酚类内分泌干扰物在长江鱼体血浆中的生物富集

内分泌干扰物(Endocrine disrupting chemicals, EDCs)广泛存在于水环境,可在生物体内富集,具有潜在生态和健康风险.本研究选取双酚A(BPA)、辛基酚(4-t-OP)、雌酮(E1)、17β-雌二醇(E2)和炔雌醇(EE2)共5种酚类EDCs,分别测定其在长江中下游地表水和鱼体血浆中的含量及生物富集因子(Bioaccumulation factor, BAF),并结合血浆BAF预测模型对其生物富集能力进行评价.结果表明,BPA、4-t-OP和E1污染最为普遍,在地表水和鱼体血浆中检出率均超过50%,而其它两种EDCs(E2和EE2)在水体和鱼体血浆中的检出率和浓度均较低.综合检出频率和含量来看:BPA 4-t-OP E1 E2、EE2,5种酚类EDCs在水体中的最大浓度分别为99.7、22.3、2.47 ng·L~(-1)、ND(未检出)和ND,在鱼体血浆中的最大浓度分别为172、91.5、21.1、5.34、5.90 ng·mL~(-1).检出的3种酚类EDCs(BPA、4-t-OP和E1)的log BAF范围分别为2.29—4.20、3.18—4.18和3.29—3.75,表明鱼体血浆对酚类EDCs具有较高的生物富集潜能.对比模型预测的log BAF,实测的log BAF值明显较高,差异的产生与模型建立的前提假设、水温和溶解氧等因素密切相关.本研究有助于认识长江中下游鱼体中酚类EDCs的生物富集特征,并为其生态风险评价和管控提供依据.     

类固醇类内分泌干扰物羟基衍生化条件的优化

以N-甲基-N-三甲基硅基三氟乙酰胺(MSTFA)为衍生化试剂,采用气相色谱-质谱联用技术(GC-MS)系统研究了4种类固醇类内分泌干扰物雌酮(E1)、17β-雌二醇(E2)、雌三醇(E3)、17α-乙炔基雌二醇(EE2)的羟基衍生化,主要考察了MSTFA用量、衍生化温度、衍生化时间对类固醇类内分泌干扰物衍生化效果的影响以及衍生化产物的稳定性、标准曲线、仪器检出限等.结果表明:100μl浓度为0.01μg.μl-1的标准混合溶液,MSTFA的最佳用量为20μl;最佳衍生化条件为70℃下反应30min;衍生化产物的稳定性较好,在-20℃下放置48h,相对响应因子(RRF)基本没有降低;在优化的实验条件下,各待测物具有良好的线性相关性,E1和E2的仪器检出限为0.1pg.μl-1,EE2和E3的仪器检出限为1pg.μl-1.     

污水处理厂中典型内分泌干扰物的去除效果研究

内分泌干扰物(Endocrine Disruption Chemicals,EDCs)在水中极低浓度下即会引起水生生物生殖发育、神经和免疫系统的异常,对人群的健康危害不容忽视。EDCs在污水厂中具有低浓度、难去除的特点,为进一步阐明污水处理厂不同处理工艺对此类EDCs的去除效果,采用化学分析法(气质联用法)和生物学方法(H295R和MVLN细胞实验法)分析东莞市2个城市污水处理厂中8种典型EDCs的分布特征和去除效果。结果显示,两污水厂进水中壬基酚(Nonlyphenol,NP)、辛基酚(Octylphenol,OP)、双酚A(Bisphenol A,BPA)浓度较高,NP质量浓度分别高达10 782 ng·L~(-1)和2 664 ng·L~(-1),而雌激素类物质雌酮(Estrone,E1)、雌二醇(17β-Estradiol,E2)与雌三醇(Estriol,E3)、17α-乙炔基雌二醇(17α-Ethinglestradiol,EE2)、己烯雌酚(Diethylstilbestrol,DES)浓度较低,经处理后,去除效率均超过90%。两污水厂进水及出水处理后H295R细胞内雌二醇(E2)水平显著升高,睾酮(Testosterone,T)水平呈下降趋势,类固醇合成基因HMGR、CYP11B2和CYP19表达量显著增加。MVLN细胞试验结果显示,两污水厂出水中雌激素当量(Estradiol Equivalency quotient,EEQ)分别达19.25ng·L~(-1)和14.21 ng·L~(-1)。本研究表明,即使化学分析结果显示EDCs去除率高达90%,且出水中雌激素化合物低于检出限,但是出水中类固醇激素干扰活性及雌激素活性依然存在。类固醇激素水平与水中酚类物质浓度没有显著相关性,A、B两个污水厂出厂水中除酚类化合物外,其他产生雌激素效应的化合物或类固醇合成干扰物也不容忽视。     

珠江三角洲两条主要河流沉积物中的典型内分泌干扰物污染状况

为了调查珠江三角洲河流沉积物中典型内分泌干扰物的污染状况,采用超声提取-衍生化-GC/MS法研究了双酚A（BPA）、雌酮（E1）、雌二醇（E2）、己烯雌酚（DES）、17α-乙炔雌二醇（EE2）、雌三醇（E3）等典型内分泌干扰物在珠江广州河段和东江东莞河段表层沉积物中的质量分数水平、分布特征,并对其来源进行了分析。结果显示,BPA在所有样品中均有检出,E1和E2的检出率分别为72%和61%,其质量分数（以干质量计）分别在14.3～429.5 ng.g-1之间（中值为79.5 ng.g-1）、〈1.3～10.9ng.g-1之间（中值为3.4 ng.g-1）和〈0.9～2.6 ng.g-1之间（中值为1.6 ng.g-1）;其余几种雌激素均未被检出。东江东莞河段沉积物中BPA的污染水平整体高于珠江广州河段,而天然雌激素的污染状况则是珠江广州河段更为严重。3种化合物的质量分数总体呈现出沿河口方向降低的分布特征,这可能与沿岸工业废水和生活污水不规则排放以及水动力条件有关。BPA、E1、E2的质量分数均与沉积物总有机碳（TOC）质量分数呈正相关,表明有机质是控制沉积物中内分泌干扰物分布的一个重要因素。     

沃特世（Waters@）环境分析园地：在线固相萃取/超高效液相色谱/质谱法测定废水样品中内分泌干扰物

开发了一种利用全自动在线固相萃取/超高效液相色谱/质谱技术（SPE-UPLC-MS/MS）检测污水样品中的27种内分泌干扰物（EDCs）的方法.该方法可在9 min内,完成一个水样的净化、提取、分离、检测,完整的流程.回收率（72%—110%）令人满意,检测限达到每升几个纳克级别（0.3—2.1 ng.L-1）,证明了它们对环境样品分析的潜力.为此,我们将该方法应用于从位于Las Palmas de Gran Canaria（加那利群岛,西班牙）的两个废水处理厂（WWTP）采集的样品.     

五氟苯甲酰氯衍生色谱法分析水体中痕量羟基多溴联苯醚

以五氟苯甲酰氯(PFBCl)作为衍生化试剂,气相色谱-电子捕获检测器(Gas chromatography-electron capture detector,GC-ECD)为检测手段,建立了水体中痕量羟基多溴联苯醚(Hydroxylated polybrominated diphenyl ethers,简称OH-PBDEs)的酰基化衍生-GC/ECD检测方法.通过实验讨论了衍生化缓冲溶液、衍生化时间以及衍生后萃取溶液异辛烷的用量对检测结果的影响,确定了最佳的衍生化条件.在最佳的衍生化条件下,4-OH-BDE 42、4’-OH-BDE 49、6-OH-BDE 47、2’-OH-BDE 68和6’-OH-BDE 99的衍生化产物均获得了良好的分离度和色谱响应值.本方法的仪器检出限为0.02—0.14 pg,方法检出限为0.61—4.67 pg.L-1,标准曲线线性相关系数的范围为0.9973—0.9996,相对标准偏差均小于10%.3种采自大连市不同地区环境水样的加标回收率检测结果表明,所建立的分析方法适于水体中痕量OH-PBDEs的检测.     

羟基多溴联苯醚(OH-PBDEs)小鼠肝脏微粒体体外代谢及对雌激素代谢的影响研究

羟基化多溴联苯醚(OH-PBDEs)是一类具有内分泌干扰效应的酚类化合物,以小鼠肝脏微粒体作为研究对象,考察了OH-PBDEs的体外代谢行为及对雌激素代谢的影响。研究结果发现,4种OH-PBDE均能够代谢,其代谢率大小为6-OH-BDE-996’-OH-BDE-996-OH-BDE-1375’-OH-BDE-99,表明羟基官能团(-OH)与醚键及溴原子处于邻位时,表现出较高的代谢率; 4种OH-PBDE对3种天然雌激素包括雌酮(E1)、17β-雌二醇(E2)、雌三醇(E3)的代谢表现出不同作用,整体来说,对E1和E2代谢随着OH-PBDE浓度的增加抑制作用逐渐增强,对E3代谢则表现出促进作用,但是对人工合成的雌激素17α-炔雌醇(EE2)无明显影响;对E2代谢产物2-羟基雌二醇(2-OH-E2)的生成量定量表征表明,除6-OH-BDE-99促进了2-OH-E2的生成量,其余3种OH-PBDE随实验添加浓度的增加,抑制了2-OH-E2的生成量,这可能与酶介导的不同代谢机制有关。     

17β-雌二醇与1-萘酚对雄性罗非鱼（GIFT Oreochromis niloticus）雌激素效应的比较

在实验室条件下,研究了暴露于不同质量浓度1-萘酚（0.064、0.8、2 mg.L-1）与17β-雌二醇（1、10、100μg.L-1）下,对雄性罗非鱼（GIFT Oreochromis niloticus）性腺系数、血清雌二醇质量浓度的影响及血清中卵黄蛋白原的诱导效应,以期对它们的环境雌激素效应有所了解。研究结果显示：暴露于1-萘酚环境下30 d后,雄性罗非鱼的性腺系数有所降低,当质量浓度为2 mg.L-1时,与对照组相比,差异显著（p〈0.05）,雄性罗非鱼血清中雌二醇的量,此时亦达到最大值,为209.3 pg.mL-1,与对照组相比,差异显著（p〈0.05）;但是,血清中卵黄蛋白原并无变化。暴露于17β-雌二醇环境中的雄性罗非鱼,30 d后,其性腺系数有所降低,当质量浓度为10、100μg.L-1时,与对照组相比,差异显著（p〈0.05）;血清中的雌二醇也随着暴露质量浓度的增多,逐渐增多,当质量浓度为100μg.L-1时,差异极显著（p〈0.01）;血清中卵黄蛋白原的质量浓度明显高于对照组,与暴露质量浓度之间呈剂量效应关系。结果显示,17β-雌二醇具有很强的雌激素效应,会使罗非鱼性腺系数下降,诱导血清中雌二醇、卵黄蛋白原的生成;而1-萘酚会使罗非鱼性腺系数下降,诱导血清雌二醇的生成,但是并不诱导卵黄蛋白原的生成,所以推测其雌激素效应可能较弱,需要进一步研究。     

超高效液相色谱串联质谱法同时测定城市污水处理厂污泥中12种抗生素

建立了一种固相萃取-超高效液相色谱-串联质谱法同时测定污泥中4类12种抗生素的方法.采用SAX串联Oasis HLB固相萃取柱净化富集污泥样品中的抗生素,选定p H 3、洗脱体积8 m L作为最佳前处理条件.采用超高效液相色谱-串联质谱检测抗生素样品,以0.3%的甲酸和乙腈作为流动相,经梯度洗脱进行分离,在多反应检测模式下进行测定.经测定,污泥样品中磺胺类抗生素、脱水红霉素和罗红霉素平均回收率为50%—80%,其余抗生素的平均回收率为82%—119%,12种抗生素在污泥中的定量限为0.001—0.008μg·kg~(-1)dw.并用该法对天津市某污水处理厂污泥样品进行检测,证明该方法可满足实际检测要求.     

加速溶剂萃取-固相萃取净化-超高效液相色谱串联质谱法测定土壤中11种全氟化合物

采用超高效液相色谱-串联质谱联用法（UPLC-ESI-MS/MS）,建立了分析土壤中11种全氟化合物（PFCs）的方法.以甲醇作为萃取剂,样品经加速溶剂萃取仪（ASE）萃取,固相萃取净化后,使用UPLC-ESI-MS/MS联用仪分析样品中11种PFCs.在6 min内就可快速稳定地将所选取的11种全氟化合物分离,且最低检测浓度为0.518—3.520 pg.g-1之间,这些化合物在土壤中的平均添加回收率在71.2%—119.2%之间.应用此方法测得宜兴市水稻土样品中所选取的PFCs含量为0.006—0.780 ng.g-1之间.     

同位素内标-高效液相色谱-串联质谱法测定畜禽粪便中6种雌激素

为了研究畜禽粪便中雌激素的污染特征及其环境影响,建立了一种同时提取、分离和检测畜禽粪便中雌酮(E1)、17α-雌二醇(17α-E2)、17β-雌二醇(17β-E2)、雌三醇(E3)、17α-炔雌醇(17α-EE2)和己烯雌酚(DES)共6种雌激素的高选择性和高灵敏性方法——同位素内标-固相萃取-高效液相色谱-串联质谱法。对提取剂、淋洗剂、洗脱剂、色谱分离条件和质谱测定条件等进行了优化。最终的优化方法为:粪便样品加入100 ng同位素内标后,先后用乙腈重复超声提取3次(每次10 mL,每次15 min),离心后合并上清液,旋转蒸发近干再复溶于1 mL甲醇,加7 mL纯水稀释并超声混匀后离心,上清液过0.45μm滤膜后用HLB固相萃取柱净化,经10 mL 15%乙腈水溶液淋洗后,用10 mL的甲醇进行洗脱,氮吹浓缩定容至1 mL,过0.22μm滤膜后测定。用2 mM氟化铵-甲醇为流动相,经TAB色谱柱分离后采用电喷雾离子源和选择反应监测负离子扫描模式进行检测,同位素内标定量。在5、50、200、1000 ng·g^?1加标水平下,6种雌激素的平均回收率为90.2%—103.2%,相对标准偏差为0.09%—9.08%,方法检出限(LOD)和方法定量限(LOQ)分别为0.25—0.95 ng·g^?1和1.00—3.82 ng·g^?1。应用此方法检测了6个猪粪、3个牛粪和3个鸡粪样品中6种雌激素的含量,结果为?1(干质量)。该方法灵敏度高,定量准确且定量范围广,适用于畜禽粪便中6种雌激素的定性定量分析。     

A high-resolution reverse-phase liquid chromatography method for the analysis of mycosporine-like amino acids (MAAs) in marine organisms

Mycosporine-like amino acids (MAAs) are a group of about 20 structurally related water-soluble compounds, widely distributed among freshwater and marine organisms. To provide a better assessment of the diversity and concentration of MAAs in aquatic environments a high-performance liquid chromatography (HPLC) method of analysis based on reverse-phase C₁₈ column and trifluoroacetic acid and an ammonium-containing mobile phase was developed. The improvements with respect to previous methods and the extraction and clean-up procedures are described here. With this method the clean-up recovery of MAAs of high polarity (shinorine), medium polarity (palythinol), and low polarity (palythene) is greater than 99% (±1%). The method is selective enough to resolve in a single run most of the characterized MAAs found in marine organisms, including the critical and highly polar compounds shinorine, mycosporine-2-glycine, and palythine-serine, the medium polarity pair palythenic acid and shinorine methyl ester (M-333), and the low polarity isomeric pair usujirene and palythene. A chromatogram of a mixture of over 20 MAAs such as might be found in complex samples of marine organisms is given. Good precision was obtained in the separations. The relative standard deviation for retention times was below 1% and the mean relative standard deviation for integrated area estimations was below 2%. A mean column recovery of standards was 99% (±1%) whereas limits of detection (signal-to-noise, S/N=2) for different MAAs varied between 0.08 and 0.47 pmol injected. The applicability of the method was tested using extracts of three microalgae cultures, three natural phytoplankton populations, two scleractinian corals, and one species of sea anemone. Results reveal the occurrence of several unknown MAAs not previously reported in the literature. The selectivity of the method toward some recently discovered MAAs makes it especially suitable not only for studying new field samples, but also for re-examining the MAA composition of previously studied organisms.Communicated by O. Kinne, Oldendorf/Luhe     

Endocrine-disrupting chemicals in the Pearl River Delta and coastal environment: sources,transfer, and implications

A study was conducted to investigate the occurrence and behavior of six endocrine-disrupting chemicals (EDCs) in sewage, river water, and seawater from the Pearl River Delta (PRD). The six EDCs under study were 4-nonylphenol (NP), bisphenol A (BPA), 17α-ethynylestradiol (EE2), estrone (E2), 17β-estradiol (E2), and estriol (E3). These EDCs, predominated by BPA, were found in high levels in the influents and the effluents of sewage treatment plants in the area. The relatively high concentrations (0.23–625 ng/L) of the EDCs detected in the receiving river water suggested that the untreated sewage discharge was a major contributor. The EDCs detected in eight outlets of the Pear River and the Pear River Estuary were in the ranges of 1.2–234 and 0.2–178 ng/L, respectively. The estrogen equivalents in the aquatic environments under study ranged from 0.08 to 4.5 ng/L, with E1 and EE2 being the two predominant contributors. As the fluxes of the EDCs from the PRD region to the nearby ocean are over 500 tons each year, the results of this study point to the potential that Pearl River is a significant source of the EDCs to the local environment there.     

Effects of estradiol-17beta and 17alpha, 20beta-dihydroxy-4-pregnen-3-one on different phospholipids metabolism and histological changes in ovary during reproductive growth in the catfish, Heteropneustes fossilis (Bloch)

Effect of cumulative doses of estradiol -17beta (E2-7, 14 and 28 mg/kg body weight) and 17alpha, 20beta-dihydroxy-4-pregnen-3-one (17alpha,20betaP-7, 14 and 28 mg/kg body weight) on total phospholipids (TP) and various phospholipids- phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidylethanolamine (PE) on liver plasma and ovary were investigated during the reproductively active preparatory and prespawning phases of the annual reproductive cycle in the freshwater female catfish, H. fossilis. The effect of E2 on TP was generally stimulatory and has pronounced effect than 17alpha,20betaP during both the phases. The levels of PC was promoted high during prespawning phase by E2 comparatively very less than by 17alpha, 20betaP in studied tissues during both the phases. The levels of PS after E2 treatments was maximum in all tissues during prespawning phase whereas 17alpha,20betaP was effective only in liver during this phase. The PI was elevated in liver during preparatory phase but its elevation was in all studied tissues during prespawning phase after E2 treatments. The levels of PI was most effective in ovary during preparatory phase in response to 17alpha,20betaP. The levels of PE was declined in liver but elevated in ovary after E2 treatments during both the phases. Treatments of E2 during preparatory phase showed greater number of vitellogenic oocytes as compared to 17alpha,20betaP treatments. The present finding has demonstrated that estradiol-17beta has more pronounced effects than the 17alpha,20beta P in regulation of different phospholipids and ovarian recrudescence during reproductively active phases and among the phospholipids the PC is the main phospholipids of vitellogenin/ovarian lipids in H. fossilis.     

环境样品中三种新型溴代阻燃剂的分析测定

采用索氏抽提、多层硅胶氧化铝柱分离净化和气相色谱质谱联用仪(GC-EI-MS和GC-ECNI-MS)分别对沉积物及土壤样品中的十溴二苯基乙烷(DBDPE),四溴舣酚A双(2,3-二溴烯丙基)醚(TBBPA-DB-PE),1,2-双(2,4,6-三溴苯氧基)乙烷(BTBPE)3种新型溴代阻燃剂进行了定性和定量分析,并建立了环境样品中这3种新型溴代阻燃剂的检测方法.在3个流程空白中,所有目标物都低于检出限;加标空白中日标化合物DBDPE,TBBPA-DBPE,BTBPE的回收率分别为74.8%-82.5%,88.9%-100.7%,86.7%-102.3%.方法检出限分别为1ng·g~(-1),0.4ng·g~(-1)和0.1ng·g~(-1).DBDPE,TBBPA-DBPE,BTBPE在沉积物和土壤样品中测定结果的相对标准偏差分别为16.29%,0.045%,0.051%.方法具有较低的方法检出限,较好的回收率和重复性,适用于一般环境样品中新型溴代阻燃剂的分析和检测.     

自动化前处理-气相色谱串联质谱法测定淡水鱼体中的有机磷阻燃剂

应用自动化前处理设备和气相色谱串联四级杆质谱仪建立了淡水鱼体中10种有机磷阻燃剂的检测方法。该方法先采用自动索氏提取仪进行样品的提取,再应用凝胶渗透色谱净化仪和florisil柱对提取液进行两级净化,最后用气相色谱串联四级杆质谱法(多反应监测模式)对样品中的有机磷阻燃剂(OPFRs)定性定量。实验表明,凝胶渗透色谱净化系统在以乙酸乙酯∶环己烷(1∶1,V/V)为流动相,流速为5.0 m L·min-1的条件下,收集OPFRs的最佳时间为900~1 680 s;方法的检出限为0.002~0.192 ng·g~(-1)湿重,加标水平为5.000 ng·g~(-1)湿重时,平均加标回收率为48.7%~122%,相对偏差除磷酸三乙酯(TEP)(17.7%)和磷酸三(1,3-二氯异丙基)酯(TDCP)(11.9%)外均小于10%。应用该方法测定采集于东江某污水处理厂入河排污口的鱼类样品时,回收率指示物(d27-TBP)的回收率为82.4%~114%,鱼类样品中∑OPFRs的浓度范围为nd~6.41 ng·g~(-1)湿重。     

Substitution of toxicologically critical solvents in the residue analysis of acetamiprid: Towards green chemistry

The article presents the reduced use of toxicologically critical solvents for the extraction and clean-up of a neo-nicotinoid pesticide, acetamiprid from vegetables. Acetamiprid was extracted from spiked vegetable samples of cabbage by the use of different techniques like microwave-assisted extraction (MWAE), sonication and by the conventional method of homogenization. The study revealed that though higher percent recoveries of 78–92 were obtained in the blending method, solvent use was high as compared to MWAE. In MWAE, the percent recoveries recorded were in the range of 46–89 depending upon the solvent used and power employed. A mixture of methanol–water and methanol–acetone at 400 W power levels gave percent recoveries of 72 and 82, respectively, as compared to solvent mixtures, namely, acetone–hexane and acetone–hexane–water, which recorded recoveries of 49 and 67%. The study indicates that the choice of solvent and power employed plays a significant role in MWAE for enhanced recoveries.     

Effect of gastric fluid on adsorption and desorption of endocrine disrupting chemicals on microplastics

• Effect of gastric fluid on EDCs adsorption-desorption to microplastics was evaluated. • The gastric fluid enhanced desorption of EDCs on the surface of microplastics. • Adsorption and desorption isotherms fitted the Freundlich model well. • Desorption ratios of EE2 (55%–59%) on PVC were larger than that of E2 (49%–55%). • Decrease in pH and increase in ionic strength in gastric fluid strengthen desorption. Microplastics and endocrine disrupting chemicals are emerging pollutants in the marine environment because of their potential hazards. The effect of gastric fluid on the adsorption and desorption of 17β-estradiol (E2) and 17α-ethynylestradiol (EE2) to microplastics was investigated. The adsorption and desorption isotherms of E2/EE2 on microplastics could be well fitted by the Freundlich model while the Gibbs free energy of these processes were negative, proving that the reaction occurred spontaneously on the heterogeneous surface of the microplastics. Desorption ratios of EE2 (55%–59%) on PVC were larger than that of E2 (49%–55%) to indicate that EE2 was less stable in gastric fluid, which could be explained by the fact that the hydrophobicity of EE2 was greater than E2. E2/EE2 were more easily desorbed from PVC in the gastric fluid and the desorption amount (5.25–12.91/7.19–17.86 μg/g) increased by 2.51 times in comparison with that in saline solution (2.22–7.81/2.87–10.80 μg/g). The decrease of pH and the increase of ionic strength in gastric fluid could further strengthen desorption of E2/EE2 from PVC. The promotion of gastric juice on desorption of PVC was achieved by reducing the hydrophobicity of the PVC surface. The desorption rate of E2/EE2 at 18°C and 38°C was respectively 44%–47%/46%–50% and 49%–55%/56%–59%, indicating that PVC loaded with E2/EE2 had a relatively greater risk of releasing pollutants in the gastric fluid of constant temperature marine organisms while higher temperatures exposed higher hazards for variable temperature animals. The interaction between microplastics and pollutants might be mainly hydrophobic interaction.     

Novel extractive colorimetric and UV spectrophotometric estimation of etizolam in bulk and tablet by forming ion association complex with methyl orange and bromocresol green

A novel, sensitive, and rapid UV spectrophotometric and colorimetric method was developed for estimation of etizolam (ETZ) in bulk and tablet. The UV spectrophotometric method (method I) is based on quantitative estimation of ETZ using 0.1N NaOH as the solvent which exhibits maximal absorption at 378 nm. Colorimetric methods (method II and III) were based on the formation of color complex in association with ions between basic nitrogen of the drug with methyl orange (MO) and bromocresol green (BCG) in acidic medium. The formed color complexes were quantitatively extracted with chloroform and measured at 509 nm for Drug–MO complex and at 442 nm for Drug–BCG complex, respectively. Beer's law was obeyed over the linear ranges 2–16 µg/ml (method I), 5–45 µg/ml (method II), and 2–20 µg/ml (method III). The correlation coefficient (r²) for ETZ was 0.999, 0.997, and 0.998 for method I, II, and III, respectively. All methods were successfully applied for the assay of the drug in tablet. The % purity was found to be 98.52 (method I), 98.72 (method II), and 99.18 (method III). These developed methods were fully validated with % relative standard deviation (RSD) for accuracy less than 2 for all methods. The % RSD of the intra-day and inter-day variations was found to be less than 2%. The limit of detection and quantitation were as follows: 0.108 µg/ml and 0.327 µg/ml (method I), 0.24 µg/ml and 0.75 µg/ml (method II), 0.1 µg/ml and 0.5 µg/ml (method III) indicating marked method sensitivity. Empirical evidence from all three methods concludes that developed methods are simple, sensitive, and reliably validated for useful routine quality control analysis of ETZ.