利用LC-MS/MS辅助优化GICA对硝基呋喃类代谢物的快速检测

针对硝基呋喃类代谢物的胶体金免疫层析法(GICA)检测方法存在的缺陷,实验以硝基呋喃类代谢物实际阳性样本和模拟阳性样本为研究对象,选择液相色谱-串联质谱(LC-MS/MS)辅助优化GICA代谢物的水解和衍生条件前处理步骤,建立了一种快速检测水产品中硝基呋喃类代谢物的方法。结果显示最佳实验条件为:盐酸浓度0.35 mol/L,0.05 mol/L 2-硝基苯甲醛0.35 mL,水浴温度90℃,水浴时间120 min。此条件下,胶体金免疫层析法的前处理时间缩短至120 min,检测时间为5 min, 4种硝基呋喃类代谢物的检出限均为0.05μg/kg且重现性好。本方法对140批次不同基质样品进行分析,检测得出12批次阳性样品,假阳性和假阴性率均为0。结果表明本方法在降低实验前处理时间同时,适用于不同的样品基质,可应用于大批量实际样品的测定。[中国渔业质量与标准,2021,11(4):31-38]     

超高压液相色谱-串联质谱测定渔业养殖环境中硝基呋喃代谢物

采用超高压液相色谱-三重串联四级杆质谱仪优化了渔业养殖水和底泥中4种硝基呋喃代谢物残留检测的方法。确定以2-硝基苯甲醛为衍生剂,样品经60℃高温振荡衍生4 h,再经乙酸乙酯提取,C_(18)色谱柱分离,在正离子反应模式下以大气压电喷雾源电离,采用多反应监测,内标法定量。方法中4种硝基呋喃代谢物的检出限为0.1 ng/mL(g),定量限为0.25 ng/mL(g),在0.25~10 ng/mL(g)线性范围良好,0.5、2.0、5.0 ng/mL(g)3个添加水平回收率范围为91.3%~102.2%,相对标准偏差均小于10%。本方法具有重复性好、灵敏度高、简便、准确以及快速的优点,可以满足渔业养殖环境中硝基呋喃类药物残留的监控要求。     

超高效液相色谱-同位素稀释串联质谱法测定水产品中硝基呋喃类代谢物残留

建立了采用Oasia PRiME HLB固相萃取柱净化-超高效液相色谱-同位素稀释串联质谱法(UPLC-MS/MS)快速测定水产品中硝基呋喃类代谢物残留的方法。样品加入同位素内标,超声衍生化后用乙酸乙酯超声提取,经Oasia PRiME HLB固相萃取柱一次性净化后上机检测,电喷雾-正离子多反应监测模式,内标法定量。该方法将测试时间缩短至4小时。结果显示,4种硝基呋喃类代谢物的检测限为0.01μg/kg,定量限为0.03μg/kg,在0.05~10ng/mL质量浓度范围内线性良好,4种硝基呋喃类代谢物相关系数r均大于0.999,5个添加浓度水平的平均回收率为88.5%~115.2%,相对标准偏差均小于8,说明该方法快速、准确并且重复性好。     

UHPLC-MS/MS测定罗非鱼中硝基呋喃类代谢物残留量的不确定度评定

为判断罗非鱼中硝基呋喃类代谢物残留量测定中不确定度的主要来源,本研究参照JJF 1059.1—2012《测量不确定度的评定与表示》等相关理论,采用农业部783号公告-1-2006的前处理方法进行样品处理,并根据公告中的结果计算公式建立相应的数学模型,对罗非鱼中硝基呋喃类代谢物残留量的测定结果中产生的各不确定度来源进行分析和计算。当硝基呋喃类代谢物的添加水平为1.00μg/kg时,4种硝基呋喃类代谢物结果可表示为:呋喃西林代谢物C_(SEM)为(0.989±0.168)μg/kg,k=2;呋喃妥因代谢物C_(AHD)为(1.009±0.165)μg/kg,k=2;呋喃它酮代谢物C_(AMOZ)为(0.991±0.169)μg/kg,k=2;呋喃唑酮代谢物C_(AOZ)为(0.986±0.180)μg/kg,k=2。研究表明,在确定的实验条件下,本方法的不确定度主要来自于标准溶液和内标溶液配制、标准曲线拟合和加标回收率。研究结果对实际操作中减少不确定度,提高检测结果的准确性和可靠性具有重要意义。     

液质质串联质谱法测定淡水及沉积物中硝基呋喃类代谢物

改进了淡水水体和沉积物中4种硝基呋喃代谢物呋喃唑酮代谢物(AOZ)、呋喃它酮代谢物(AMOZ)、呋喃妥因代谢物(AHD)、呋喃西林代谢物(SEM)的高效液相色谱串联质谱(LC-MS/MS)检测方法。方法采用内标法定量,提高了定量的准确性。水及沉积物样品直接进行衍生化后乙酸乙酯提取,化合物采用Thermo C18(5.0μm,100mm×2.1mm)色谱柱进行分离,以0.1%甲酸/甲醇-5mmol/L乙酸铵水溶液(含0.1%甲酸)为流动相梯度洗脱。方法的线性范围为0.1~200ng/mL,r2≥0.999。空白水体在0.02、1.0、10.0ng/L 3个加标水平下的平均回收率为75.4%~82.7%,相对标准偏差(RSD)为3.2%~8.7%,检出限(LOD)和定量下限(LOQ)分别为0.01ng/mL和0.02ng/mL。沉积物样品在0.3、1.0、10μg/kg 3个加标水平下,平均回收率为77.9%~93.6%,RSD为2.5%~7.8%,检出限(LOD)和定量下限(LOQ)分别为0.1μg/kg和0.3μg/kg。该方法灵敏度高、选择性好,适用于实际淡水养殖环境水体和底泥中4种硝基呋喃代谢物呋喃唑酮代谢物的残留测定。     

高效液相色谱—串联质谱法测定鲍鱼中硝基呋喃类代谢物残留量

本文用高效液相色谱-串联质谱法测定鲍鱼中3-氨基-2-噁唑烷基酮(AOZ)、5-甲基吗啉-3-氨基-2-噁唑烷基酮(AMOZ)、氨基脲(SEM)、1-氨基-2-内酰脲(AHD)4种硝基呋喃类代谢物残留量的方法。0.2mol/L盐酸溶液水解鲍鱼组织中与蛋白结合的硝基呋喃类代谢物,用0.05mol/L的2-硝基苯甲醛溶液(2-NBA)37℃衍生16小时。用1.0mol/L磷酸氢二钾溶液调节pH至7.0~7.5,乙酸乙酯萃取。采用电喷雾电离,正离子扫描,选择反应监测模式(SRM)监测,内标法定量。本方法对4种硝基呋喃类代谢物标准曲线的线性回归系数均在0.99以上,线性范围为0.5μg/kg~20μg/kg。方法定量限为0.5μg/kg。在添加浓度0.5μg/kg~5μg/kg,回收率在75.2%~102.1%之间,相对标准偏差为3.56%~12.6%。说明本方法简单、灵敏,结果可靠,可满足实验室大量、快速分析的需求。     

HPLC-APCI(+)MS/MS内标法测定水产品中的3种硝基咪唑类药物残留量

建立了水产品中3种硝基咪唑类残留量的HPLC-APCI(+)MS/MS测定方法.以氘代二甲硝唑(DMZ-D3)作为内标,以乙酸乙酯提取水产品中残留的硝基咪唑类药物,采用HPLC-MS/MS多反应监测(MRM)正离子模式测定,可一次对水产品中的MTZ、DMZ和RNZ进行定性和定量.该法简便快捷,适合大批量样品处理.方法简化了样品前处理过程,省去固相萃取步骤,具有操作简便和有机试剂消耗量少等优点.DMZ、MTZ和RNZ的检出低限均为0.5 μg/kg,DMZ、MTZ和RNZ的定量下限均为1.0 μg/kg.     

超高效液相色谱串联质谱法对水产品中硝基呋喃代谢物的测定

建立了使用Waters UPLC Xevo TQ超高效液相色谱串联质谱仪检测水产品中4种硝基呋喃代谢物的分析方法。匀浆后的样品在酸性环境下水解,并用2-硝基苯甲醛衍生16h,经磷酸氢二钾提取,LMS固相萃取小柱净化后,在三重四极杆多反应监测模式下进行测定。方法采用同位素内标法定量,四种代谢物的检出限(LOD)均低于0.25μg·kg-1,线性范围在0.5μg·L~(-1)到10μg·L~(-1)间,相关系数大于0.99。在0.5、1.0和5μg·kg-1浓度下分别进行添加回收试验(n=5),回收率在75.2%~123.3%之间,相对标准偏差(RSD)小于9.4%。     

超高效液相色谱-串联质谱法测定凡纳滨对虾中的硝基呋喃代谢物残留

采用超高效液相色谱-串联质谱（UPLC-MS/MS）法,测定了凡纳滨对虾（Litopenaeus vannam ei）中氨基脲（SEM）、1-氨基-2-内酰脲（AHD）、3-氨基-2-唑烷基酮（AOZ）和5-甲基吗啉-3-氨基-2-唑烷基酮（AMOZ）4种硝基呋喃类代谢物。采用2-硝基苯甲醛作为衍生化试剂,氘、碳-13、氮-15同位素标记的SEM1-3C1-5N2、AHD1-3C3、AOZ-D4和AMOZ-D5为内标物,样品经衍生、乙酸乙酯提取、正己烷净化,以甲醇0.5 mmol.L-1乙酸铵水溶液（含0.1%甲酸）为流动相进行梯度洗脱,8 m in可将4种目标化合物完全分离并测定。结果表明,4种待测物在0.4～20.0 ng.mL-1范围内线性关系良好（R〉0.999）,检出限为0.03～0.15μg.kg-1。在0.5μg.kg-1、1.0μg.kg-1和2.0μg.kg-13个水平上进行加标回收试验,平均回收率为92.0%～107.3%,RSD为3.1%～8.7%。采用该法参加FAPAS国际水平测试,获得了满意的结果。     

水产品中硝基呋喃类药物及其代谢物残留检测技术的研究进展

综述了水产品中硝基呋喃类药物代谢规律,分别介绍了硝基呋喃原药及其代谢物的检测方法,比较了各种方法在实际应用中的优缺点,并对检测方法进行了展望,以期为水产品中硝基呋喃类药物及其代谢物检测的研究提供帮助。     

同位素稀释液质联用法测定鱼体中氯丙嗪的残留

建立了高效液相色谱-串联质谱(HPLC/MS/MS)分析鱼体中氯丙嗪残留的方法.样品用乙腈提取,MCX固相萃取柱净化,氨化甲醇洗脱.在正离子模式下以电喷雾串联质谱仪进行测定,采用内标法定量,氘代氯丙嗪为内标物.以欧洲鳗鲡、鳜鱼和大口黑鲈为研究对象,在1.0、5.0、25.0μg/kg添加水平下,方法平均回收率为90.1...     

Rapid and sensitive determination of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone by biotin–streptavidin-amplified time-resolved fluoroimmunoassay

A competitive time-resolved fluoroimmunoassay (TRFIA) using a biotin–streptavidin system was developed for the detection and quantification of the furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ) in aquatic tissues. AMOZ-bovine serum albumin was coated to a solid phase and then competed with a free nitrophenyl derivative of AMOZ (2-NP-AMOZ) in standards or samples for binding with biotin-anti-AMOZ-ovalbumin polyantibody. Later, the complex was recognized by europium-labelled streptavidin, which was used as a novel tracer to amplify the signal, and it also reacted faster than europium-labelled second antibody in traditional TRFIA. After optimizing the reaction conditions, the method showed high sensitivity and specification to 2-NP-AMOZ with a half maximal inhibitory concentration of 0.190 μg/l and a sensitivity of 0.019 μg/l with a working range from 0.025 to 10 μg/l. The data from fish and shrimp samples indicated that the limit of detection was 0.021 μg/kg, and the recovery rates were 84.1–107.0% and 80.9–98.4%, respectively, with an average RSD below 10%. High correlation rates were observed in TRFIA, high performance liquid chromatography and universal enzyme-linked immunosorbent assay methods. The experiment above confirms that biotin–streptavidin-amplified TRFIA could be an ultrasensitive and accurate tool for screening large numbers of aquatic products for the determination of AMOZ residues.     

Determination of selected antibiotics in farmed fish species using LC‐MS/MS

The aim of the study was to monitor the occurrence of 37 antibiotics from different classes including fluoroquinolone, tetracycline, macrolide, sulphonamide, penicillin, amphenicol and cephalosporin in three different farmed fish species (n = 75; rainbow trout, see bass, gilthead see bream) from five cities in Turkey. A method for LC‐MS/MS was optimized and validated to assess these antibiotics. As a result, 25 samples showed contamination with at least one antibiotic residue. The most detected antibiotics were norfloxacin (NOR) (18.7%), marbofloxacin (MAR) (12%), tetracycline (TC) (10.7), danofloxacin (DAN) (9.3%), oxytetracycline (OTC) (9.3%), tilmicosin (TLM) (8%) and enrofloxacin (ENR) (6.7%). A total of 17.3% of samples examined were found to be contaminated with residues exceeding the maximum residue limits (MRLs). The results indicate that antibiotics are frequently used in aquaculture production without the lack of knowledge. This study also revealed that the consumption of farmed fish could produce a public health concern as the level of residues for some antibiotics was much higher than MRLs.     

HPLC—MS／MS法测定水产品中硫酸粘菌素、杆菌肽及维吉尼霉素M1的残留量

建立了水产品中硫酸粘菌素（CS）、杆菌肽（BTC）及维吉尼霉素M1（VBGMM1）3种多肽类抗生素残留量检测的HPLC-MS/MS法.样品经水溶液[V（甲醇）∶V（0.1％甲酸水溶液）=2∶5]提取,4％三氯乙酸乙腈除蛋白,乙腈饱和正己烷除脂,过OASIS HLB（60 mg）小柱净化后,利用HPLC-MS/MS法,以选择反应监测模式检测,外标法进行定量分析.CS和BTC在0.01～10.00 mg·L^-1质量浓度范围内线性良好,VGMM1在0.002～2.000 mg·L^-1质量浓度范围内线性良好,R2均大于0.995;3种多肽类抗生素的检出限分别为CS 10 μg· kg^-1、BTC 10 μg·kg^-1、VGMM1 2μg·kg^-1,定量限分别为20 μg ·kg^-1、20 μg ·kg-和4 μg·kg-;选择3个不同浓度水平做加标回收,平均回收率在72.3％ ～ 103.9％,相对标准偏差为1.10％～10.92％.该方法具有操作简便、准确性高、灵敏度高和重现性好等优点,可为检测水产品中这3种药物的残留提供相关技术支持.     

固相萃取-液相色谱串联质谱法同时测定养殖海水中17种喹诺酮类药物

建立了固相萃取-液相色谱串联质谱法(SPE-LC-MS/MS)同时检测养殖海水中17种喹诺酮类药物残留的分析方法。海水经酸化处理后,采用HLB固相萃取柱富集、净化目标化合物,通过对比水样在不同上样pH、淋洗液与洗脱液等条件下的回收率,以此对前处理方法进行优化。收集到的洗脱液经氮气吹干后用流动相定容至1 ml,待测。色谱流动相A相为0.1%甲酸水溶液,B相为乙腈,所有药物经梯度洗脱进行分离,在LC-MS/MS正离子模式的多反应监测模式下进行定性定量分析。17种化合物可以在10 min中内得到较好的分离,线性范围为1–200 ng/ml,线性相关系数均大于0.999,检出限均在2–10 ng/L范围内,定量限均在5–20 ng/L范围内。以空白海水为基质,在20、100、200 ng/L三个不同添加水平下采用内标法定量的加标回收率均在71.3%–125.0%范围内,相对标准偏差(RSD)为2.44%–12.27% (n=5)。采用该方法对黄海灵山湾近岸4个养殖场进行海水采集并检测,共检测出4种喹诺酮类药物,分别是恩诺沙星、氧氟沙星、诺氟沙星和环丙沙星,其中,恩诺沙星浓度最高。研究表明,该方法快速、可靠,适用于养殖海水中喹诺酮类药物的检测。     

运输时间和MS-222浓度对翘嘴鲌皮质醇、乳酸及氧气袋内水质的影响

釆用MS-222浓度为0(C0组)、5 mg/L(C1组)、10 mg/L(C2组)、15 mg/L(C3组)、20 mg/L(C4组),运输时间为0、2、4、6、8、10 h,运输密度为33 g/L,采用5×6双因素实验,氧气袋运输翘嘴鲌(Culter alburnus)稚鱼,研究了运输时间和MS-222浓度对翘嘴鲌全鱼皮质醇、乳酸含量及氧气袋内水质的影响。结果显示:翘嘴鲌全鱼皮质醇、乳酸水平随运输时间呈现先升高后降低的趋势,随麻醉剂浓度的增加而显著降低;C1-C4组的DO、p H随运输时间呈现先降低后升高的趋势,C0组则随运输时间的增加显著降低,各组的DO和p H随MS-222浓度的增加而显著增加;C1-C4组的氨氮(TAN)和游离CO_2浓度随运输时间呈现先升高后降低的趋势,而C0组则随运输时间的增加显著升高,各组的TAN和游离CO_2浓度随MS-222浓度的增加而显著降低。时间和浓度的交互作用都显著。运输时间T6 h时,MS-222刺激翘嘴鲌,不利于运输,运输时间T6 h时,MS-222能显著降低其应激水平及代谢强度,其中C4组皮质醇、乳酸含量最低,C3、C4组水质条件最好,其适宜的麻醉剂浓度为15~20 mg/L。     

Nutrients apparent digestibility coefficients of selected protein sources for juvenile Siberian sturgeon (Acipenser baerii Brandt), compared by two chromic oxide analyses methods

Apparent digestibility coefficients (ADCs) of dry matter (ADC_d), crude protein (ADC_p), energy (ADC_e) and amino acids in selected feedstuffs were determined for juvenile Siberian sturgeon (8.38 ± 0.20 g). The tested feedstuffs were fishmeal (FM), meat and bone meal (MBM), poultry by-product meal, hydrolysed feather meal, fermented feather meal solvent-extracted cottonseed meal and soybean meal. ADCs were determined using a reference diet and test diets at 7 : 3 ratios with 5 g kg⁻¹ chromic oxide (Cr₂O₃) as an inert marker. Fish were reared in a recirculating system and fed to apparent satiation five times daily. Cr₂O₃ in diets and faeces samples were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES) and acid-digestion colorimetry (AC) methods, respectively. The results showed that ICP-AES method was more accurate for Cr₂O₃ determination than AC method, and the results determined by ICP-AES method were used in this study. ADC_d and ADC_p of seven tested ingredients were lowest for MBM (59.1 and 84.5%) and highest for FM (79.9 and 94.5%); ADC_e of tested ingredients were from 71.8% for SECM to 93.2% for FM. ADCs of amino acid in test ingredients followed similar trend to the ADC_p. The ADCs of individual amino acids varied from 61.6% (histidine in MBM) to 98.8% (valine in FM).     

南极磷虾脂肪酸组成及多不饱和脂肪酸质谱特征分析

以10％浓硫酸－甲醇溶液为甲酯化试剂,采用气相色谱/质谱(GC/MS)联用技术分析南极磷虾(Euphausua superba)的脂肪酸组成.根据GC/MS标准质谱数据库检索,结合有机质谱学规律,对多不饱和脂肪酸甲酯的裂解规律和质谱特征进行分析归纳,建立了特征离子确定其碳数和双键数,α离子和ω）离子分别确定脂肪链羰基端和甲基端双键位置的方法.通过气相色谱/质谱分析,从南极磷虾中鉴定出27种脂肪酸,其中多不饱和脂肪酸13种,二十碳五烯酸(EPA)和二十二碳六烯酸(DHA)占总脂肪酸含量的40.64％,高于一般海洋鱼虾类,表明南极磷虾具有较高的营养价值和脂质开发潜力.本研究旨在为南极磷虾营养评价和南极磷虾油等产品的研制开发以及多不饱和脂肪酸甲酯的鉴定提供理论和参考依据.     

Screening of quinalphos,trifluralin and dichlorvos residues in fresh water of aquaculture systems in Mekong Delta,Vietnam

To develop an easy and reliable method for detecting pesticides and their residues in the Mekong Delta, a GC‐MS analytical method was developed and validated according to European guidelines (SANTE/11945/2015) for the determination of residues of three pesticides (quinalphos, trifluralin and dichlorvos) in water. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.002 and 0.007 μg/L, respectively, for quinalphos and trifluralin, and 0.016 and 0.053 μg/L, respectively, for dichlorvos and quinalphos. The repeatability, the within‐laboratory reproducibility as well as the trueness met the European criteria. The recovery rate ranged between 72% (for dichlorvos and quinalphos) and 82% (for trifluralin). The developed method was then applied for the analysis of 33 water samples, collected in April 2013, at the beginning of the rainy season in the Mekong Delta in Vietnam. Thirteen samples were from rice field, 10 were collected from cat fish ponds and from red tilapia cages. Results showed that only 9% of total water samples analysed contained residues of pesticides, but only in water from rice fish systems. From the 13 samples taken in these systems, quinalphos was detected in three samples. The other two pesticides were not detected. A comparison between analytical results obtained from GC‐MS and an alternative method, that is GC‐ECD indicated that GC‐ECD is less sensitive than GC‐MS, with LOQ ranging from 0.37 to 1.18 (depending on the pesticide). However, for samples with concentrations above these LOQ, no significant difference was observed between the results obtained from the two analytical methodologies.