土霉素在鸡蛋中的残留消除研究

研究临床使用常规剂量及养殖环节加大盐酸土霉素可溶性粉剂量后其在鸡蛋中的残留消除规律。对产蛋期蛋鸡分别给予500 mg/L和667 mg/L 50%盐酸土霉素可溶性粉,集中饮水给药,每日1次,连续5 d。分别采集停药后1～12 d的鸡蛋样品,采用UPLC-MS/MS测定鸡蛋中土霉素的残留量。按上述方法给药后,低、高剂量组分别在停药第1天和停药第2天达到土霉素残留量最高。低、高剂量组的最高残留量分别为142. 72、82. 84μg/kg,均低于国家规定的最高残留限量(200μg/kg),不会造成土霉素残留超标。本试验通过研究土霉素可溶性粉在鸡蛋中的残留情况,确定弃蛋期前后土霉素残留量,以期为蛋鸡产蛋期安全用药,保障食品安全提供科学依据。     

牛奶中多西环素残留消除及最大残留限量

为制定牛奶中多西环素最大残留限量标准,首先开展盐酸多西环素子宫注入剂在牛奶中的残留消除试验,盐酸多西环素子宫注入剂经子宫给药1次,给药前及停药后6,12,24,48,72,96,120,144和168 h共10个时间点采集牛奶,采用液相色谱-串联质谱测定多西环素残留量。然后在查阅国际食品法典委员会、欧洲药品管理局和我国制定动物性食品中多西环素最大残留限量研究报道基础上,制定牛奶中多西环素最大残留限量标准。结果表明,停药后168 h,牛奶中多西环素的残留量均低于10μg/kg。多西环素每日允许摄入量为0～3μg/kg,建议参照欧盟动物性食品中多西环素最大残留限量制定方法,将每日允许摄入量按比例分配到全部组织+奶,推荐牛奶中多西环素的最大残留限量为10μg/kg,盐酸多西环素子宫注入剂在牛奶中弃奶期为9 d。     

猪肉中四环素类抗生素残留的ELISA检测

采用间接竞争ELISA检测猪肉及内脏组织中土霉素、四环素、金霉素的残留量,在50~300μg/kg浓度范围内,标准添加样品回收率81.3%~122.4%,变异系数2.7%~11.1%。本方法的建立为四环素类抗生素残留的快速检验试剂盒的研制奠定了基础。     

牛奶中抗生素残留规律研究

为了解新疆地区奶牛生产中常用抗生素类药物在牛奶中的残留情况,本试验按照新疆地区奶牛生产中抗生素类药物的使用方法及剂量进行投放,并采集牛奶样品进行检测。结果表明,青霉素钾在牛奶中的残留规律为：停药1d〉5d〉3d;头孢噻呋钠为：停药3d〉5d〉1d;链霉素为：停药1d〉3d〉5d;长效土霉素在牛奶中的残留量在停药1～11d一直维持在175μg／kg左右。本试验中除了长效土霉素外,其他药物的残留量均低于中国和欧共体（EEC）规定的标准残留限量。     

恩诺沙星微囊制剂在猪体内残留消除规律研究

采用高效液相色谱法研究恩诺沙星微囊制剂在猪体内各组织中的残留消除规律。恩诺沙星微囊制剂混饲,连续给药7 d,宰杀猪,取组织。组织样品经磷酸盐缓冲液提取,C18固相萃取柱净化,过膜,用流动相:0.05 mol/L磷酸溶液/三乙胺-乙腈(82+18)溶解,微孔过滤,进行HPLC分析。结果表明,当样品浓度为0.02～1μg/g时,线性关系良好。该法最低检测限为20μg/kg,回收率在72%～90%之间,变异系数小于10%。残留在肌肉、脂肪中的恩诺沙星和环丙沙星消除较快,第5天总残留量已下降至检测限以下;肝和肾脏中的残留药物消除较缓慢,第7天肾中药物总残留量为91.48μg/kg。综合各组织中总残留量和MRL规定,建议恩诺沙星微囊制剂休药期为7 d。     

牛奶中氯唑西林残留量的高效液相色谱法测定

为建立高效液相色谱法测定牛奶中氯唑西林残留的方法,牛奶样品用乙腈沉淀蛋白,三氯甲烷反萃取,有机相旋干用流动相溶解,正己烷除脂。0.02mol/L磷酸二氢钾溶液(pH 5.0)-乙腈(68∶32)比例为流动相,C18反相色谱柱分离,紫外检测。结果氯唑西林在15μg/kg～1 000μg/kg范围内呈现良好的线性关系,相关系数大于0.999 0;在15、30、60μg/kg三个添加水平,氯唑西林在牛奶中的平均回收率为70.25%～94.38%,批内变异系数小于8.34%,批间变异系数小于3.52%。方法检出限为5μg/kg,定量限为15μg/kg。表明该检测方法简单、灵敏、可靠,适用于牛奶中氯唑西林残留的分析检测。     

奶及奶粉中4种阿维菌素类药物残留检测高效液相色谱法

建立一种适用于牛奶、羊奶和奶粉中乙酰氨基阿维菌素、阿维菌素、伊维菌素和多拉菌素的残留检测方法。奶及奶粉中的4种阿维菌素类药物用20%乙醇乙腈溶液提取,加水和微量三乙胺稀释后经C18固相萃取柱净化,氮气吹干后,65℃避光衍生化反应15 min。用高效液相色谱—荧光检测法分析,外标法定量。4种药物在0.5~1 000 ng/m L浓度范围内线性关系良好,在牛奶、羊奶中的定量限为0.5μg/kg;在奶粉中的定量限为5μg/kg。一定浓度范围内,4种药物在牛奶、羊奶和奶粉中的平均回收率为89.2%~113%;批内变异系数为0.450%~9.73%,批间变异系数为2.90%~10.6%。该方法操作简单、灵敏度高、准确度和精密度好,适用于牛奶、羊奶及奶粉中阿维菌素类药物的残留检测。     

液相色谱-串联质谱法检测牛奶中的四环素类药物

本文以液相色谱-串联质谱法检测牛奶中的四环素类药物残留,试料中残留的四环素、土霉素、金霉素和多西环素经Mcllvaine-Na2EDTA缓冲液提取,HLB柱净化。用液相色谱-串联质谱法测定,外标法定量。经实验,制得10、20、50、100、200、500 ng/m L的系列标液。以系列标液浓度为横坐标,对应峰面积为纵坐标进行线性回归,四环素、土霉素、金霉素和多西环素在10~500 ng/m L范围内呈现良好的线性关系,R2均大于0.990。四环素、土霉素、金霉素和多西环素在牛奶中的检测限为5 ng/g,定量限为10 ng/g。在10~200 ng/g添加浓度的回收率为70%~120%。批内变异系数≤20%,批间变异系数≤20%。     

阿莫西林在鸡组织中的残留消除规律

本试验旨在研究阿莫西林在鸡组织中残留消除规律。鸡组织样品经磷酸二氢钠溶液提取,乙腈去蛋白,正己烷去脂肪,饱和二氯甲烷萃取,上清液经水杨醛在酸性条件下沸水浴衍生化后,在激发波长358nm、发射波长440nm处用高效液相色谱荧光检测器检测。该方法测定鸡组织中阿莫西林的检测限为5μg/kg、定量限为13μg/kg。阿莫西林在鸡组织样品中平均回收率为73.64%~88.82%,相对标准差为6.21%~9.63%。各试验组京海黄鸡分别按体质量以30、60mg/kg剂量内服阿莫西林,每天1次,连续给药7d,休药4h后(零休药期)各组织中阿莫西林残留量最高,休药第3天各组织中阿莫西林残留量均迅速降低;休药后第5天阿莫西林残留量低于最高残留限量(50μg/kg);休药第9天所有组织中阿莫西林残留量均低于检测限;休药后相同时间点鸡肌肉中药物残留量最低,肝脏中的药物残留量最高;阿莫西林在鸡肾脏中残留比鸡肌肉、肝脏消除缓慢且消除时间长;阿莫西林在鸡组织中的残留量与给药剂量呈正相关。根据WT1.4软件计算所得,建议黄羽肉鸡按体质量以30、60mg/kg剂量给药,每天1次,连续7d后,其休药期(WT)分别为3、4d。     

动物组织中四环素类抗生素残留的HPLC检测研究

本试验建立了动物组织中四环素类药物残留的高效液相色谱测定方法。试验分别采用50 mL/L高氯酸、Mcllvaine缓冲液为样品提取液;乙腈/磷酸二氢钠、甲醇乙腈0.01mol/L草酸(1.5 1.5 2.0,pH 2.0)为流动;流速1.0 mL/min,组合测定。结果显示,50 mL/L高氯酸为样品提取液,流动相甲乙腈0.01 mol/L草酸,检测灵敏度较高在此色谱条件下,土霉素、四环素、金霉素的最低检测限为100μg/kg,在100μg/kg~1 000g/kg浓度范围内,标准添加回收率为74.3%~97.1%,变异系数为0.43%~9.54%。本方法适用于四环素类抗生素残留的确证性定量分析。     

Study on Optimizations of Oxytetracycline Residues Detection Method in Milk and its Clinical Elimination

Here an improved HPLC-PAD method was developed and demonstrated suitable for studying the clinical residue elimination of oxytetracycline in milk from postpartum cows with uterine diseases after intrauterine treatment with oxytetracycline.In this study, 0.1 mol/L Na₂EDTA-Mcllvaine buffer solution was used to extract oxytetracycline from milk, n-hexane for degreasing, and a 3 CC Oasis HLB solid-phase extraction column for purification and concentration.A reversed phase chromatographic column Symmetry Shield RP C18 (250 mm×4.6 mm, 5 μm) with acetonitrile and 0.048% phosphoric acid solution (pH 2.5) as mobile phase by gradient eluting was applied to as the chromatographic separation condition.The results showed that the retention time of oxytetracycline was 6.5 min with a symmetrical and sharp peak, which was well separated from the impurity in the milk sample.Oxytetracycline standard solutions within the concentrations ranging from 5 to 2 000 μg/kg, had a good linearity between peak areas and the concentrations, R²=0.9999.The recoveries of oxytetracycline at three levels of 10, 20, 100 μg/kg were 87.9%, 90.5% and 87.8%, respectively, with the coefficient of variation ranged from 2.2% to 5.8% in intra-day and 4.0% to 5.1% in inter-day.The LOD was 5 μg/kg and the LOQ was 10 μg/kg.All of those parameters suggested that the optimized method was suitable for the study on the clinical residue elimination of oxytetracycline.29 postpartum cows with uterus disease were randomly divided into four different groups in terms of dosage, 2, 3, 4 and 5 g, and treated with oxytetracycline by intrauterine infusion administration, and the residues of oxytetracycline in milk were detected to study its clinical residue elimination.The results showed the oxytetracycline in milk fell to the MRL during 72 h after treatment at 4 g and below dose group.However, in 5 g dose group, residue levels of oxytetracycline in milk were above the MRL at 72 h after treatment.Therefore, 5 g and the higher were not recommended for intrauterine infusion administration.     

抗菌药物治疗奶牛子宫内膜炎后牛奶中药物残留及其对酸奶发酵的影响

用TTC法检测44例经过抗菌药物治疗的患子宫内膜炎荷斯坦奶牛。其中，41例采用子宫内灌注药物治疗，主要药物有土霉素、金霉素和庆大霉素；每个病例治疗1～5次不等，3例采用静脉注射，主要药物有磺胺嘧啶钠和头孢噻啶。结果表明，31例经土霉素子宫内灌注的奶牛，停药后第24h所采的奶样均可检出抗生素；其中6头奶牛在第6d仍然抗生素阳性，残留时间最长的可达8d。8例用金霉素子宫灌注治疗的奶牛，停药后第48h所采的奶样，抗生素检出率100％，其在牛奶中的残留时间为2～5d不等。2例经庆大霉素子宫灌注治疗的患病牛，药残时间分别为1～2d。3头经静脉滴注治疗的病牛，牛奶中的药残时间约达到9d以上。此外，从3头用金霉素子宫内灌注的奶牛采集牛奶用于乳酸菌发酵试验。结果表明，牛奶TTC阳性时，发酵产酸较低。在治疗后6h采集的牛奶都不能正常发酵产酸、凝固。3例试验牛牛奶发酵产生的酸度分别于治疗后第13、24和48h恢复到治疗前的水平。     

Disposition kinetics of gentamicin in normal and endometritic cows using a microbiological assay

Gentamicin concentrations in serum, urine and milk were assayed microbiologically after intramuscular and intrauterine administrations in normal and endometritic cows. Following intramuscular injections of 5 mg gentamicin/kg b. wt. 3 times daily for three consecutive days, the highest serum concentrations occurred 1 h post administration of each dose with absorption half-lives [t0.5(ab)] ranging from 0.23 to 0.30 h for normal cows and from 0.21 to 0.29 h for endometritic cows. The elimination half-lives [t0.5(beta)] ranged from 2.51 to 2.95 h (normal cows) and from 2.71 to 3.29 h (endometritic cows). Following intrauterine administration of 4 mg gentamicin/kg. b. wt. once daily for three consecutive days, the drug peaked in serum 2 h after each dose with [t0.5(ab)] ranging from 0.47 to 0.52 h (normal cows) and from 0.57 to 0.68 h (diseased cows), while the drug was eliminated faster in endometritic cows than in normal cows. The mean systemic bioavailability were (70%) and (30%) after intramuscular and intrauterine administration, respectively. To compare serum concentrations, gentamicin was assayed in urine and milk in high and low concentrations, respectively.     

Tilmicosin antibacterial activity and pharmacokinetics in cows

The minimal inhibitory concentration (MIC) of tilmicosin for 90% of 112 Staphylococcus aureus isolates from the bovine udder was 0.78 μg/mL and 149 of 164 (90.8%) other gram-positive udder pathogens were inhibited by tilmicosin concentrations < 3.12 μg/mL. The MIC of the drug for 19 of 22 S. aureus isolates was < 0.78 μg/mL when the test was conducted using Mueller-Hinton (MH) agar or MH agar containing 7.5% skimmed milk. Acute cardiac toxicity followed intravenous (i.v.) injection of the drug at 10 mg/kg to 3 cows, but animals appeared clinically normal within 30 min after treatment. The pharmacokinetics of i.v.-administered tilmicosin is typical for the macrolide class of antibiotics, i.e. low serum drug concentrations and a large volume of distribution (> 2.0 L/kg). The elimination half-life (t_1/2β values for 3 cows were 46.4. 56.0 and 72.8 min. The drug was administered subcutaneously (s.c.) to 5 cows at 10 mg/kg; the elimination half-life (t_1/2el) was 4.18 ± 0.55 h and the mean s.c. bioavailability was 22%. Rapid and extensive penetration of tilmicosin from blood into milk, and slow elimination from the milk were among the characteristic kinetic features of the drug after i.v. and s.c. administration. Tilmicosin was injected s.c. at 10 mg/kg once to 9 cows after the last milking of lactation; dry udder secretion samples were collected daily for 11 consecutive days and assayed microbiologically. Concentrations of drug > 0.78 μg/mL were found in the secretion for 8–9 days after dosing. Systemic side-effects were not observed after s.c. drug administration.     

超高效液相色谱-串联质谱法测定牛乳、发酵乳和乳粉中四环素类药物残留

建立超高效液相色谱-串联质谱法同时检测牛乳、发酵乳和乳粉3 种基质中4 种四环素类药物残留的检测方法。对前处理方法和仪器上机条件进行研究。以EDTA-Mcllvaine缓冲液进行提取,经HLB固相萃取柱净化,甲醇-乙酸乙酯（1∶9,V/V）洗脱,氮吹后复溶,采用电喷雾离子源正离子模式、多反应监测模式进行检测。结果表明：乳粉中四环素、土霉素、金霉素和强力霉素的定量限均为10 μg/kg;液态乳和发酵乳中4 种药物的定量限均为2 μg/kg;四环素类药物添加量为2～100 μg/kg时,回收率为63.1%～1     

Drug residues in milk after intrauterine injection of oxytetracycline, lincomycin-spectinomycin, and povidone-iodine in cows with metritis

A study was conducted to document the maximum retention times of antimicrobial residues in milk after their use in intrauterine treatment of metritis in lactating cows and to evaluate several risk factors hypothesized to influence the retention time of these drugs. Oxytetracycline (3 g), lincomycin-spectinomycin (2 g of one-third lincomycin and two-thirds spectinomycin), or povidone-iodine (6 g) were given to cows with metritis by intrauterine route. The Bacillus stearothermophilus var calidolactis disk assay was performed on each milk sample. Of the 61 cows treated with oxytetracycline, 30 had residues in their postinjection milk for variable periods (range, 12.5 to 44.0 hours; mean, 26.6 +/- 10.3). Of the 47 cows treated with lincomycin-spectinomycin, 17 had residues in their postinjection milk for various periods (range, 14.5 to 24 hours; mean, 19.5 +/- 8.9). Povidone-iodine was not detected in milk. Because a high number of cows (n = 61) were treated with oxytetracycline, only data from these cows were used in testing the influence of 3 factors (severity of metritis, time after parturition when cows with metritis were treated, and parity) on maximum retention of the drug in milk. Severity of metritis did not have a significant influence (P greater than or equal to 0.1) on the maximum retention time of the drug. The retention time decreased linearly with the increase of time after parturition when the cow with metritis was treated. First lactation cows had a significantly (P less than or equal to 0.01) shorter retention time than did older cows.     

Pregnancy rate following post-insemination intrauterine treatment of endometritis in dairy cattle

Summary Of 1500 examined Holstein cows in milk, there were 315 cows with mucopurulent discharges at the time of insemination (21%). Twenty-four hours after insemination, the infected animals randomly received no treatment (group I, control, n = 93), intrauterine infusion of 1 g oxytetracycline (group II, n = 63) and intrauterine infusion of 5 million IU procain penicillin G sodium (group III, n = 159). First service conception rate following treatment was 48.3, 49.2 and 47.7% in groups I, II and III, respectively. In conclusion, treatment with either antibiotic had no advantage relative to the control on first service conception rate.     

Strategy for the treatment of puerperal metritis and improvement of reproductive efficiency in cows with retained placenta

The objective of this study was to improve the reproductive efficiency of dairy cows with puerperal metritis (PM) subsequent to retained placenta (RP) using a two-step treatment strategy. A total of 188 postpartum cows, aged from 2 to 8 years, were utilised for 2 experiments. In Experiment 1, cows affected with RP/PM were randomly assigned to two treatment groups. Cows in Group A (n = 17) were treated with 600 mg of ceftiofur intramuscularly for 3 days followed by intrauterine lavage with 0.1% chlorhexidine and infusion with 0.5% povidoneiodine, while cows in Group B (n = 16) received two intrauterine infusions, first with 5 g of oxytetracycline and then with 0.5% povidone-iodine. Cows with normal postpartum findings were regarded as the healthy control group (n = 26). Ultrasonographic examination revealed that the ovarian activities including the appearance of a dominant follicle and days to first ovulation of the cows in Group A during the early postpartum period differed from those of Group B (P < 0.05), which coincided with the results of uterine swabbing for bacteriology. In Experiment 2, cows with normal postpartum findings were allocated to Group D (n = 78), which received an ovulation protocol (GnRH - 7 d PGF2α - 48 h hCG - 24 h AI) on day 50 ± 2 postpartum. Cows affected with PM were randomly divided into two groups, Group E (n = 25) combined the treatments applied in Groups A and D, while Group F (n = 26) repeated the treatment administered in Group E except for uterine lavage. The results indicated that the pregnancy rate within 150 days postpartum and the mean days open in Group E (76.0% and 106.3 ± 4.6 days, respectively) were significantly different from those in Group F (38.5% and 137.9 ± 10.9 days, respectively) (P < 0.05). This study suggests that reproductive efficiency could be improved by using the two-step treatment to regulate uterine involution and an early resumption of ovarian function in cows with RP/PM.     

乳房炎牛奶中复方阿莫西林纳米乳的残留检测及消除试验

利用高效液相色谱仪建立复方阿莫西林纳米乳（AMX-LH-NE）在牛奶中的残留监控高效液相色谱法（HPLC）,并通过残留消除试验,研究AMX-LH-NE在乳房炎牛奶中的残留消除规律,确定其弃奶期。结果显示,阿莫西林（AMX）和盐酸左氧氟沙星（LH）分别在7.5～400μg/L和40～600μg/L线性关系良好;平均回收率为（99.72±1.46）%和（99.65±1.53）%;相对标准偏差（relative standard deviation,RSD）为1.46%和1.54%;平均保留时间为（11.59±0.21）min和（6.87±0.02）min;日内精密度RSD为1.59%和1.39%,日间精密度RSD为3.17%和3.29%。在乳房炎牛奶中,AMX和LH的残留量均随着休药时间的延长而迅速降低;AMX的弃奶期为停药后35.96h,LH的弃奶期为停药后32.94h。结果表明,所建立的HPLC方法专属性好,回收率、重复性和精密度高,可为牛奶中AMX-LH-NE的残留监控提供检测方法;利用此检测方法确定的AMX-LH-NE的弃奶期为停药后36h。