牛奶中磺胺类抗生素的快速检测分析

建立牛奶中磺胺类抗生素残留检测的放射免疫方法。通过对磺胺嘧啶、磺胺甲基嘧啶、磺胺二甲基嘧啶、磺胺甲氧哒嗪、磺胺甲基异恶唑、磺胺间甲氧嘧啶、磺胺间二甲氧嘧啶和磺胺喹恶啉8种磺胺类药物在空白牛奶样品中的加标试验,确定该方法的筛选水平。结果发现,采用放射免疫法检测牛奶中的磺胺类抗生素,筛选水平可达到10μg/L,变异系数在1.79%￣5.56%之间;对8种磺胺类药物的混合标准溶液进行检测,100%可以被检测出,检测限达5μg/L。放射免疫法检测牛奶样品中的磺胺类抗生素,方法简便、快速,灵敏度和精确度能够达到检测要求。     

HPLC法同时测定禽肉中氯羟吡啶和多种磺胺类药物残留

建立了用于禽肉中氯羟吡啶和磺胺嘧啶、磺胺甲基嘧啶、磺胺二甲基嘧啶、磺胺甲氧嘧啶、磺胺-6-甲氧嘧啶、磺胺甲基异噁唑、磺胺二甲氧嘧啶、磺胺喹噁啉等残留测定的高效液相色谱法。样品以乙腈-氯仿提取,正己烷脱脂净化,以Symmetry C18 5μm(4.6 mm×150 mm)色谱柱进行分离,二极管阵列检测器检测,梯度洗脱测定氯羟吡啶和8种磺胺。回收率在71.7%~99.7%,相对标准偏差为1.5%~3.8%,最低检测限除磺胺二甲氧嘧啶、磺胺喹噁啉为0.02 mg/kg外,其余都可达到0.01 mg/kg。该方法已成功应用于禽类样品多兽药残留的检测。     

高效液相色谱法检测鸡可食性组织中的磺胺氯吡嗪残留

对建立的一种鸡组织中磺胺氯吡嗪钠残留检测的高效液相色谱法进行复核验证。鸡组织样品中磺胺氯吡嗪钠残留经乙腈提取,正己烷去脂肪,MCX固相萃取小柱净化。采用高效液相色谱紫外检测器检测,流动相为乙腈-0.02mol/L磷酸二氢钾水溶液(35:65,V/V),紫外检测波长为272nm,外标法定量。结果显示,磺胺氯吡嗪钠标准工作液在0.04~20.0μg/mL范围内具有良好的线性关系(R^2=0.9999)。在鸡肌肉、肝脏、肾脏、皮脂各组织中添加浓度分别为40、100、200μg/kg时,各组织样品中磺胺氯吡嗪钠的回收率范围为74.70%~93.67%。磺胺氯吡嗪钠的检测限为20μg/kg,定量限为40μg/kg。复核验证的鸡组织中磺胺氯吡嗪钠残留检测高效液相色谱法可行,方法简便快捷,回收率高,适合鸡组织中磺胺氯吡嗪的残留检测。     

猪肉中磺胺类药物残留检测操作要点

目前我国磺胺类药物残留检测对象主要是猪肉,使用的检测方法是(以下简称方法).该检测方法用于测定动物可食性组织中磺胺间甲氧嘧啶(SMM)、磺胺二甲基嘧啶(SM2)、磺胺甲恶唑(SMZ)、磺胺二甲氧嘧啶(SDM)、磺胺喹恶啉(SQ)单个或混合物的残留量.     

鸡组织中磺胺氯吡嗪和磺胺氯哒嗪残留的HPLC检测方法

建立了一种鸡组织样品中磺胺氯吡嗪和磺胺氯哒嗪残留量的HPLC测定方法.鸡组织样品中的药物残留经乙腈和丙酮提取,正己烷去脂肪和MCX固相萃取小柱净化.洗脱液经浓缩后,其样品中的药物残留采用HPLC紫外检测器检测,流动相为乙腈/0.017 mol/L磷酸溶液(32/68),紫外检测波长为270 nm.2种磺胺药对照品溶液在0.02～1 mg/L范围内呈良好的线性相关(r＞0.999 7).在50、100,200 μg/kg的添加水平上,磺胺氯吡嗪和磺胺氯哒嗪残在鸡组织(肌肉、肝脏和肾脏)中的回收率在70%%～85%之间,日内和日渐变异系数分别小于8%和10%.在该检测条件下,上述两种磺胺药物在鸡组织中的检出限为20μg/kg,定量限为50 μg/kg.该检测能够满足鸡组织中磺胺氯吡嗪和磺胺氯哒嗪残留的检测要求.     

应用胶体金免疫层析法测定牛奶中的磺胺类药物残留

应用胶体金免疫层析法对牛奶中的磺胺类药物残留进行检测,研究该方法的灵敏度、假阳性率、假阴性率及特异性。结果表明,用胶体金免疫层析法测定牛奶中的磺胺类药物,检测限分别为磺胺嘧啶60μg/L、磺胺二甲基嘧啶20μg/L、磺胺喹噁啉80μg/L、磺胺氯哒嗪30μg/L、磺胺间二甲氧嘧啶50μg/L、磺胺间甲氧嘧啶50μg/L、磺胺甲噁唑80μg/L、磺胺苯酰50μg/L、磺胺对甲氧嘧啶30μg/L、磺胺异噁唑10μg/L、磺胺甲氧哒嗪70μg/L、磺胺多辛70μg/L,假阳性率为2%,假阴性率为0;方法的特异性较好,对其他抗生素无交叉反应;检测实际样本的结果与液相色谱-质谱/质谱法基本一致。可见,该方法简便、快速、准确、稳定,适用于牛奶中磺胺类药物残留的现场快速检测。     

动物源食品中磺胺对甲氧嘧啶残留检测方法——高效液相色谱法

农业部于2001年11月1日发布38号文,发布10种动物源食品中兽药残留检测方法.方法之二:动物源食品中磺胺对甲氧嘧啶残留检测方法--高效液相色谱法,全文如下: 动物源食品中磺胺对甲氧嘧啶残留检测方法--高效液相色谱法     

鸡血浆中磺胺氯吡嗪钠和甲氧苄啶含量HPLC检测方法的建立

建立了鸡血浆中磺胺氯吡嗪钠和甲氧苄啶含量测定的HPLC方法。对鸡血浆中磺胺氯吡嗪和甲氧苄啶采用乙腈提取和净化,梯度洗脱,高效液相色谱紫外检测法测定,外标法定量。流动相为甲醇-0.02 mol/L磷酸二氢钾水溶液。紫外测定时采用双波长检测:磺胺氯吡嗪检测波长为268 nm,甲氧苄啶检测波长为240 nm。空白血浆添加磺胺氯吡嗪和甲氧苄啶分别在0.1～70.0μg/m L和0.05～5.00μg/m L范围内线性关系良好(R~2均大于0.9999),磺胺氯吡嗪和甲氧苄啶的检测限分别为0.05μg/m L和0.025μg/m L,定量限分别为0.1μg/m L和0.05μg/m L。空白血浆添加磺胺氯吡嗪和甲氧苄啶在各自线性范围内,批内变异系数分别小于6.84%和7.55%,批间系数分别小于5.23%和6.81%。本研究建立的鸡血浆中磺胺氯吡嗪和甲氧苄啶的提取和净化方法,适用于鸡血浆中磺胺氯吡嗪和甲氧苄啶含量的单个或同时测定。     

氨苯磺胺多克隆抗体的制备

采用重氮化法将氨苯磺胺与人血清白蛋白偶联形成免疫原，免疫新西兰大白兔制备多克隆抗体。通过紫外扫描分析，免疫原中药物分子与蛋白质分子的偶联比为32：1，改良辛酸-饱和硫酸铵法纯化抗体，多克隆抗体效价达1：10^5，间接ELISA建立的氨苯磺胺检测的线性范围为1ng／mL～100μ/mL，70％抑制率可检测最低浓度为0．09μ/mL，以抗体与氨苯磺胺的反应率为100％，抗体与其他7种磺胺药交叉反应率很低。结果表明，制备的氨苯磺胺多克隆抗体具有很高的特异性，可用于动物性食品中氨苯磺胺残留的检测。     

恩诺沙星注射液中非法添加磺胺嘧啶HPLC-PDA检查法的建立

对恩诺沙星注射液中非法添加磺胺嘧啶的检测方法进行了研究.通过比较受试样品中未知峰与对照品色谱图保留时间的一致性和光谱指数图的匹配,建立了HPLC-PDA测定法,并进行相应的方法学研究.结果显示,磺胺嘧啶回收率为99.5％,RSD=0.4％;线性方程为y=873577x＋98154,R2 =1;检测限为5μg/mL,表明该检测方法准确可靠,可用于恩诺沙星注射液中非法添加磺胺嘧啶的检测.     

山茶蜂花粉粗多糖的脱蛋白研究

对山茶蜂花粉粗多糖的脱蛋白方法进行了初步研究.考察了不同体积乙醇沉淀多糖试样的效果和不同体积三氯乙酸沉淀多糖试样中蛋白质的效果,并经Sephadex G-75的洗脱.研究结果表明:乙醇体积为多糖液体积的3.5倍时,沉淀效果最好;多糖液中加入等倍体积的10%三氯乙酸时,脱蛋白效果最佳;多糖经Sephadex G-75的洗脱,出现两个洗脱峰.     

Methoxydextran as an indicator of gastrointestinal transit in Japanese quail

The recovery of polyethylene glycol, a substance frequently used as an indicator of the dynamics in the liquid phase of intestinal chyme, is considerably influenced by the presence of trichloroacetic acid in certain applications. The suitability of (methoxy-14C)methoxydextrane as an intestinal indicator in the Japanese quail was tested in the study. After the administration of this indicator, the bird expires less than 1% of the substance in form of 14CO2. The recovery of labelled methoxydextrane is 98 +/- 7%. The distribution of the indicator within the segments of the gastrointestinal tract does not differ significantly from the distribution of (14C)polyethylene glycol 4000, measured in the absence of trichloroacetic acid. The transit of the labelled methoxydextrane into the jejunum did not reduce the radio-chemical purity of the isotope, as compared with its original purity before administration. A slight decrease was only recorded during the measurement of the cumulative excretion of the indicator for 72 hours, but this decrease has no influence upon the suitability of methoxydextrane as an intestinal indicator. (Methoxy-14C)methoxydextrane does not interact with trichloroacetic acid, whereas when polyethylene glycol is used as an indicator, the substance precipitates from the liquid phase just at the concentration commonly used for the removal of protein from biological samples.     

An assay to quantitate the binding of Rhodococcus equi to macrophages.

A Rhodococcus equi radiobinding assay has been developed using organisms labeled with 3H-uracil. These labeled organisms resemble their unlabeled counterparts with respect to colony morphology, viability, and buoyant density. Bacteria routinely incorporate between 5 x 10(-3) and 5 x 10(-2) counts per minute per colony forming unit (cfu) which in this assay allows the detection of fewer than 0.2 cfu per macrophage. Once incorporated, greater than 90% of the label remains bacterial associated for at least 4 h postlabeling. The majority of the label is trichloroacetic acid precipitable, partitions into the aqueous phase following phenol/chloroform extraction and is ethanol precipitable. RNAse treatment of the ethanol precipitate abolishes label trichloroacetic acid precipitation. This radiolabeling technique has been used to quantitate the attachment of R. equi to both murine peritoneal and equine alveolar macrophages adherent to 13 mm glass coverslips. R. equi binding is dose dependent, saturable, and specific to macrophages. Further, binding is enhanced in the presence of fresh serum. Inhibition of radiolabeled bacterial binding can be obtained by competition with cold R. equi. This radiolabeled binding assay represents a crucial step in identifying the receptors on macrophages involved in the recognition of R. equi and may help to provide information on how macrophages recognize intracellular bacteria in general.     

塞曼效应石墨炉原子吸收法测定生物样品中钼含量的研究

首次对比研究了塞曼效应石墨炉原子吸收法测定生物样品中钼含量。以硝酸钯 (Pd(NO3 ) 2 )、三氯醋酸 (TCA)、及Pd(NO3 ) 2 TCA作为基体改进剂可使吸光度分别增加 5倍、12 .4倍及 19倍 ,检出下限分别为 2 5ng/mL、12ng/mL、10ng/mL。以塞曼效应扣除背景吸收 ,能有效地克服Fe、Cu、Mn等共存离子的干扰。通过对奶牛、雏鸡血清及组织中钼含量的测定可知 ,混合型的基体改进剂能够提高钼含量测定的灵敏度及回收率     

Detection of uremic peaks in dogs by anion exchange high performance liquid chromatography.

Sera of dogs with gentamicin-induced uremia were analyzed by high performance liquid chromatography system with strongly basic macroreticular anion exchange resin. Satisfactory separation of peaks was achieved with good reproducibility after deproteinization of sera with trichloroacetic acid at a final concentration of 3%, confirming that the system was suitable for qualitative analysis of uremic serum. The chromatograms showed that the number of peaks and the peak area had relation to concentrations of serum urea nitrogen or creatinine and severity of uremia. Four peaks were selected as suspected canine uremic peaks with high correlation to serum creatinine concentrations which were hardly influenced by extrarenal factors. The results suggested that these four fractions might contain uremic toxins.     

Urinary excretion of N-acetyl-beta-D-glucosaminidase and its isoenzymes in cats with urinary disease

The present study was designed to assess the clinical usefulness of measurement of urinary N-acetyl-beta-D-glucosaminidase (NAG) activity and its isoenzymes in cats with urinary disease. Thirty-five healthy cats and 9 cats with renal disease were used. Furthermore, a 5-year-old female cat was administered a large amount of sulfonamide in order to induce acute renal failure, and urine samples were collected for the assay of NAG activity and its isoenzymes. Urinary NAG activity was measured using p-nitrophenyl N-acetyl-beta-D-glucosaminide, and expressed as units per gram of urinary creatinine (NAG index). Urinary NAG isoenzymes were assayed by use of the mini-column method and electrophoresis. The overall mean value of urinary NAG index in healthy cats was 1.6+/-1.5 U/g. Urinary NAG index varied from 6.2 to 35.5 U/g in cats with chronic renal failure. There was no significant correlation between BUN, serum creatinine concentration and urinary NAG index. In cats with feline lower urinary tract disease, normal values of urinary NAG index were observed. In the urine samples of healthy cats, the proportions of NAG isoenzyme A (NAG-A) and isoenzyme B (NAG-B) were 79.1+/-4.4% and 21.0+/-4.4%, respectively, as assayed by the mini-column method. In the assay of NAG isoenzymes by electrophoresis, the proportions of NAG-A and NAG-B in healthy cats were 66.6+/-5.8% and 33.4+/-5.8%, respectively. The proportion of NAG-B as measured by electrophoresis was significantly larger (p<0.05) than that obtained with the mini column method. A feline case of acute renal failure experimentally-induced by sulfonamide showed elevation of urinary NAG index, NAG-A and NAG-B after injection of sulfonamide. The increase in NAG-B was larger than that of NAG-A. From the results reported here, measurement of urinary NAG and its isoenzymes seems to yield information about tubular damage at an early stage in cats with urinary disease.     

饲料和鸡肉中利巴韦林的测定--超高效液相色谱-串联质谱法

本研究建立了饲料和鸡肉中利巴韦林的UPLC-MS/MS分析方法。样品经三氯乙酸-乙腈溶液提取,离心后提取液经PBA固相萃取小柱净化,0.1 mol/L甲酸水溶液溶解,进行UPLC-MS/MS分析。结果显示,利巴韦林在10.0～500.0 g/L浓度范围内线性关系良好,r2=0.9991。以浓缩饲料、配合饲料、预混料和鸡肉等为添加基质,其回收率在90.0%～114.5%,检出限为25.0 g/kg,定量限为50.0 g/kg。表明本方法灵敏度高、专属性好、操作简单、结果可靠,可满足饲料和鸡肉中利巴韦林的分析检测。     

Method for determining serum pepsinogen concentration, using pepsin standards and ultraviolet absorbance

A simplification of the traditional hemoglobin methods for determining serum pepsinogen concentration was developed. In this method, 10% trichloroacetic acid solution was added to control samples, and hemoglobin substrate was added to controls and active enzyme samples; standards and samples were incubated for 18 hours, the proteins in the active tubes were precipitated with trichloroacetic acid and removed by filtration, and the absorbances of the supernatant of each standard and sample at 280 nm were measured. The major differences between this method and other methods for determining pepsinogen values are that the preacidification of serum with hydrochloric acid was eliminated, the incubation period was reduced to 18 hours (down from 24 hours), the relative pepsinogen concentration was determined by measuring the concentration of hydrolysis products, using ultraviolet, rather than visible absorbance, and a pepsin standard curve was used to determine the serum pepsinogen concentration. Comparison of freshly prepared pepsinogen and pepsin standard curves indicated that the pepsinogen preparations were slightly more active than the pepsin preparations (on a weight-to-weight basis) on the same substrate. Pepsin standards are used because they are more stable than pepsinogen standards. Three linear standard curve ranges were used: O 10 to 100, 50 to 300, and 100 to 500 ng of pepsinogen/ml of serum. The use of pepsin standard curves permits some variability of the incubation conditions without altering the results. For best results, the hemoglobin substrate solution should be prepared daily. This method may be useful in diagnosing ostertagiasis.     

Establishment of Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Determination Method of Amantadine in Chicken and Preparation of Positive Samples

An ultra performance liquid chromatography (UPLC)-tandem mass spectrometry analysis method was established in this experiment for determination of amantadine in chicken.1% trichloroacetic acid with methanol as extraction liquid, the ultrasonic assisted solvent extraction was used to extract amantadine in chicken, extract liquid was purified and enriched in MCX solid-phase extraction column.Using C18 chromatographic column as separation column, in the positive ion mode, we detected the samples by electrospray ionization tandem mass spectrometry.Mass spectrometry and chromatographic conditions, the type of extract liquid, ultrasonic extraction time, the types of solid-phase extraction column and eluent were optimized.Under optimal conditions, a good linearity (r=0.9998) in the ranges of 0.1 to 20.0 ng/mL was obtained, the recoveries were 89.7% to 101.4% in the additive levels of 5.0, 10.0 and 20.0 μg/kg, the limit of detection (LOD) of amantadine was 0.07 μg/kg, the limit of quantiation (LOQ) of amantadine was 0.23 μg/kg, and the relative standard deviations (RSD) were below 7.0%.This novel approach had high sensitivity and accuracy, and was successfully applied to rapid and high sensitive detection of amantadine residues in chicken.The positive samples of amantadine in chicken were successfully prepared with concentrations of 11.16 and 7.18 mg/kg, which could be used for the research of ELISA kits of amantadine.     

鸡肉中金刚烷胺超高效液相色谱-电喷雾串联质谱检测方法的建立及阳性样品的制备

本试验建立了一种超高效液相色谱-电喷雾串联质谱测定鸡肉中金刚烷胺的分析方法。以甲醇-1%三氯乙酸作提取剂,采用超声波辅助溶剂萃取法萃取鸡肉中金刚烷胺,萃取液用MCX固相萃取柱进行净化浓缩。以BEH C18色谱柱为分离柱,在正离子模式下以电喷雾电离串联质谱仪进行测定。对质谱和色谱条件、提取剂的种类、超声提取时间、固相萃取柱及洗脱液的种类进行了优化,在0.1~20.0 ng/mL范围内线性关系良好(r=0.9998)。样品在5.0、10.0 和20.0 μg/kg添加水平的回收率为89.7%~101.4%,相对标准偏差(RSD)小于7.0%;方法的检测限为0.07 μg/kg,定量限为0.23 μg/kg。本方法灵敏度高、准确度高,能满足公司及相应鸡肉行业的客户进行鸡肉中金刚烷胺残留量的快速、高灵敏检测分析。本试验还根据金刚烷胺在动物体内的代谢动力学成功制备了浓度为11.16和7.18 mg/kg的金刚烷胺阳性鸡肉样品,为研发金刚烷胺ELISA试剂盒提供了相应的阳性样品。