高效液相色谱-串联质谱法同时测定鸡肉、 鸡蛋中喹乙醇和卡巴氧及代谢物残留

目的建立高效液相色谱-电喷雾电离串联质谱法测定鸡肉、鸡蛋中喹乙醇、卡巴氧及其代谢物残留量的分析方法。方法组织样品中的卡巴氧和喹乙醇用乙腈-乙酸乙酯(1:1, V:V)溶液提取;代谢物的提取用蛋白酶酶解后经盐酸溶液酸化,采用阴离子交换固相萃取柱净化和富集。分析样品以0.1%甲酸溶液-甲醇进行梯度洗脱,经Waters Xbridge C_(18)色谱柱分离,以液相色谱串联质谱多反应监测(multiple reaction monitoring,MRM)正离子模式进行检测。结果 5种喹喔啉类药物在0.5～50ng/mL浓度范围内线性良好,相关系数r~2为0.9956～0.9988;该方法的检出限和定量限分别为0.1～0.5μg/kg和1.0～2.0μg/kg;鸡蛋和鸡肉样品中,喹乙醇、卡巴氧、脱氧卡巴氧、喹喔啉-2-羧酸和3-甲基喹喔啉-2-羧酸在0.2～2.0μg/kg加标水平的平均回收率为85.1%～108.2%,日内精密度为4.9%～8.7%,日间精密度为6.2%～11.7%。结论该方法简单有效、回收率好、灵敏度高、特异性好,能满足日常检测的需求。     

超高效液相色谱串联质谱法测定鸡蛋中喹乙醇及卡巴氧代谢物

目的建立超高效液相色谱串联质谱法检测鸡蛋中喹乙醇代谢物3-甲基-喹恶啉-2-羧酸以及卡巴氧代谢物喹恶啉-2-羧酸的分析方法。方法样品经酶解后,用乙酸乙酯提取,PAX固相萃取柱净化,用色谱柱WATERS ACQUITY UPLC HSS T_3(2.1 mm×100 mm, 1.8μm)分离,同位素内标法定量。结果 3-甲基-喹恶啉-2-羧酸及喹恶啉-2-羧酸的最低检出限均为0.2μg/kg,定量限均为0.6μg/kg,平均回收率为80.9%～106.4%,相对标准偏差为2.6%～7.2%。结论该方法净化效果好,灵敏度高,准确性好,适用于鸡蛋中喹乙醇以及卡巴氧代谢物的同时检测。     

超高压液相色谱-串联质谱法测定鱼组织中卡巴氧及喹乙醇代谢物

目的 建立鱼组织中卡巴氧代谢物喹恶啉-2-羧酸(QCA) 及喹乙醇代谢物3-甲基-喹恶啉-2-羧酸(MQCA)的超高压液相色谱-串联四极杆质谱(UPLC-MS/MS)残留分析方法。方法 样品在一定温度、Tris/HCL缓冲溶液作用下, 经蛋白酶酶解, 浓盐酸酸化, 乙酸乙酯提取, 提取液吹干后加入20%甲醇水溶解, 过PAX固相萃取柱净化、富集; 以乙腈-0.1%甲酸水为流动相, 经HSS T3色谱柱分离, 采用多反应监测(MRM)正离子模式进行检测。结果 QCA和MQCA定量限(LOQ)均为0. 5 μg/kg , 在0.5~5.0 μg/kg添加水平的平均回收率在93.1%~101.2%之间, 相对标准偏差为1.4%~5.5%。结论 本方法适用于鱼组织中卡巴氧及喹乙醇代谢物残留检测。     

高效液相色谱-串联质谱法检测猪肉中喹乙醇代谢物

本文建立了猪肉中喹乙醇代谢物3-甲基-喹恶啉-2-羧酸(MQCA)的高效液相色谱-串联四极杆质谱(UPLCMS/MS)的分析方法。样品经过2%偏磷酸:甲醇(4:1)初步提取,乙酸乙酯再次提取,通过正己烷除油,旋转蒸发浓缩至干,乙腈-0.1%甲酸水复溶解,经过C18色谱柱分离,多重反应监测(MRM),内标法定量。结果表明:本方法MQCA检出限为0.1μg/kg,在0.5～10.0μg/kg范围内线性关系良好,r~2=0.999,平均回收率在71%～90%,相对标准偏差为0.9%～2.5%。结论:本方法适用于猪肉中喹乙醇代谢物残留的检测。     

碱水解-SPE-LC-MS/MS法快速测定动物源食品中喹乙醇代谢物残留量

建立了一种快速准确定性定量检测动物源性食品中喹乙醇代谢物3-甲基-喹喔啉-2-羧酸(MQCA)残留量的液相色谱—串联质谱(LC-MS/MS)检测方法。样品组织通过碱水解提取MQCA,阴离子固相萃取柱净化,LCMS/MS检测,内标法定量。结果显示,在0.5～50.0ng/mL浓度范围内,MQCA线性关系良好,相关系数R2为0.999 7。MQCA检出限为0.1μg/kg,方法在0.1,0.2,1.0μg/kg的添加水平下,回收率为95.6%～108.2%,相对标准偏差为3.4%～14.3%(n=6)。该检测方法准确、快速、灵敏度高,适用于动物源食品中MQCA残留量的检测和确证。     

硫胺素对鲁氏接合酵母高盐适应性的影响

采用同位素内标-超高效液相色谱串联三重四极杆质谱（UPLC-MS/MS）法测定金沙江攀枝花江段6种常见鱼中卡巴氧代谢物喹恶啉-2-羧酸（QCA）和喹乙醇代谢物3-甲基-喹恶啉-2-羧酸（MQCA）的残留量。样品经过预处理,采用ZORBAX SB-C18色谱柱分离,以0.1%甲酸水-乙腈为流动相进行梯度洗脱;采用电喷雾-正离子多反应监测模式（MRM）检测,同位素内标法定量。结果表明,QCA和MQCA的质量浓度为2.0～100.0 ng/mL时,线性关系良好（R＞0.999）,回收率为91.4%～100.4%,精密度试验结果相对标准偏差（RSD）为6.3%～11.5%,定量限为0.2 μg/kg,检出限为0.05 μg/kg。该方法灵敏度及准确度良好,能满足6种常见鱼的组织中卡巴氧及喹乙醇代谢物残留分析的要求。     

液相色谱-串联质谱法测定猪肝中喹乙醇残留标示物3-甲基喹噁啉-2-羧酸

目的 建立猪肝中喹乙醇残留标志物3-甲基喹噁啉-2-羧酸(MQCA)的液相色谱-串联质谱(HPLC-MS/MS)快速检测方法。 方法 猪肝匀浆,加乙腈提取。提取液用Oasis MAX固相萃取柱净化,2%的甲酸乙酸乙酯溶液洗脱。洗脱液氮气吹干,用0.1%甲酸-甲醇(19+1)溶液定容,溶液经滤膜过滤后,供液相色谱-串联质谱仪测定。采用0.1%甲酸(A)-乙腈(B)作为流动相进行梯度洗脱,C18色谱柱分离,采用电喷雾电离,正离子扫描,多离子检测模式对MQCA的定量离子和定性离子进行监测,基质匹配内标(MQCA-D4),标准曲线法定量。结果 喹乙醇残留标志物3-甲基喹噁啉-2-羧酸(MQCA)在0.5-100μg/L浓度范围内线性良好,相关系数r²大于0.99;该方法的检出限和定量限分别为0.3μg/kg和 0.5μg/kg;3-甲基喹噁啉-2-羧酸在1.0、5.0、20μg/kg添加水平的回收率为93.9%-101.3%,相对标准偏差小于5.8%。结论 该方法简单快速,准确,灵敏,适用于猪肝中喹乙醇残留标志物3-甲基喹噁啉-2-羧酸的快速检测。     

快速高分离液相色谱- 串联质谱法对肌肉组织中糖皮质激素残留的检测

建立采用快速高分离液相色谱- 串联质谱(UHPLC-MS2)技术检测肌肉组织中糖皮质激素残留的方法。样品经乙酸乙酯提取,固相萃取法净化,以C18 色谱柱(150mm × 2.1mm,3.5μm)为分离柱,水- 乙腈- 甲酸溶液为流动相进行梯度洗脱,快速高分离液相色谱- 串联质谱ESI 负离子模式进行检测。该方法线性范围为0.5～5ng/mL,线性相关系数R2 ≥ 0.9963,检测限：倍氯米松为1.0μg/kg,氢化可的松为2.0μg/kg,其余均为0.5μg/kg。平均回收率为82.75%～91.87%,相对标准偏差≤ 4.43%(n=5)。     

液相色谱串联质谱法测定动物组织中卡巴氧和喹乙醇代谢物残留量

本文建立了猪肉中卡脱氧巴氧和喹乙醇代谢物的液质联用检测方法,样品经甲酸溶液消化,蛋白酶溶液酶解后经盐酸溶液酸化,用waters混合阴离子固相萃取柱净化处理。脱氧卡巴氧用二氯甲烷洗脱,喹乙醇代谢物用甲酸-乙酸乙酯溶液洗脱,收集液经氮气吹干后用甲酸-甲醇溶液定容,用液相色谱-串联质谱仪测定,内标法定量。结果显示:该方法 0~100μg/L范围内线性良好,3种残留的回收率在80%~110%,相对偏差在2.03%~4.94%。     

丝织物中酸性金黄G的液相色谱-质谱法测定

建立了固相萃取-超快速液相色谱-电喷雾串联三重四级质谱(SPE-UFLC-ESI-MS/MS)联用技术测定丝织物中的酸性金黄G。样品经SPE提取和净化后,采用ACQUITY UPLC BEH C18柱(100 mm×2.1 mm,1.7μm)色谱柱,乙腈(含0.1%甲酸)和水(含0.1%甲酸)为流动相进行梯度洗脱,电喷雾离子源电离,负离子多反应监测模式进行定性定量分析。酸性金黄G染料在0.5~100.0μg/L范围内线性良好,相关系数0.999 5,方法检出限为0.17μg/kg,回收率为97.2%~102.3%。     

Identification of potential markers in blood for the development of subclinical and clinical mastitis in dairy cattle at parturition and during early lactation

Our objective was to identify specific blood markers as risk factors for the development of mastitis during early lactation. We used a subset of cows from a larger experiment that consisted of a total of 634 lactations from 317 cows. Cows were of 3 breeds and ranged from parity 1 to 4. Blood samples were collected weekly from 56 d before expected calving date through 90 d in milk (DIM). Blood was analyzed for several hormones, metabolites, and enzymes, and energy intake and energy balance were calculated. Veterinary treatment records and daily composite milk somatic cell counts were analyzed and used to determine incidence and severity of mastitis in early lactation. Cows were separated into 2 groups: 1) WK0, consisting of cows that developed clinical mastitis (CM), cows that developed subclinical mastitis (SM), or cows that were healthy (H) during the first 7 DIM; and 2) EL, consisting of CM, SM, or H cows during wk 2 through 13 of lactation. Data were adjusted for numerous fixed effects (e.g., parity, breed, season, and DIM) before statistical analysis. The time of mastitis (TOM) was recorded as the DIM in which the first rise in somatic cell count was observed and was recorded as TOM = 0. The time before and after TOM was distinguished as ± n wk relative to TOM = 0. Healthy cows were paired with either a SM or CM cow and the TOM for each H cow was equal to the TOM for its paired SM or CM cow. Data from wk −1 and −2 relative to TOM were analyzed for group WK0 and EL, respectively. For all parameters, SM cows did not differ from H cows from either group. The CM cows had higher nonesterified fatty acid levels and a tendency toward higher β-hydroxybutyrate levels than H cows before mastitis for both groups. For group WK0, glucose was higher −1 wk relative to calving in CM than H cows. For group EL, aspartate aminotransferase was higher −2 wk relative to mastitis in CM than H cows during 8 to 90 DIM. All other variables were similar among CM, SM, and H cows for both groups. Our results indicate that substances in blood, especially nonesterified fatty acids and aspartate aminotransferase, may be potential markers for the risk of mastitis in early lactation.     

Characteristics of spontaneous revertants in haploid yeast

The appearance dynamics of spontaneous revertants in adenine- or leucine-auxotrophic haploid Saccharomyces cerevisiae strains has been studied using mathematical simulation methods. In the case of adenine auxotrophs an increase in the number of revertants with decreasing metabolite content is found to result mainly from increasing the rate of intragenic suppressor mutations. In the case of leucine auxotrophs revertants result from increasing the appearance of mutants formed at similar rates under different cultivation conditions. In the latter case the appearance of mutant colonies increases with decreasing size of colonies of the initial auxotrophic cells. The last can simulate so-called adaptive mutagenesis. The heterogeneity of revertants appearing in different time periods is described.     

Performance, metabolic, and endocrine responses of periparturient Holstein cows fed 3 sources of fat

The objective of this study was to examine the effect of feeding diets containing fat supplements enriched in either saturated fatty acids (n = 10), Ca salts of trans-octadecenoic fatty acids (tFA, n = 10) or Ca salts of safflower oil fatty acids (SFL, high in linoleic acid, n = 9) on performance, metabolic, and endocrine responses of periparturient Holstein cows. Dietary treatments were initiated at approximately 28 d before calculated calving dates and continued through 49 d postpartum. Blood samples for metabolite and hormone analyses were collected weekly beginning 1 wk before estimated calving date through 7 wk postpartum. Incorporation of tFA or SFL into the peripartum diet had no detectable effects on body weight or body condition score. Cows fed the SFL-enriched diet produced less milk fat and established a positive energy balance sooner after calving than those fed the tFA supplement. Analysis for individual fatty acids resulted in increased concentrations of trans 18:1 fatty acid and conjugated linoleic acid isomers in milk fat from cows supplemented with SFL. Across weeks, the average nonesterified fatty acids concentration in plasma was lower in cows fed the SFL-enriched diet than in those consuming the tFA-supplemented diet. Mean concentrations of plasma glucose, insulin-like growth factor-I, and progesterone were greater in cows fed the SFL-enriched diet compared with those fed the saturated fatty acid-supplemented diet. Feeding fat supplements that can suppress milk fat production during the early postpartum period may help minimize negative energy balance, reduce adipose tissue mobilization, and improve circulating concentrations of insulin-like growth factor-I and progesterone. Whether the SFL supplement would have similar effects without a decrease in milk fat production remains to be determined and warrants further investigation.     

气质联用法分析动物肌肉、肝脏和肾脏中喹喔啉-2羧酸的含量

本文研究了用气质联用仪检测动物肌肉、肝脏及肾脏中喹喔啉-2-羧酸的含量，该方法灵敏度较高，检测低限为1．0μg／kg，干扰较少，并具有较好的重现性，可达到60％～125％，相关线性系数为0．998。     

迟缓芽孢杆菌固态发酵代谢产物研究新策略初探

运用Plackett-Burman实验筛选因素,确定温度对迟缓芽孢杆菌的生长影响显著,为其主要影响因素。然后对不同温度下迟缓芽孢杆菌固态发酵代谢产物进行提取分析,发现随着温度的升高,代谢产物的种类增加,同时代谢产物的更迭也存在一定规律。     

Alterations in milk metabolome and coagulation ability during the lactation of dairy cows

Milk composition has been known to change during lactation. To help understand the changes in metabolic profile throughout the whole lactation, liquid chromatography mass-spectrometry was used to analyze 306 milk samples from 82 primi- and multiparous dairy cows. Changes in metabolic profile common to all cows throughout lactation were ascertained based on principal component and general linear model analysis. Sets of specific markers; for instance, 225, 397, and 641–642 m/z (positive mode), and 186, 241, and 601–604 (negative mode), with at least a 1.5-fold higher intensity during the first 60 d compared with the last 60 d of lactation were observed. The metabolome was affected by parity and milking time. Markers, identified as peptides differentiating parity, were observed. A significant increase for citrate was observed in evening milk. Milk coagulation traits were strongly animal specific. The curd firmness values were influenced by milking time. Sets of markers were associated with curd firmness in positive (197 m/z) and negative (612, 737, 835, 836, 902, 1000, 1038, and 1079 m/z) ion mode.     

呋喃唑酮代谢物抗体的制备与筛查

利用呋喃唑酮的4种代谢物合成免疫原以及包被原,用4种免疫原各免疫两只新西兰大白兔,得到8份抗血清,再纯化抗血清。采用间接ELISA方法,筛选出OD值明显高于阴性样本的血清用于以后的实验,同时对抗体的效价进行测定。实验检测结果表明,当用包被原A和包被原B包被酶标板时,7号和8号抗血清对该包被抗原有很好的亲和性,呋喃唑酮代谢物衍生物对抗体有很好的抑制。通过效价检测,7号和8号抗血清均具有高效价、高特异性的特点,其中8号抗血清的效价更高,亲和力更强。再通过进一步对两种包被原和两份抗血清进行筛查,选择B包被原和8号兔子的抗体清用于ELISA法的建立。     

黏细菌及其次级代谢物生物学性质的初步研究

目的研究分离到的黏细菌So ce shu-1及其次级代谢物的生物学特性.方法用烘干法和DNS法测定So ce shu-1利用纤维素的能力,用滤纸片法和溴化四唑蓝(MTT)法测定So ce shu-1利用纤维素次级代谢物抑制细菌和肿瘤细胞的效果.结果 So ce shu-1能利用玉米秸秆和滤纸等纤维素,纤维素酶活性为12.30 UFPA/mL,其次级代谢物有广谱抗菌活性,对K562等肿瘤细胞有较好的抑制效果.结论 So ce shu-1在能源利用方面,其次级代谢物在药物和食品保鲜方面具有良好的开发前景.     

Blood metabolites and hormone-based programmed breeding treatments in anovular lactating dairy cows

The objectives were to (1) compare blood metabolites and reproductive outcomes in lactating dairy cows not inseminated before (early) and after (late) 100 d in milk (DIM) because of prolonged anovulation or anestrus; and (2) evaluate reproductive responses of cows ≤100 DIM to GnRH + PGF_2α treatments after a fixed-time artificial insemination (AI; Ovsynch) or after induced estrus (Select Synch). In blood samples collected before initiating hormone-based breeding programs, anovular cows ≤100 DIM had the greatest serum total protein and globulin concentrations and the lowest tri-iodothyronine concentrations. Anovular and ovular cows >100 DIM had the greatest serum urea concentrations. Ovaries in cows (n = 40) >100 DIM were examined by transrectal ultrasonography, and those without a detectable corpus luteum (CL; anovular) were given GnRH and then PGF_2α 7 d later (Select Synch), whereas cows with a CL (ovular) were given 2 PGF_2α injections 12 d apart. Cows were inseminated at observed estrus after the second or only PGF_2α injection. More ovular (79%; 15/19) than anovular cows (24%; 5/21) were detected in estrus. No differences were detected between ovular and anovular cows in DIM at first AI, conception rate to first AI, cumulative pregnancy rates, number of services per conception, or days open. Cows (n = 93) ≤100 DIM were assigned randomly to 3 treatments: (1) control (n = 20) AI at estrus; (2) GnRH and then PGF_2α on 7 d (Select Synch; n = 42) and monitored for signs of estrus for 5 d and AI accordingly; or (3) 2 GnRH injections 9 d apart with PGF_2α given 48 h before second GnRH injection and AI at 16 h after the second GnRH injection (Ovsynch; n = 31). Among cows ≤100 DIM, controls had more days to first service (149 ± 16 d) than Select Synch cows (117 ± 7 d). Ovsynch cows had the fewest days to first service (84 ± 10 d) compared with control (149 ± 16 d) and Select Synch (117 ± 7 d) cows. Conception rates in control (25%) and Select Synch (26.2%) cows did not differ from those in Ovsynch cows (29%). Ovsynch cows had greater cumulative pregnancy rates and fewer days open than control (161 ± 20 vs. 258 ± 29 d), but did not differ from Select Synch (233 ± 19 d). Timed AI produced comparable fertility and superior cumulative pregnancy rates, fewer days to first service, and fewer days open than AI at observed estrus in cows inseminated ≤100 DIM.