饲料中聚醚类抗生素的柱后衍生同时测定方法研究

采用高效液相色谱柱后衍生化法同时检测饲料中莫能菌素和盐霉素的含量。试验采用两个柱后衍生反应器,使莫能菌素和盐霉素在柱后分离后与衍生试剂1(硫酸)和衍生试剂2(香草醛)在不同的反应器中先后分别发生反应,经检测莫能菌素和盐霉素的保留时间分别在13min和17min左右。在饲料中添加莫能菌素和盐霉素浓度分别为20、60、120μg/mL时,莫能菌素的平均回收率为93.0%～97.7%,变异系数为1.05%～1.54%;盐霉素的平均回收率为94.0%～96.5%,变异系数为0.78%～1.01%。     

高效液相色谱柱后衍生化法测定饲料中盐霉素

对高效液相色谱柱后衍生化测定饲料中盐霉素的方法作验证研究.采用Cl8反相柱,甲醇—冰乙酸—水为流动相,香草醛为衍生剂.方法检测极限为0.5μg/ml,添加回收率为85.6%,变异系数为4.92%.     

饲料中T-2毒素的高效液相色谱-荧光法检测研究

以甲醇和水(体积分数8∶2,v/v)为提取溶剂,采用硅镁吸附剂层析柱结合T-2毒素免疫亲和柱两阶段净化处理、柱前衍生,建立了饲料中T-2毒素的高效液相色谱法结合荧光检测方法。衍生液选用2-萘甲酰氯(2-NC),4-甲基氨基吡啶(DMAP)作为反应促进剂。确立的色谱检测条件为:流动相是乙腈和水(体积分数75∶25,v/v),激发波长(Ex)381 nm,发射波长(Em)470 nm,流速0.6 mL/min,柱温30℃。方法的检测限1.8 ng/g,定量限为6×103 ng/kg,标准方差(RSD)低于4.25%,回收率72.24%～83.31%,T-2毒素在25～800 ng/mL范围内线性回归良好,线性方程为:y=2186.2x-31953,标准方差R2=0.9992。本方法简单快速、灵敏、准确、重复性好,能满足饲料中检测T-2毒素的需要。     

饲料中甲砜霉素GC-MS检测方法的研究

用气相色谱-质谱法(GC-MS)测定饲料中甲砚霉素的含量,检测限为0.5μg/kg.通过色谱分离,保留时间与标准一致,选择离子相对丰度与标准均不大于20%,结果表明,该方法适用于饲料中的甲砜霉素含量测定.     

高效液相色谱法测定盐霉素预混剂中盐霉素A的含量

建立了测定盐霉素预混剂中盐霉素A含量的高效液相色谱分析方法。采用C18色谱柱,甲醇-水-冰醋酸(94︰6︰0.1)等度洗脱,甲醇-硫酸-香草醛(95︰2︰3)为衍生试剂,流速0.7 mL/min;衍生温度98 ℃;检测波长520 nm;进样量20 μL。结果表明,盐霉素A在0.4～0.6 mg/mL范围内线性关系良好（r2=0.9998）,平均回收率100.6%。该方法简便、准确、可行。     

HPLC测定混合型饲料添加剂中L-苹果酸含量

用HPLC法测定混合型饲料添加剂中L-苹果酸(malic acid,LMA)的含量。采用C18(250 mm×4.6 mm×5μm)色谱柱,以0.02 mol/l磷酸二氢钾:乙腈=982(v:v),流速0.8 ml/min,柱温25℃,检测波长210 nm,进样量10μl。结果表明:L-苹果酸对照品和样品的保留时间均是3.9 min,其线性方程分别为Y=0.390 9X-4.578 6,R=0.999 8(n=5),在126.25~2 020μg/ml的范围内,线性关系良好,其最低检测量为12.23μg/ml。精密度的RSD为0.43%,表明此方法精密度较好;重现性的RSD为0.17%,表明重现性良好;回收率的RSD为0.61%。用此方法检测出其混合饲料添加剂中L-苹果酸含量为(99.01±0.11)%,符合产品规定的质量要求。该实验建立的HPLC方法可快速、准确测定混合饲料中L-苹果酸含量,为其质量控制提供科学依据。     

预混料中越霉素A的反相液相色谱分析

用0.1mol/LpH7.8磷酸盐缓冲液提取样品中的越霉素A,过CG-50弱酸性阳离子交换树脂浓缩和纯化,邻苯二甲醛(o-phthalaldehyde,以下简称OPA)衍生、在32℃条件下衍生30min,进样。色谱柱采用HypersilBDS系列C18,250mm×4.6mmi.d,粒径10μm,柱温30℃,流速1.0ml/min,以甲醇∶磷酸盐缓冲液0.033mol/LpH7.0=53∶47为流动相,波长333nm条件下,用紫外检测器检测。在该条件下,各组分得到良好的分离,越霉素A保留时间为16.0min左右,依次进样,30min完成色谱分离。该方法的线性范围为9.355～748.52mg/L,回收率为86.34%～94.48%,批内变异系数小于4.84%,批间变异系数小于6.07%。     

微生物抑制法检测饲料中盐霉素含量的研究

从几种标准工作菌株中筛选出对盐霉素敏感的菌株,以敏感菌株为工作菌株测定饲料中盐霉素的含量。结果显示:枯草芽孢杆菌、藤黄微球菌对盐霉素不敏感,地衣芽孢杆菌、嗜热脂肪芽孢杆菌对盐霉素敏感。以嗜热脂肪芽孢杆菌敏感菌株为工作菌株测定饲料中盐霉素含量,最低检出浓度为0.25μg/ml,饲料中最低检出限为1.0mg/kg,标准曲线相关系数为0.99,回收率在60%～80%,均高于标准中规定的参数。试验结果表明:微生物抑制法检测饲料中盐霉素含量,灵敏度高,快速,简便。     

莫能菌素和盐霉素在鸡组织中的残留分析方法研究

本文报道用高效液相色谱柱后衍生化检测肉鸡肌肉、肝脏和脂肪组织中莫能菌素和盐霉素的残留。肉鸡肌肉、肝脏和脂肪组织经异辛烷提取,用硅胶柱净化分离,洗脱液浓缩后用甲醇／水溶解。以甲醇／冰乙酸／水（943／30／30,v/v）作为流动相,香草醛为衍生剂,用RP－C18柱在可见波长520nm处检测。将莫能茵素和盐霉素分别以0．10,0．20,0．40和0．20,0．40,0．800644g/g分别添加到空白肉鸡组织中,测得莫能菌素和盐霉素在肌肉、肝脏和脂肪组织中的平均回收率分别为97．7％、91．1％、92．1％和94．1％、85．4％、90．7％,变异系数范围在2．7％－16．8％之间。用该方法测定肉鸡组织中莫能菌素和盐霉素残留的最低检测限分别为0．05μg/g0．1μg/g。     

固相萃取-高效液相色谱法测定饲料中的地西泮

本文建立了固相萃取-高效液相色谱法测定饲料中地西泮的方法。在碱性条件下,以二氯甲烷和正己烷(3∶7,v/v)提取饲料中的地西泮,再经SLB固相萃取柱净化,以甲醇和水为流动相,利用反相高效液相色谱-紫外检测法检测。此方法平均回收率为94.0%,变异系数为7.0%。     

动物源性食品中莫能菌素残留ELISA试剂盒的研制

利用碳化二亚胺法合成抗原,进行动物免疫,制备特异性强的单克隆抗体。在筛选莫能菌素单克隆抗体的基础上,建立了莫能菌素ELISA检测方法,并研制出利用单克隆抗体对动物源性食品中残留的莫能菌素进行检测的试剂盒。本试剂盒的最低检测限为1μg/kg,试剂盒标准品变异系数为5.4%～13.1%,肌肉、肝脏样本的变异系数为5.1%～14.2%,肌肉和肝脏样本添加回收率分别为67.5%～87.4%和64.7%～86.7%。该方法的建立为莫能菌素的残留检测提供了可靠的分析检测手段。     

Detection of Salmonella by using the colorimetric DNA/rRNA sandwich hybridization in microtiter wells

A rapid and readily available DNA probe kit was developed for the detection of Salmonella spp. This kit utilized the colorimetric DNA/rRNA sandwich hybridization method in microtiter wells. Within 3 hr Salmonella spp. in selective enrichment broth cultures were detected by the DNA probe kit. The kit effectively identified all of 187 strains of Salmonella tested and yielded no false-positive reactions in the examination of 674 pure cultures of non-salmonellae. The DNA probe kit could detect 10(5) cfu/ml in pure culture. A total of 379 naturally contaminated samples (raw chicken meat, liquid egg, animal feeds, poultry feces and frozen foods) were tested, both by the standard culture method and the DNA probe kit. The 169 of these samples were culture positive and 210 were culture negative. The sensitivity of the DNA probe kit was 98.2% (166/169) and the specificity was 99.5% (209/210). These results show that the DNA probe kit is a useful tool to examine a large number of various samples for contamination by Salmonella spp. in food and livestock industry.     

Effectiveness of the use of momensin for cattle fattening

Two groups of fattened bulls (125 bulls in each group) were investigated for the effect of monensin (125-175 mg per head/day) on live weight gains and for the effectiveness of monensin administration. After 160 days of fattening, the average daily weight gain was 713 g in the control group and 800 g in the monensin-treated group (an increase by 12.2%). After 11 months of fattening the daily weight gain was 702 g in the control group and 768 g in the monensin-treated group (an increase by 9.4%). Besides the control and experimental groups, monensin was administered to 1500 head of fattened cattle on the whole. Greater differences in the daily live weight gains (higher gains in the monensin-treated animals) were recorded mainly in the period when the feed ration contained high-quality bulk feeds. When the bulls were given feeds of lower quality (mainly late in winter), the differences in the average daily live weight gains decrease and the effect of monensin treatment is not so great. Throughout the fattening period, monensin had a favourable influence on the live weight gains and its use was economically advantageous.     

Effect of three polyether ionophores on pharmacokinetics of florfenicol in male broilers

Drug–drug interactions (DDIs) may adversely affect the prevention and cure of diseases. The effects of three polyether ionophore antibiotics, salinomycin (SAL), monensin (MON), and maduramycin (MAD) on the pharmacokinetics of florfenicol (FFC) were investigated in broilers. The chickens were fed rations with or without SAL (60 mg/kg feeds), MON (120 mg/kg feeds), or MAD (5 mg/kg feeds) for 14 consecutive days. FFC was given to the chickens either intravenously (i.v.) or orally (p.o.) at a single dose of 30 mg/kg body weight. Blood samples were taken from each chicken at 0–24 h postadministration of FFC. The plasma concentration of FFC was detected by high‐performance liquid chromatography. The plasma concentration of FFC decreased with i.v. or p.o. co‐administration of SAL, MON, or MAD in broilers, implying occurrence of DDIs during the co‐administration of FFC with these ionophores. Our findings suggest that more attention should be given to the use of FFC to treat bacterial infections in chickens supplemented with polyether ionophore antibiotics.     

食糜样本中11种生物胺同步检测方法的建立

旨在建立一种利用液质联用（liquid chromatography mass spectrometry,LC-MS）技术实现对猪肠道食糜中多种生物胺进行同步检测的方法。试验首先优化了丹磺酰氯（dansyl chloride,DNS）对生物胺的衍生条件,并进一步确定了肠道食糜样品中生物胺的最佳净化方法。结果显示,衍生条件在衍生剂浓度为8 mg·mL^-1,衍生温度为40℃的条件下,衍生90 min为最佳。生物胺净化方法确定为先用乙酸乙酯进行提取,衍生结束后再用乙酸乙酯进行萃取的组合方式提取生物胺的效果最好;11种生物胺在5～1 000 μg·L^-1的质量浓度范围内定量结果呈良好的线性关系（R²>0.99）,检出限和定量限分别为0.01～0.23 μg·L^-1和0.03～0.76 μg·L^-1;各生物胺的日内相对标准偏差在0.73%～ 7.92%,日间相对标准偏差在0.07%～8.67%。最后采用本方法对仔猪回肠、盲肠、结肠和直肠食糜样品中的生物胺进行检测,初步确定了11种生物胺在不同肠段食糜中的含量分布。通过对仔猪回肠、盲肠、结肠和直肠中的食糜样品进行检测,发现食糜中的生物胺主要为亚精胺、腐胺、尸胺、精胺和组胺,而酪胺、1,7-二氨基庚烷、5-羟色胺和胍基丁胺4种生物胺含量较少,色胺和苯乙胺未检出。综上所述,本试验建立的方法具有结果准确、灵敏度高、重现性好等优点,可用于仔猪不同肠道段食糜中多种生物胺的同步检测。     

多壁碳纳米管分散固相萃取结合LC-MS/MS测定饲料中11种β-受体激动剂

本研究建立了采用多壁碳纳米管(MWCNTs)为吸附剂的分散固相萃取(dSPE)净化、液相色谱串联质谱测定饲料中11种β-受体激动剂(克伦特罗、莱克多巴胺、沙丁胺醇、苯乙醇胺A、氯丙那林、妥布特罗、西马特罗、特布他林、马布特罗、班布特罗和喷布特罗)的方法。饲料样品经甲酸水溶液提取后,调节pH值至10,加入50 mgMWCNTs进行分散固相萃取,被吸附的化合物经1.0%甲酸水甲醇(91)洗脱,洗脱液过滤膜后进行LC-MS/MS分析。采用Acquity UPLC BEH C18色谱柱分离,以0.1%甲酸水溶液和甲醇为流动相进行梯度洗脱,电喷雾正电子(ESI+)模式电离,多反应监测(MRM)模式监测,内标法校准进行定量。结果表明:11种β-受体激动剂在0.1～20μg/L浓度范围内呈良好的线性,线性相关系数均大于0.997,猪配合饲料样品中最低定量限为0.05～0.48μg/kg。猪配合饲料中添加0.5～500μg/kgβ-受体激动剂的回收率在89.6%～112.9%,相对标准偏差RSD均小于15%。     

Surveillance of Toxigenic Fungi and Ochratoxin A in Feedstuffs from Córdoba Province,Argentina

The aim of this work was to evaluate the incidence of potential ochratoxigenic mycoflora and ochratoxin A (OA) in poultry, pig and rabbit feeds. Eighty poultry, pig and rabbit feed samples were taken at random from factories located from Córdoba province, Argentina, over a period of 8 months. Isolation and quantitative enumeration of fungal propagules were done on DRBC and DG18 media. The predominant species wereA. candidus, A. flavus,A. terreus,A. parasiticus,P. implicatum P. minioluteum,P. crustosum andP. citrionigrum. The distribution of sectionNigri species varied according to the feedstuffs analysed. The frequency ofA. niger var.niger was noticeably high in poultry feed samples on DRBC medium. TheNigri section species was present at moderate mean colony counts (CFU/g) from three feeds. Mycotoxin analysis of these samples showed that OA was detected in 15%, 10% and 12% of pig, poultry and rabbit feed samples, respectively. The mean levels detected ranged between 15 and 25 ng/g from three feeds. The presence of ochratoxigenic species ofNigri section and OA in feeds indicates the risk of potential exposure of poultry, pigs and rabbits through the ingestion of feeds.     

微生物检定法测定莫能菌素的含量

试验建立一种新的抗生素微生物检定法测莫能菌素预混剂含量的方法。以无水甲醇为溶剂,超声浸提30min,以枯草芽孢杆菌为试验菌,用pH值6.0灭菌缓冲盐溶液为稀释液,按抗生素微生物检定法检测,莫能菌素浓度在2~10U/ml范围内抑菌圈直径与浓度的对数之间线性关系良好。结果表明:其相关系数为0.9983(n=8),准确度为90.0%～110.0%,分析精密度为RSD2.8%～2.9%,人员精密度为RSD为2.9%。此方法与国标法相比,甲醇用量少,降低环境污染,快速、准确、可靠。     

超高效液相色谱―串联质谱法测定饲料中的万古霉素

试验旨在建立饲料中万古霉素的超高效液相色谱—串联质谱检测方法,以0.05 mol/L磷酸盐缓冲液—乙腈(85:15,V/V)提取样品中的万古霉素,提取液经过强阳离子交换固相萃取柱净化,采用液相色谱—串联质谱多反应监测模式进行测定,外标法定量。不同饲料基质中,万古霉素在25~1000 ng/mL浓度范围内呈良好的线性关系。本方法对万古霉素在饲料中的检测限为0.05 mg/kg,定量限为0.1 mg/kg。在不同饲料中添加浓度范围为0.1~100 mg/kg,其平均回收率为86.7%~117.1%,批内变异系数在1.0%~17.4%之间,批间变异系数在2.9%~10.1%之间。本方法分析速度快、灵敏度高、重现性好,各项技术指标均满足国内外相关法规要求,可用于饲料中万古霉素含量的测定。