌和内侧腓肠肌内ATP·ADP及AMP含量的研究[期刊论文]-安徽农业科学 2008(24)2.洪平.赵鹏.杨奎生不同强度运动时大鼠骨骼肌能量代谢产物的变化[期刊论文]-中国运动医学杂志 2002(3)3.孙淑贞.汲平.来庆国.王惠.祁冬.王喜军.王梁.王明臻咬合创伤致兔咬肌线粒体钙离子和二、三磷酸腺苷含量的改变[期刊论文]-华西口腔医学杂志 2004(4)4.戴志远.张燕平.张虹.洪詠平.宋广磊紫贻贝低温无水保活过程中的生化变化[期刊论文]-中国食品学报 2004(3)5.刘林丰.刘忠芳.刘绍璞.段慧维多利亚蓝4R褪色分光光度法测定辅酶Ⅰ[期刊论文]-西南大学学报（自然科学版） 2009(1)6.孙蕊.贾鹏禹.俞龙浩.何淑清.张盟反相高效液相色谱法测定小鼠心肌中ATP、ADP和AMP含量及分析[期刊论文]-黑龙江八一农垦大学学报 2012(2)7.任立杰.佟慧丽.李庆章.高学军奶牛乳腺发育与泌乳过程中能量代谢的变化[期刊论文]-东北农业大学学报2010(2)8.王翠华.李友元.陈长华.李啸Torulopsis glabrata 620在丙酮酸生物合成中的能荷变化和氧化-还原态趋势[期刊论文]-华东理工大学学报（自然科学版） 2006(5)9.樊生华.邢莉丽.管玉霞以反相高效液相色谱法测定生物组织及细胞中的能量代谢变化[期刊论文]-中国组织工程研究与临床康复2008(34)10.邢莉丽.管玉霞反相高效液相色谱对生物中ATP和ADP的检测[期刊论文]-科技信息(科学·教研) 2007(34)11.崔艳.江莉.包建强波纹巴非蛤无水低温保活过程中的生化变化[期刊论文]-安徽农业科学 2008(26)12.凌良飞.戈梅.付磊.黄为一.陈代杰偶发分枝杆菌MF2和MF96生物转化差异的机理研究[期刊论文]-微生物学报2005(4)13.贾鹏禹.孙蕊.俞龙浩.何淑清.张盟.张宇宁反相高效液相色谱法检测鱼肉中的ATP关联物[期刊论文]-肉类研究2012(4)14.孙明娣.史锋.王小元酿酒酵母NAD(H)激酶Pos5p在细胞抵抗氧化胁迫中的作用[期刊论文]-微生物学通报2010(12)15.门小明.邓波.徐子伟.刘敏华.齐珂珂猪宰后肌肉非乳酸供能特点及其与肉质性状相关性[期刊论文]-中国农业科学 2011(7)16.孙彩霞左旋卡尼汀对离体鼠心缺血-再灌注损伤的保护作用[学位论文]硕士 200517.张永强左旋卡尼汀心脏停搏液对大鼠离体心肌保护作用的实验研究[学位论文]硕士 2006引用本文格式：洪平.刘虎威.靳光华.黎燕.杨奎生高效液相色谱法测定骨骼肌ATP、ADP、AMP、NAD+、NADH含量[期刊论文]-中国运动医学杂志 2002(1)。

高效液相色谱仪法检测乳酸链球菌素含量的研究

《高效液相色谱仪法检测乳酸链球菌素含量的研究》一、引言在当今的生物医药领域，乳酸链球菌素（Lactobacillus）作为一种重要的生物活性物质，具有抗菌、抗氧化和抗炎等多种生物功能，因此备受关注。

为了更好地研究和应用乳酸链球菌素，科研人员需要一种准确、快速、稳定的检测方法。

而高效液相色谱仪法（HPLC）作为一种常用的分析方法，在乳酸链球菌素含量检测中具有广泛的应用前景。

二、乳酸链球菌素的生物活性及检测方法综述1. 乳酸链球菌素的生物活性乳酸链球菌素是一种由乳酸链球菌产生的多肽类物质，具有多种生物活性，包括抗菌、调节免疫功能、抗氧化等，因此被广泛应用于食品、药品和保健品等领域。

2. 乳酸链球菌素检测方法综述目前常用的乳酸链球菌素检测方法包括生物学法、免疫学方法和色谱法等。

其中，色谱法中的HPLC方法因其检测灵敏度高、分离效果好、定量准确等优点，成为乳酸链球菌素检测的首选方法之一。

三、HPLC方法在乳酸链球菌素检测中的应用1. 样品前处理在使用HPLC方法检测乳酸链球菌素含量时，首先需要对样品进行前处理。

常见的前处理方法包括提取、浓缩和净化等，通过前处理可有效提高样品中乳酸链球菌素的浓度，提高检测灵敏度。

2. 色谱条件优化在乳酸链球菌素的HPLC检测中，色谱条件的优化对于检测结果的准确性和稳定性至关重要。

包括流动相的选择、柱温、流速、检测波长等参数的优化，都能够有效地提高检测效果。

3. 标准曲线的建立为了定量分析样品中乳酸链球菌素的含量，需要建立标准曲线。

通过制备不同浓度的乳酸链球菌素标准溶液，利用HPLC方法测定其峰面积，建立标准曲线并进行定量分析。

四、乳酸链球菌素HPLC检测方法的优势与挑战1. 优势HPLC方法在乳酸链球菌素检测中具有灵敏度高、分离效果好、定量准确等优点，能够满足科研和生产中的实际需求。

2. 挑战在实际应用中，乳酸链球菌素的HPLC检测也面临着样品前处理复杂、色谱条件优化难度大等挑战，需要科研人员不断进行改进和优化。

细胞ATPADPAMP含量高效液相色谱法HPLC定量检测

细胞ATP/ADP/AMP含量高效液相色谱法（HPLC）定量检测试剂盒产品说明书（中文版）主要用途细胞A TP/ADP/AMP含量高效液相色谱法（HPLC）定量检测试剂是一种旨在通过高氯酸酸性处理，碱性中和后，在高效液相色谱仪和紫外光度仪下（254nm波长）检测分析，分离出ATP、ADP或AMP波峰，以定量测定样品中腺嘌呤核苷酸含量的权威而经典的技术方法。

该技术经过精心研制、成功实验证明的。

其适用于各种动物或人体细胞裂解样品中A TP、ADP、AMP的含量检测。

产品严格无菌，即到即用，操作简捷，性能稳定。

技术背景腺嘌呤核苷酸（adenine nucleotides），简称腺苷，包括单磷酸腺苷、二磷酸腺苷和三磷酸腺苷，是一种单体单位，为核糖核酸（RNA）和脱氧核糖核酸（DNA）的组成成分，并提供化学能量，在能量代谢通路中维护诸如肌肉、胞膜等组织细胞结构的生化和生理功能，调节细胞糖酵解、三羧酸循环、电子传递系统、氧化磷酸化活动。

腺嘌呤核苷酸在细胞中浓度低，且不稳定，通常通过其类型不同、浓度差异、分布状况，来评价细胞的代谢情况和能量状态。

单磷酸腺苷（Adenosine monophosphate；AMP），又称为5'-腺嘌呤核苷酸或腺苷酸（5'-adenylic acid），是一种在（脱氧）核糖核酸中发现的核苷酸。

它是一种磷酸及核苷腺苷的酯，并由磷酸基团、戊糖核酸糖及碱基腺嘌呤所组成。

分子式为C10H14N5O7P，分子量347。

AMP可以通过腺苷酸激酶（adenylate kinase）催化2分子ADP生成，或通过ATP以及ADP水解产生。

AMP常以腺苷-3',5'-环化一磷酸（cAMP）形式存在于细胞中。

AMP与A TP之比反映细胞ATP生成状况以及脂肪酸氧化程度。

二磷酸腺苷（adenosine diphosphate；ADP），参与ADP-ATP循环，提供热能转换和动态平衡，尤其在线粒体需氧呼吸、光合成等实现。