化学发光酶免疫分析测定鱼肉中呋喃它酮代谢物方法研究

建立了呋喃它酮代谢物5-吗啉甲基-3-氨基-2-6唑烷基酮(AMOZ)间接竞争化学发光酶免疫分析(icCLEIA)检测方法,通过单因素实验优化了包被原浓度、抗体稀释倍数、反应缓冲体系及浓度、竞争反应时间等参数,结果表明icCLEIA最佳反应条件为:包被抗原浓度为10 ng/mL,抗体稀释60000倍,最佳竞争时间为50 min,体系缓冲液0.01 mol/L PBS(pH 7.4).在优化的条件下,本方法的线性检测范围为0.026 ～3.52 μg/L,IC50为0.29 μg/L,检出限(LOD,IC10)为0.012 μg/L.对鱼肉样品的平均添加回收率在101.4％～115.5％之间.建立的icCLEIA方法可用于实际样品中AMOZ残留检测.     

酶联免疫吸附分析法测定食品中的苏丹红Ⅰ号

本研究建立了测定食品中苏丹红Ⅰ号含量的间接竞争酶联免疫分析法。首先对苏丹红Ⅰ号分子作了的修饰,再与载体蛋白交联制得免疫原和包被抗原,经动物免疫制得抗苏丹红Ⅰ号的抗体。在包被抗原为100μg/L,抗体为1:100,000倍稀释,标记二抗为1:15000倍稀释的优化条件下,测得检出限为0.12μg/L,IC50值(标准曲线中吸光度抑制至最大吸光度值的50%时所对应的待测物浓度)为0.74μg/L。苏丹红Ⅰ号在番茄酱和辣椒面中的回收率分别为106%和110%。样品仅需甲醇萃取再用缓冲液简单稀释就可以直接进行ELISA测定。     

流动注射光度法测定药物中的盐酸氯丙嗪

在pH 4.04的HAc-NaAc缓冲液中, 刚果红与盐酸氯丙嗪在室温下迅速结合生成缔合物, 且缔合物在480 nm处有最大吸收. 基于此建立了流动注射光度法测定药物中盐酸氯丙嗪的含量. 方法线性范围为0.25～50.0 μg/mL, 检出限为0.082 μg/mL, 测定频率达80次/h.     

酶联免疫吸附分析法测定食品中的苏丹红I号

本研究建立了测定食品中苏丹红I号含量的间接竞争酶联免疫分析法。首先对苏丹红I号分子作了的修饰，再与载体蛋白交联制得免疫原和包被抗原，经动物免疫制得抗苏丹红I号的抗体。在包被抗原为100μg／L，抗体为1：100，000倍稀释，标记二抗为1：15000倍稀释的优化条件下，测得检出限为0．12μg／L，IC50值（标准曲线中吸光度抑制至最大吸光度值的50％时所对应的待测物浓度）为0．74μg／L。苏丹红I号在番茄酱和辣椒面中的回收率分别为106％和110％。样品仅需甲醇萃取再用缓冲液简单稀释就可以直接进行ELISA测定。     

细交链孢菌酮酸酶联免疫吸附分析方法研究

采用水合肼和乙醛酸依次对细交链孢菌酮酸(Tenuazonic acid,TeA)进行衍生化,设计合成了含有氮杂共轭双键偶联手臂,可增强免疫效果的半抗原TeAHGA.通过偶联载体蛋白BSA后的免疫原TeAHGABSA免疫新西兰大白兔,成功制备了特异性识别TeA水合肼衍生物TeAH的多克隆抗体;优化确立了ELISA最佳反应条件(TeAH-OVA为异源包被原、包被浓度0.156 μg/L、药物稀释及反应缓冲液为PBS、一抗反应时间40 min、二抗反应时间20 min),建立了TeA间接竞争ELISA(icELISA)检测方法,其抑制中浓度(IC50)为1.61 μg/L,检出限(LOD)为0.08 μg/L,定量线性检测范围为0.19～12.89 μg/L (IC20～IC80).番茄、面粉样品平均添加回收率分别为67.2％～89.8％和74.8％～93.7％.     

食品中罂栗碱金标快速检测试剂条的研究

首先以1-乙基-3-(3-二甲氨基丙基)碳二亚胺盐酸盐(EDC·HCI)方法合成罂粟碱完全抗原;通过改变还原剂加入量,确定纳米金合成条件为每50.0 ml,氯金酸加入1.5 mL柠檬酸三钠;合成金标保护剂量为20.0 mL胶体金溶胶加入1100稀释抗体(12 mg/L)2.0 mL,标记pH值为8.65.以罂粟碱多克隆抗体-纳米金复合物作为分析探针,罂粟碱完全抗原作为竞争抗原,构建分析体系.点样条件抗原质量浓度为1.0 g/L(以蛋白浓度计算);抗原点样量1 μL/条;金标点样量5 μL/条;二抗浓度(羊抗兔)3.3 g/L稀释至13 500;二抗点样量1μL/条.同时在实验中确定了样品提取方法.检测的灵敏度达到10 μg/L,检测时间不超过20 min.与ELISA方法对照结果,对比检测123份样品,准确率100%.     

免疫化学方法测定动物组织中呋喃唑酮残留标示物

以自制特异性抗体为核心试剂,建立了以苯甲醛为衍生试剂的呋喃唑酮残留标示物间接竞争ELISA方法.通过方阵滴定法和间接竞争法确定ELISA方法最佳反应条件:抗原最佳包被浓度200 μg/L,抗体最佳稀释倍数1:2.5×105,抗体与药物最佳工作配比40 μL: 60 μL,最佳竞争时间1 h.检出限为0.1 μg/L,检测范围0.1～25.6 μg/L,线性关系良好.在空白组织中(猪肌肉、猪肝脏、鸡肌肉、鸡肝脏、鱼肉)添加0.4、1.0和5.0 μg/kg AOZ,回收率55.8%～96.6%,相对标准偏差均小于20%.测定20份不同组织的空白样品,方法的检出限为0.4～0.5 μg/kg.以100 mg/kg呋喃唑酮饲喂动物,其组织样品同时经HPLC方法和本方法测定,数据表明本方法具有实际检出能力,测定结果与仪器方法的测定结果相近.通过与德国同类试剂盒比较实验表明,本方法的灵敏度、精密度、准确度接近于进口试剂盒水平,可用于动物可食性组织肝脏和肌肉中AOZ筛选.     

食品中罂粟碱金标快速检测试剂条的研究

首先以1-乙基-3-(3-二甲氨基丙基)碳二亚胺盐酸盐(EDC·HCI)方法合成罂粟碱完全抗原;通过改变还原剂加入量,确定纳米金合成条件为每50.0 ml,氯金酸加入1.5 mL柠檬酸三钠;合成金标保护剂量为20.0 mL胶体金溶胶加入1:100稀释抗体(12 mg/L)2.0 mL,标记pH值为8.65.以罂粟碱多克隆抗体-纳米金复合物作为分析探针,罂粟碱完全抗原作为竞争抗原,构建分析体系.点样条件:抗原质量浓度为1.0 g/L(以蛋白浓度计算);抗原点样量:1 μL/条;金标点样量:5 μL/条;二抗浓度(羊抗兔)3.3 g/L稀释至1:3 500;二抗点样量:1μL/条.同时在实验中确定了样品提取方法.检测的灵敏度达到10 μg/L,检测时间不超过20 min.与ELISA方法对照结果,对比检测123份样品,准确率100%.     

荧光偏振免疫分析方法快速检测沙拉沙星残留

以异硫氰酸荧光素(FITC)标记沙拉沙星合成荧光标记物,采用薄层色谱法提纯,优化了反应时间、标记物和抗体的工作浓度,建立了沙拉沙星的快速荧光偏振免疫分析法( FPIA).本方法测定沙拉沙星在缓冲液中的半数抑制浓度(IC50)为43.2 μg/L;检测范围为5.7～327 μg/L,可以达到国家规定的动物性食品中兽药最高残留限量(80 μg/kg)的要求.本研究考察了FPIA测定沙拉沙星的动力学过程及对其它4种喹诺酮类药物的交叉反应.结果表明,环丙沙星、恩诺沙星、加替沙星及氧氟沙星的交叉反应率分别为3.3％,1.8％,1.7％和0.7％.在牛奶和猪尿中沙拉沙星的回收率分别在71％～94％和74％～102％之间.本方法操作简单快捷,整个检测过程只需5 min、而且灵敏度较高、特异性强,适用于动物性食品中沙拉沙星残留的快速筛选检测.     

液相色谱-串联质谱法测定牛奶中伏马菌素FB1和FB2及其水解代谢物

建立了MAX混合阴离子固相萃取柱净化-高教液相色谱-串联质谱法测定牛奶中伏马菌素FB1和FB2及其水解代谢产物HFB1和HFB2的方法.牛奶样品经水稀释后,经MAX柱直接净化,甲醇洗脱得到FB1和FB2,经液相色谱-串联质谱负离子扫描测定,1％乙酸甲醇洗脱得到HFB1和HFB2,经液相色谱-串联质谱正离子扫描测定.结果表明,添加浓度为0.1～5.0 μg/L,牛奶中FB1和FB2及其水解代谢产物的回收率为76.4％～92.3％;相对标准偏差(RSD,n=5)为5.9％～12.5％;方法检出限(LOD)均为0.03 μg/L;定量限(LOQ)均为0.1 μg/L.本方法操作简单,灵敏度、回收率和重复性均良好.     

增强化学发光酶免疫法对猪肉中盐酸克伦特罗的检测

建立了猪肉中克伦特罗(CLB)的直接竞争化学发光酶免疫检测(dc-CLEIA)方法。通过优化离子强度、pH值确立了化学发光酶免疫法的标准曲线,优化后dc-CLEIA的检出限可达0.02μg/L。猪肉中的盐酸克伦特罗用高氯酸提取、SPE净化后绘制基质曲线,基质曲线与标准曲线吻合,说明基质影响基本消除。测定0.10、1.0、5.0μg/kg 3个水平的添加回收率,平均回收率为83%~98%,批内与批间的相对标准偏差均小于15%。进一步研究了dc-CLEIA与HPLC两种方法测定猪肉样品的相关性,结果显示两者测定结果相关性良好,r=0.964 7,说明所建立的直接化学发光酶免疫方法可用于实际样品的检测,结果准确可靠。     

Determination of microcystin-LR in drinking water using UPLC tandem mass spectrometry-matrix effects and measurement

A simple detection method using ultra-performance liquid chromatography electrospray ionisation tandem mass spectrometry (UPLC-ESI-MS-MS) coupled with the sample dilution method for determining trace microcystin-LR (MC-LR) in drinking water is presented. The limit of detection (LOD) was 0.04 μg/L and the limit of quantitation (LOQ) was 0.1 μg/L. Water matrix effects of ionic strength, dissolved organic carbon (DOC) and pH were examined. The results indicate that signal detection intensity for MC-LR was significantly suppressed as the ionic strength increased from ultrapure water condition, whereas it increased slightly with solution pH and DOC at low concentrations. However, addition of methanol (MeOH) into the sample was able to counter the signal suppression effects. In this study, dilution of the tap water sample by adding 4% MeOH (v/v) was observed to be adequate to compensate for the signal suppression. The recoveries of the samples fortified with MC-LR (0.2, 1, and 10 μg/L) for three different tap water samples ranged from 84.4% to 112.9%.     

Electrochemically controlled fiber‐in‐tube solid‐phase microextraction method for the determination of trace amounts of antipsychotic drugs in biological samples

In this study, a novel ‘fiber‐in‐tube’ configuration was applied to electrochemically controlled fiber‐in‐tube solid‐phase microextraction of antipsychotic drugs (perphenazine and chlorpromazine) from biological samples. To prepare an electrochemically controlled fiber‐in‐tube solid‐phase microextraction column, first eight stainless‐steel wires were placed into the stainless‐steel column. Then, a nanostructured Cu‐Cr‐Al ternary layered double hydroxide/polythiophene coating was prepared on the inner surface of the stainless‐steel tube and on the surfaces of the stainless‐steel wires by a facile in situ electrodeposition method. The nanostructured coating exhibited enhanced long lifetime, good mechanical stability, high porosity, and large specific surface area compared with polythiophene and Cu‐Cr‐Al layered double hydroxide coatings. Under the optimal conditions, the limits of detection were in the range of 0.07–0.8 μg/L. This method showed good linearity for perphenazine and chlorpromazine in the ranges of 0.3–300 and 0.2–300 μg/L, respectively, with coefficients of determination more than 0.9982. The inter‐ and intra‐assay precisions (RSD%, n = 3) were in the ranges of 3.0–5.1 and 2.5–4.5% at three concentration levels of 5, 25 and 50 μg/L, respectively. Finally, the method was applied for the analysis of the drugs in human urine and plasma samples.     

S,S-氰戊菊酯间接竞争酶联免疫吸附分析方法研究

合成了S,S-氰戊菊酯的半抗原S-2-(4-氯苯基)-3-甲基丁酸-α-S-(N-丁酸基)-甲酰氨-(3-苯氧基)苄酯(Efvb).半抗原通过混合酸酐法与卵清蛋白(OVA)偶联作为包被抗原,活泼酯法与牛血清蛋白(BSA)偶联作为免疫抗原.用免疫抗原免疫新西兰大白兔,得到抗S,S-氰戊菊酯多克隆抗体.通过异源分析及检测条件优化,确立了间接竞争酶联免疫分析方法的最佳检测条件为pH 7.4,0.4 mol/L Na+、40％甲醇-PBS溶液,建立了S,S-氰戊菊酯酶联免疫分析方法.方法的抑制中浓度(IC50)值为(3.16±0.01)mg/L;检出限(IC10)为(0.0053±0.0012) mg/L.对甲氰菊酯、溴氰菊酯、氯氰菊酯、三氟氯氰菊酯及其代谢物戊菊酸没有明显交叉反应.在自来水、河水和土壤样品中添加S,S-氰戊菊酯,其回收率分别为82.3％～108.2％,83.1％～109.2％和72.0％～91.2％.     

Oriented antibody immobilization for atrazine determination by a flow-through fluoroimmunosensor

An atrazine flow-through fluoroimmunosensor was developed, based on an oriented antibody covalently bound to Protein-A (Prot-A) immobilized on Controlled Pore Glass (CPG). Atrazine was detected “in-situ” by placing the immobilized antibody in the optical path of the flow cell. Immobilization of 30 μg of polyclonal anti-atrazine antibody on 0.5 g of Prot-A-CPG provided the highest sensitivity. The effect of several solvents on the covalently immobilized antibodies regeneration was evaluated, the optimum conditions being achieved by pumping 5% acetonitrile (pH = 3) at 0.15 mL/min for 100 s. The detection limit of the immunosensor was 0.7 μg/L and the reproducibility was 2% and 4% for 5 μg/L and 40 μg/L, respectively, in the optimum working concentration range (0.7–50 μg/L). This device allowed 12 samples per hour to be analyzed and had a life-time of 200 assays. Simazine and desisopropylatrazine (DIA) were not cross-reactive, desethylatrazine (DEA) has a cross-reactivity of 8% and propazine and prometryn of 44% and 27%, respectively. The immunosensor was applied to the determination of atrazine in tap and ground water samples spiked at the ¶10 and 30 μg/L concentration level.     

A validated stability‐indicating ultra performance liquid chromatography method for the determination of potential process‐related impurities in eplerenone

A simple, sensitive, and accurate stability‐indicating analytical method has been developed and validated using ultra high performance liquid chromatography. The developed method is used to evaluate the related substances of eplerenone (EP). The degradation behavior of EP under stress conditions was determined, and the major degradants were identified by ultra high performance liquid chromatography with tandem mass spectrometry. The chromatographic conditions were optimized using an impurity‐spiked solution, and the samples, generated from forced degradation studies. The resolution of EP, its potential impurities, and its degradation products was performed on a Waters UPLC BEH C₁₈ column (50 × 2.1 mm, 1.7 μm) by linear gradient elution using a mobile phase consisting of 10 mmol/L ammonium acetate adjusted to pH 4.5, methanol and acetonitrile. A photo‐diode array detector set at 245 nm was used for detection. The flow rate was set at 0.3 mL/min. The procedure had good specificity, linearity (0.02–3.14 μg/mL), recovery (96.1–103.9%), limit of detection (0.01–0.02 μg/mL), limit of quantitation (0.03–0.05 μg/mL), and robustness. The correction factors of the process‐related substances were calculated.     

β-cyclodextrin-bonded silica particles as novel sorbent for stir bar sorptive extraction of phenolic compounds

A stir bar coated with β-cyclodextrin-bonded-silica (CDS) as novel sorbent has been developed and used to analyze seven phenolic compounds in aqueous samples, followed by thermal desorption and gas chromatography-mass spectrometric detection. Significant parameters affecting sorption process such as the time and temperature of sorption and desorption, ionic strength, pH and stirring rate have been optimized and discussed. The coating has a high thermal stability up to 300°C and long application lifetime (80 times). The porous structure of CDS coating provides high surface area and allows high extraction efficiency. Under the selected conditions, linearity range of 0.1-400 μg/L, limit of quantifications of 0.08-3.3 μg/L and method detection limits of 0.02-1.00 μg/L have been obtained. A satisfactory repeatability (RSD ≤ 6.5, n = 7) with good linearity (0.9975 ≤ r(2) ≤ 0.9996) of results illustrated a good performance of the present method. The recovery of different natural water samples was higher than 81.5%.     

Determination of triallate and its metabolite 2,3,3-trichloro-prop-2-en-sulfonic acid in soil and water samples

A method based on solid phase extraction was developed for the determination of the herbicide triallate and its metabolite 2,3,3-trichloro-prop-2-en-sulfonic acid (TCPSA). Soil samples were extracted with methanol and diluted with water to yield a methanol/water ratio of 1?:?4. Triallate was adsorbed on C₁₈ cartridges while TCPSA was enriched on quaternary amine anion exchange resins. Cartridges were eluted with methanol/ethyl acetate and methanol/sulfuric acid mixture, respectively. TCPSA methyl ester was formed using trimethyl orthoformate and subsequently analyzed by GC/ECD. Determination limits of both target compounds were 5 μg/kg soil with recoveries of 100 ± 12% for triallate and 57 ± 5% for TCPSA. In water analysis, determination limits were 0.05 μg/L with recoveries of 84 ± 14% for triallate and 100 ± 22% for TCPSA. In laboratory batch experiments, concentration of triallate decreased from 2690 to 1550 μg/kg soil within 59 days. 14 days after triallate application, TCPSA was determined to be 14 μg/kg which increased to 98 μg/kg soil at the end of the incubation period. Soil/water distribution coefficients in loamy sand soil were 102 for triallate and 0.02 for TCPSA which indicated a higher leaching tendency of the polar metabolite.     

Large volume sample stacking with EOF and sweeping in CE for determination of common preservatives in cosmetic products by chemometric experimental design

This study proposes a capillary electrophoresis method incorporating large volume sample stacking, EOF and sweeping for detection of common preservatives used in cosmetic products. The method was developed using chemometric experimental design (fractional factorial design and central composite design) to determine multiple separation variables by efficient steps. The samples were loaded by hydrodynamic injection (10 psi, 90 s), and separated by phosphate buffer (50 mM, pH 3) containing 30% methanol and 80 mM SDS at -20 kV. During method validation, calibration curves were found to be linear over a range of 5-100 μg/mL for butyl paraben and isobutyl paraben; 0.05-10 μg/mL for ethyl paraben; 0.2-50 μg/mL for dehydroacetic acid; 0.5-70 μg/mL for methyl paraben; 5-350 μg/mL for sorbic acid; 0.02-450 μg/mL for p-hydroxybenzoic acid and 0.05-10 μg/mL for salicylic acid and benzoic acid. The analytes were analysed simultaneously and their detection limits (S/N = 3) were down to 0.005-2 μg/mL. The analysis method was successfully used for detection of preservatives used in commercial cosmetics.     

胶束电动毛细管色谱法检测红曲米中的莫纳可林K

建立了测定红曲米中莫纳可林K含量的胶束电动毛细管色谱(MEKC)方法。考察了运行缓冲液的种类、pH及其浓度、有机添加剂、十二烷基硫酸钠(SDS)的浓度和分离电压等实验条件对电泳分离效果及检测灵敏度的影响。在优化的实验条件下,以20 mmol/L硼砂(pH 10.6,含10%(体积分数)乙醇和40 mmol/L SDS)作为缓冲溶液,莫纳可林K能在23 min内实现很好的基线分离,线性范围为5.00～100.00 mg/L,线性相关系数为0.9976,检出限(以信噪比(S/N)为3计)为0.13 mg/L,加标回收率为98.5%～99.5%。精密度和稳定性试验中,峰面积和迁移时间的相对标准偏差均小于3%,表明重复性良好。该方法简便、快速、灵敏,可用于红曲米中莫纳可林K含量的测定。