Isolation of 6-mercaptopurine in human plasma by aluminum ion complexation for high-performance liquid chromatographic analysis

A sample preparation technique and a high-performance liquid chromatographic method for 6-mercaptopurine (6-MP) that is simple, sensitive and without interference from its metabolites is described. 6-Thioguanine (6-TG) is added as an internal standard to the plasma sample, which is then treated with an aqueous solution of aluminum perchlorate to denature the plasma proteins and form complexes with 6-TG, 6-MP and its major metabolite, 6-thiouric acid (6-TUA). These complexes coprecipitate with proteins on centrifugation. 6-MP and its analogues are then extracted from the precipitate with perchloric acid containing sodium hydrosulfite and the extract is chromatographed on an Ultrasphere ODS column eluted with 0.1 M phosphoric acid and 0.001 M dithiothreitol in deionized water. The eluate is monitored at 340 nm. No interfering peak was encountered in over 300 clinical plasma samples. 6-TUA was separated from 6-MP and was found to be present in much higher concentration than 6-MP itself throughout the sampling time (6 h) following oral administration of the drug.     

Quantification of Thiopurine/UVA-Induced Singlet Oxygen Production

Thiopurines were examined for their ability to produce singlet oxygen ((1)O(2)) with UVA light. The target compounds were three thiopurine prodrugs, azathioprine (Aza), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), and their S-methylated derivatives of 6-methylmercaptopurine (me6-MP) and 6-methylthioguanine (me6-TG). Our results showed that these thiopurines were efficient (1)O(2) sensitizers under UVA irradiation but rapidly lost their photoactivities for (1)O(2) production over time by a self-sensitized photooxidation of sulfur atoms in the presence of oxygen and UVA light. The initial quantum yields of (1)O(2) production were determined to be in the range of 0.30-0.6 in aqueous solutions. Substitution of a hydrogen atom with a nitroimidazole or methyl group at S decreased the efficacy of photosensitized (1)O(2) production as found for Aza, me6-MP and me6-TG. (1)O(2)-induced formation of 8-oxo-7,8-dihydro-2'-dexyguanosine (8-oxodGuo) was assessed by incubation of 6-methylthiopurine/UVA-treated calf thymus DNA with human repair enzyme 8-oxodGuo DNA glycosylase (hOGG1), followed by apurinic (AP) site determination. Because more 8-oxodGuo was formed in Tris D(2)O than in Tris H(2)O, (1)O(2) is implicated as a key species in the reaction. These findings provided quantitative information on the photosensitization efficacy of thiopurines and to some extent revealed the correlations between photoactivity and phototoxicity.     

An electrochemical DNA sensor for determination of 6-thioguanine using adsorptive stripping voltammetry at HMDE: An anticancer drug DNA interaction study

In the present study, the electrochemical behavior of 6-Thioguanine (6-TG) and its interaction with double-strand DNA (ds-DNA) were investigated at the surface of hanging mercury drop electrode (HMDE) in neutral media. The interaction of 6-TG with ds-DNA in neutral buffer was clearly demonstrated by the elimination of 6-TG signal and the advent of a new reduction peak. To investigate the interaction, various parameters including accumulation time and potential as well as ds-DNA concentration were optimized using a combination of adsorptive stripping voltammetry (AdSV) and square wave voltammetry (SWV) techniques. As a consequence a low detection limit of 1.1 μM was obtained in a dynamic range of 16.0 to 360.0 μM. To better understand the interaction mechanism between 6-TG anti-cancer drug and ds-DNA, cyclic voltammetry and UV-Vis spectroscopy measurements were carried out and the intercalation of 6-TG into ds-DNA was proposed as the plausible mechanism. The application of this screening assay in real sample analysis was investigated by using the procedure for determination of 6-TG in 6-Thioguanine tablets and also in spiked 6-TG blood serum. Overall, the results were indicative of a DNA sensor which could be applied effectively in the analysis of 6-TG in vitro.     

Electroanalysis and simultaneous determination of 6-thioguanine in the presence of uric acid and folic acid using a modified carbon nanotube paste electrode

The present work describes the preparation and characterization of a carbon nanotube paste electrode modified with 2,7-bis(ferrocenyl ethyl)fluoren-9-one (2,7-BF). This electrode showed an efficient catalytic activity for the electro-oxidation of 6-thioguanine (6-TG), which leads to lowering 6-TG overpotential by more than 610 mV. Also, the values of catalytic rate constant (k = 2.7 × 10(3) mol(-1) L s(-1)), and diffusion coefficient (D = 2.7 × 10(-5) cm(2) s) were calculated. In 0.1 M phosphate buffer solution of pH 7.0, the oxidation current increased linearly with two concentration intervals of 6-TG, one is 0.06 to 10.0 μmol L(-1) and the other is 10.0 to 160.0 μmol L(-1). The detection limit (3σ) obtained by differential pulse voltammetry (DPV) was 22.0 nmol L(-1). DPV was used for simultaneous determination of 6-TG, uric acid (UA) and folic acid (FA) at the modified electrode, and for quantification of 6-TG, UA and FA in some real samples by the standard addition method.     

Voltammetric studies of the interaction of 6-mercaptopurine with cucurbit[7]uril and DNA

The interaction of 6-mercaptopurine (6-MP), an antitumor drug, with cucurbit[7]uril (Q[7]) and DNA in an acetate buffer solution was studied by differential pulse voltammetry (DPV) and cyclic voltammetry(CV). The electrochemical data indicated a 1:1 complex formation of 6-MP with Q[7] and DNA. The formation constants of these complexes were determined based on the variations in the current. Moreover, the interactions of the 6-MP-Q[7] inclusion complex with DNA have been investigated by means of voltammetry. The results suggested that 6-MP displayed a high affinity for Q[7] and that the inclusion complex did not decompose when it bound to DNA. It can be inferred from the experimental data that the binding model of 6-MP to DNA may be ??electrostatic binding??. In addition, the formation of inclusion complexes between Q[7] and 6-MP was confirmed by UV-Vis spectroscopy and the ¹H NMR technique.     

A gas chromatographic method for measuring 6-mercaptopurine in serum.

A method is presented for the measurement of 6-mercaptopurine (6-MP) in serum. Serum samples containing the drug were treated with dithioerythritol and saturated with ammonium carbonate. 6-MP was then extracted from the serum with a mixture of isopropanol-ethyl acetate (1:1, v/v) containing 0.01% ethanethiol. The solvent was evaporated, the residue dissolved in dilute hydrocholoric acid, washed with chloroform, and extracted with ethyl acetate. 6-MP was derivatized with trimethylanilinium hydroxide and measured gas chromatographically. The sulfhydryl-protecting reagents, dithioerythritol and ethanethiol, were added to prevent the decomposition of 6-MP. The extraction and clean-up method recovered 78 +/- 2% (S.E.) of the 6-MP present. Tracer amounts of [8-14C]6-MP served as the internal standard during the extraction part of the method. Theophyline was used as the internal standard during the gas chromatographic analysis. The standard curve obtained from the gas chromatograph was linear between 0.5 and 20 mug/ml of 6-MP. Serum samples were stored in the freezer for two weeks without significant loss of drug. No interference was encountered from normal serum constituents or xanthines, such as caffeine or theophylline added to serum.     

Interactions between PAMAM−NH2 and 6-Mercaptopurine: Qualitative and Quantitative NMR studies

Despite being used as an anti-leukemic drug, the poor solubility of 6-mercaptopurine (6-MP) limits its use in topical and parenteral applications. Dendrimers are commonly used as drug carriers to improve their solubility in aqueous solution. In this work, the interactions between 6-MP and the amine-terminated poly(amidoamine) dendrimers (PAMAM−NH₂) were investigated by various NMR technology. The chemical shift titrations disclosed that the 6-MP interacted with the surface of PAMAM−NH₂ mainly through electrostatics. The determination of diffusion coefficient and relaxation measurements further confirmed the presence of interactions in 6-MP/PAMAM−NH₂ complexes. In addition, the encapsulation of 6-MP within the cavity of PAMAM−NH₂ was revealed through nuclear Overhauser effect spectroscopy and Saturation Transfer Double Difference analysis. Finally, the binding strength (H-8 is 100% and H-2 is 70%) of 6-MP to PAMAM−NH₂ was quantitatively expressed using epitope maps. This study provides a systematic methodology for qualitative and quantitative studies of the interactions between dendrimers and drug molecules in general.     

高效液相色谱法分析人血清中硫唑嘌呤和巯嘌呤的浓度

建立了用ＨＰＬＣ同时测定人血清中硫唑嘌呤（ＡＺＰ）和６－巯基嘌呤（６－ＭＰ）浓度的方法。血清样品经乙腈除蛋白后在常压和３７℃下用氮气吹干,残余物用１００μＬ洗脱液溶解,离心后上清液直接进样。以ＳｐｈｅｒｉｓｏｒｂＣ１８固定相,采用梯度洗脱,从Ｖ（乙腈）∶Ｖ（０．０１ｍｏｌ／Ｌ磷酸二氢钾）＝３∶９７经５ｍｉｎ时变为１８∶８２,１５ｍｉｎ后再变为５０∶５０,检测波长开始为３２５ｎｍ,１０ｍｉｎ后为２７８ｎｍ,甲硝唑（ＭＮＺ）为内标。ＡＺＰ和６－ＭＰ平均回收率分别为（１００．６±４．２）％和（１０２．４±４．５ ）%。     

Photo-oxidation of 6-Thioguanine by UVA: The Formation of Addition Products with Low Molecular Weight Thiol Compounds

The thiopurine, 6-thioguanine (6-TG) is present in the DNA of patients treated with the immunosuppressant and anticancer drugs azathioprine or mercaptopurine. The skin of these patients is selectively sensitive to UVA radiation—which comprises >90% of the UV light in incident sunlight—and they suffer high rates of skin cancer. UVA irradiation of DNA 6-TG produces DNA lesions that may contribute to the development of cancer. Antioxidants can protect 6-TG against UVA but 6-TG oxidation products may undergo further reactions. We characterize some of these reactions and show that addition products are formed between UVA-irradiated 6-TG and N-acetylcysteine and other low molecular weight thiol compounds including β-mercaptoethanol, cysteine and the cysteine-containing tripeptide glutathione (GSH). GSH is also adducted to 6-TG-containing oligodeoxynucleotides in an oxygen- and UVA-dependent nucleophilic displacement reaction that involves an intermediate oxidized 6-TG, guanine sulfonate (G^SO3). These photochemical reactions of 6-TG, particularly the formation of a covalent oligodeoxynucleotide–GSH complex, suggest that crosslinking of proteins or low molecular weight thiol compounds to DNA may be a previously unrecognized hazard in sunlight-exposed cells of thiopurine-treated patients.     

The use of formic acid in carrier gas. Rapid method for identification and determination of phytanic acid by gas chromatography-mass spectrometry-chemical ionization

A rapid gas chromatographic method to determine phytanic acid in plasma from Refsum's disease is described. After a brief alkaline hydrolysis of lipids, the biological sample is directly injected into a glass pre-column; an acid carrier gas (formic acid in nitrogen) is used to displace the long-chain fatty acids from their sodium salts and from their binding to proteins. Formic acid introduced through the column may also be used as a reagent gas for chemical ionization in combined gas chromatography--mass spectrometry; fatty acids (C14 to C18:2 and phytanic acid) are easily identified by their M + 1 (base peak) and M - 17 peaks. The described procedure is also suitable for studying normal fatty acids from plasma lipids.     

微透析采样荧光法研究6-硫鸟嘌呤与蛋白结合作用

将微透析技术与荧光分析相结合,测定了6-硫鸟嘌呤（Thioguanine,6-TG） 与牛血清白蛋白的相互作用。在NaOH(1.0 * 10~(-3)mol·L~(-1))介质中,6-TG可 被KMnO_4氧化,氧化产物具有较强的荧光,且荧光强度与6-TG浓度可在2.0 * 10~ (-11) ～ 2.0 * 10~(-6)mol·L~(-1)范围内分段拟合呈良好的线性关系,检测限 为4.0 * 10~(-12)mol·L~(-1)。当灌流速度为5μL·min~(-1)时,6-TG的透析率 为(7.2 ± 0.2)%。利用Scatchard方程处理数据,得到6-TG-BSA的结合常数和结合 位点分别为1.02 * 10~4 (mol·L~(-1))~(-1)和1.63。     

Application of ion-exchange separations to determination of trace elements in natural waters-IX: simultaneous isolation and determination of uranium and thorium

A method is described for the determination of uranium and thorium in samples of natural waters. After acidification with citric acid the water sample is filtered and sodium citrate and ascorbic acid are added. The resulting solution of pH 3 is passed through a 4-g column of Dowex 1 x 8 (citrate form) on which both uranium and thorium are adsorbed as anionic citrate complexes. Thorium is eluted with 8M hydrochloric acid and separated from co-eluted substances by anion-exchange in 8M nitric acid medium on a separate 2-g column of the same resin in the nitrate form. After complete removal of iron by washing with a mixture consisting of IBMK, acetone and 1M hydrochloric acid (1:8:1 v v ) and treatment of the resin with 6M hydrochloric acid, the uranium is eluted from the 4-g column with 1M hydrochloric acid. In the eluate thorium is determined spectrophotometrically (arsenazo III method) while fluorimetry is employed for the assay of uranium. The procedure was used for the determination of uranium and thorium in numerous water samples collected in Austria, including samples of mineral-waters. The results indicate that a simple relationship exists between the uranium and thorium contents of waters which makes it possible to calculate the approximate thorium content of a sample on the basis of its uranium concentration and vice versa.     

Microchip electrophoretic protein separation using electroosmotic flow induced by dynamic sodium dodecyl sulfate-coating of uncoated plastic chips

Separation of sodium dodecyl sulfate (SDS)-protein complexes is difficult on plastic microchips due to protein adsorption onto the wall. In this paper, we elucidated the reasons for the difficulties in separating SDS-protein complexes on plastic microchips, and we then demonstrated an effective method for separating proteins using polymethyl methacrylate (PMMA) microchips. Separation difficulties were found to be dependent on adsorption of SDS onto the hydrophobic surface of the channel, by which cathodic electroosmotic flow (EOF; reversed flow) was generated. Our developed method effectively utilized the reversed flow from this cathodic EOF as a driving force for sample proteins using permanently uncoated but dynamic SDS-coated PMMA microchips. High-speed (6 s) separation of proteins and peptides up to 116 kDa was successfully achieved using this system.     

Determination of 6-Mercaptopurine in Rat Blood by Microdialysis Coupled with High Performance Liquid Chromatography on a Functionalized Multi-wall Carbon Nanotubes Modified Electrode

Introduction Sincecarbonnanotubeswerediscoveredin 1991,theyhaveattractedmuchattentionbecause oftheirremarkablenanostructureswithhighsur- facearea,highelectricalconductivity,goodchemi- calstabilityandsignificantmechanicalstrength[1]. Reportshavealreadybeenpublishedonthecata- lyst[2],nanoscaleelectronicandmechanicalsys- tems[3],andscannedprobemicroscopesandelec- tronfieldemissiontips[4].Itwasreportedthatcar- bonnanotubescouldbemadeintocarbonnan- otubeselectrodesandhavebeensuccessfullyused intheo…     

光谱法研究硫鸟嘌呤与七元瓜环及牛血清白蛋白的超分子相互作用

利用紫外-可见吸收光谱法和荧光光谱法研究了抗癌药物硫鸟嘌呤(6-TG)与七元瓜环(Q[7])及牛血清白蛋白(BSA)的相互作用. 结果表明, 6-TG与Q[7]及BSA可形成三元复合物, 且6-TG与Q[7]及BSA均可形成1:1的超分子配合物, 6-TG能引起BSA的荧光猝灭, 猝灭机制为静态猝灭. 此外, 还用同步荧光法和三维荧光法考察了6-TG对BSA构象的影响, 结果表明6-TG的加入使BSA的构象发生了变化, 而同步荧光光谱结果表明结合位点更接近于色氨酸.     

Hydroxymethylation and aminomethylation of 6-mercaptopurine and its derivatives

Labile aminomethyl and hydroxymethyl derivatives of 6-mercaptopurine (I) (6-MP) and S⁶-acyloxymethyl-6-MP have been converted to stable acetyloxymethyl derivatives by their reaction with acetic anhydride. Analysis of the reaction products and comparison of their 'H nmr spectra and hplc chromatograms with those of acetyloxymethyl derivatives of known structures suggested 1) that the aminomethyl derivatives of 6-MP were 7-substituted derivatives, 2) that the aminomethyl derivative of S⁶-acetyloxylmethyl-6-MP was a 9-derivative, 3) that the hydroxymethyl derivative of 6-MP was a mixture of 7-substituted and S⁶,3-disubstitu-ted derivatives, and 4) that the hydroxymethyl derivative of S⁶-pivaloyloxymethyl-6-MP was a 9-substituted derivative. In addition, a previously unreported dialkyl derivative of 6-MP VI was isolated from its reaction with aminomethylating agent and characterized. Analyses of the 'H nmr spectra and hplc chromatograms of the reaction of VI with acetic anhydride suggested that VI was a 1,7-disubstituted derivative.     

Determination of thioguanine in pharmaceutical preparations by paper substrate room temperature phosphorimetry

A room temperature phosphorimetric (RTP) procedure was used for the determination of 6-thioguanine (6-TG). The method is based on paper substrate room temperature phosphorimetry (PS-RTP) using indium sulfate, In2(SO4)3 as a heavy atom perturber. Various factors affecting the room temperature phosphorescence of 6-TG are discussed. The linear dynamic range for 6-TG is from 3.3 to 334.3 ng per spot with a detection limit of 4.6 ng per spot and a relative standard deviation (RSD) of 2.38%. The recovery of standard 6-TG added to commercial tablets is in the range 96.39-98.44%. The method is simple, rapid and sensitive and can be applied to the analysis of commercial tablets without interference.     

旋转铂盘电极上Cu(phen)_2~(2 )与6-巯基嘌呤的相互作用

在Tris-NaC1*(pH=7.2)缓冲溶液中,应用循环伏安法,微分脉冲伏安法、旋转圆盘电极实验、交流阻抗法及其数据模拟等技术研究了Cu(phen)2 25(phcn=1.10-邻菲咯啉)与6-巯基嘌呤(6-MP)的相互作用.结果显示.Cu(phen)2 2MP与6-MP无论在扩散控制过程或电化学控制过程都发生了相互作用.Cu(phen)2 2及其与6-MP的作用产物于铂电极上均呈现一对氧化还原峰,但后者呈现的氧化还原峰负移.峰电流减小.交流阻抗结果显示,无论6-MP存在与否,Cu(phen)2 2在交流阻抗谱上均呈现两个清晰的电容弧,但当6-MP存在时,电化学反应电阻和电化学吸脱附电阻均增大.Cu(phen)2 2在不同转速下的阻抗拟合结果显示.随转速增大.电化学反应电阻和电化学吸脱附电阻均减小.双电层电容呈增大趋势,而吸脱附电容呈减小趋势:当6-MP存在时.仍然呈现此变化规律.     

Decrease of dynamic range of proteins in human plasma by ampholine immobilized polymer microspheres

A novel protein sample pretreatment method based on ampholine immobilized polymer microsphere (ampholine@PM) was developed for the fractionation of intact proteins prior to protein digestion and peptide analysis to reduce the dynamic range of human plasma proteome. After incubation with our prepared ampholine@PM, the captured plasma proteins were successively desorbed by 2 M NaCl, 100 mM glycine-hydrochloric acid, and 30% (v/v) acetonitrile with 0.1% (v/v) trifluoroacetic acid. The SDS-PAGE results showed the protein dynamic range in such three fractions was obviously reduced as compared with the native plasma. On-particle digestion was ultimately performed to release all proteins retained on ampholine@PM. Followed by MuPIT analysis, the number of identified proteins in plasma was improved by 75% after ampholine@PM treatment. Furthermore, the spectral count of 9 high abundance proteins was decreased by 37.6–97.2%, and the identified low abundance protein (<100 ng mL⁻¹) number was increased from 4 to 17. These results demonstrated that the fractionation by ampholine@PM could efficiently decrease the protein dynamic range in abundance, beneficial to achieve the deep coverage identification of human plasma proteome.     

Isotope dilution analysis of selenite and selenate in natural water using microwave-induced nitrogen plasma mass spectrometry.

Isotope dilution analysis of the sub-microg l(-1) levels of selenite and selenate in natural water samples by microwave-induced nitrogen plasma mass spectrometry (MIP-MS) was performed. An appropriate amount of a spike solution containing 78Se-selenite and 78Se-selenate was added to the natural water sample to be analyzed. Both analytes in the water were then concentrated simultaneously by passing the sample through a column that was filled with an anionic exchange resin. After the concentration process, all of the selenite and some of the selenate on the resin were eluted by 0.03 M nitric acid. The residual selenate was eluted by 0.13 M nitric acid. The eluted sample solutions were injected into MIP-MS, and isotope dilution analyses were carried out. Selenite and selenate concentrations as low as 0.01 microg l(-1) in the natural water sample were successfully determined by the proposed method.