液相色谱-串联质谱法检测贝类产品中腹泻性贝类毒素

建立了贝类组织中2种腹泻性贝毒(Diamletic shellfish Poisobing,DSP)聚醚类毒素-大田软海绵酸(Okadaic acid,OA)和鳍藻毒素(Dinophysistoxin-1,DTX-1)的高效液相色谱-串联质谱分析方法.贝类样品经80%甲醇溶液提取,经正己烷脱脂和HLB固相萃取柱净化,采...     

UPLC-MS/MS测定海水、悬浮颗粒物及沉积物中18种藻毒素

采用固相萃取结合超高效液相色谱-串联质谱（UPLC-MS/MS）同时检测18种海洋藻毒素,包括原多甲藻酸贝类毒素（AZAs）、裸藻毒素（BTXs）、太平洋雪卡毒素（P-CTXs）、鳍藻毒素（DTXs）、米氏裸甲藻贝类毒素（GYM）、刺尾鱼素（MTX3）、大田软骨酸毒素（OA）、扇贝毒素（PTX2）、螺环内酯毒素（SPX1）及虾夷扇贝毒素（YTXs）。海水（1 L）或悬浮颗粒物及沉积物（1 g）的超声萃取液经Agilent Bond Elut C18(500 mg/6mL)固相萃取柱净化萃取后,在洗脱液中加入10%丙三醇-甲醇溶液以减少目标物损失。以95%乙腈水和水（两相均含有0.1%甲酸和2 mmol/L甲酸铵）为流动相,目标物经Phenomenex Kinetex C18色谱柱（100 mm×2.1mm i. d.,1.7μm）分离,采用电喷雾串联质谱多反应监测模式下正、负离子同时检测,外标法定量。18种藻毒素在6 min内分离良好,在线性范围内的相关系数为0.991 1～0.999 9,定量下限为0.05～250 pg/L（或pg/g）。三水平六平行的加标回收率为77.4%～119...     

辛基功能化离子液体键合硅胶固相萃取海水中贝类毒素的方法研究

贝类毒素(Shellfish toxins)是重点监控的海洋污染物。本研究通过将辛基功能化离子液体接枝到硅胶表面,制备了一种混合模式的共价键合硅胶材料(Silica-[SOIM][PF₆]),利用红外光谱、核磁共振和元素分析进行了表征。采用自制材料填制固相萃取柱,通过固相萃取-液相色谱-质谱联用(SPE-LC-MS/MS)技术对海水中贝类毒素(大田软海绵酸毒素(OA)、鳍藻毒素-1 (DTX-1)和鳍藻毒素-2 (DTX-2))进行富集检测。研究发现,此固相萃取材料与目标贝类毒素可能存在疏水作用和离子交换作用等多重相互作用。分别对进样溶液的体积和pH值、淋洗剂和洗脱剂的种类、用量以及pH值范围等因素进行了优化。结果表明,此固相萃取材料对海水中3种贝类毒素具有良好的萃取效果,优于或与商用化萃取材料性能相当,检出限(LOD)为0.01μg/L,定量限(LOQ)为0.05μg/L,在0.02～2.50μg/L范围内线性关系良好(R²>0.995),回收率在93.0%～116%之间。同时,材料具有良好的重现性,批内和批间的相对标准偏差均小于15...     

液相色谱-串联质谱检测贝类组织中5种脂溶性贝毒素

建立了液相色谱-串联质谱分析贝类组织中米氏裸甲藻(GYM)贝毒素、螺环内酯毒素(SPX1)、大田软骨酸(OA)贝毒素、蛤毒素(PTX2)、原多甲藻酸(AZA1)贝毒素的方法.用甲醇-水(4: 1, V/V)溶液对贝类组织中GYM, SPX1, OA, PTX2和AZA1进行提取,MAX阴离子交换柱净化后,采用液相色谱分离,除OA以负离子选择反应监测外,GYM, SPX1, PTX2和AZA1以电喷雾离子源正离子选择反应监测模式进行质谱分析.5种脂溶性贝毒素GYM, SPX1, OA, PTX2和AZA1在各自相应浓度范围内线性良好,相关系数＞0.99.扇贝闭壳肌空白样品添加5种贝毒素的提取率均为78.6%～94.4%(n=6); 精密度(RSD)为6.8%～14.9%.贝类组织中5种贝毒素GYM, SPX1, OA, PTX2和AZA1的检出限分别为0.10, 0.21, 2.00, 0.32和0.04 μg/kg.     

超高效液相色谱-串联质谱法测定水产中10种麻痹性贝类毒素

建立超高效液相色谱–串联质谱法测定贝类中麻痹性贝类毒素的方法。样品经0.5%甲酸加热提取,石墨化炭黑固相萃取柱净化后,用超高效液相色谱–串联质谱法测定。采用TSK–Gel Amide–80色谱柱(150mm×2.0mm,5μm),以水溶液(含2mmol/L甲酸铵,50mmol/L甲酸)A,95%乙腈水溶液(含2mmol/L甲酸铵,50 mmol/L甲酸)B为流动相,梯度洗脱,流量为0.3 mL/min,进样体积为10μL,多反应监测(MRM)模式检测。藤沟藻毒素3、4(GTX3、GTX4)、脱氧藤沟藻毒素3(dcGTX3)的检出限为8μg/kg,藤沟藻毒素5(GTX5)、新石房蛤毒素(neoSTX)、石房蛤毒素(STX)、脱氧甲酰基类毒素(dcSTX)的检出限为20μg/kg,藤沟藻毒素1,2(GTX1,GTX2)的检出限为24μg/kg,脱氧藤沟藻毒素2(dcGTX2)的检出限为28μg/kg。GTX3,dcGTX3,GTX4的线性范围为4～80μg/L,GTX5,neoSTX,STX, deSTX的线性范围为10～200μg/L,GTX1的线性范围为12～240μg/L,GTX2的线性范围为11～220μg/L,dcGTX2的线性范围为14～280μg/L,线性相关系数均大于0.99,平均回收率为82.5%～115.1%,测定结果的相对标准偏差为0.6%～7.5%(n=6)。该方法检出限低,精确度高,适用于水产品中麻痹性贝类毒素的检测。     

生物样品中大田软海绵酸类毒素代谢规律及检测方法的研究进展

近年来,中国赤潮污染日趋严重,因此引发多起贝类毒素中毒事件,威胁消费者的食用安全。大田软海绵酸（okadaic acid,OA）及其衍生物鳍藻毒素（dinophysistoxins,DTXs）是分布最广、危害最大的一类腹泻性贝类毒,具有急性腹泻毒性及多种慢性毒性。建立生物体液样品中OA类毒素残留的检测方法对辅助诊断患者的中毒情况极为必要。文章简要介绍了OA类毒素的理化性质、中毒事件、毒理作用,并详细总结了生物样品中OA类毒素代谢规律及检测方法的研究进展。     

酶联免疫吸附分析检测鳍藻毒素DTX1和DTX2

利用BALB/c小鼠腹水大量制备抗大田软海绵酸(Okadaic Acid,OA)的单克隆抗体,并以此抗体为探针建立了检测鳍藻毒素(Dinophysistoxins,DTXs)DTX1和DTX2的间接竞争酶联免疫吸附分析方法(Enzyme Linked Immunosorbent Assay,ELISA)。DTX1和DTX2各组分的标准曲线在一定范围内都有良好的线性范围和相关系数,最低检出浓度为0.53μg/L和0.45μg/L;批内和批间平均变异系数为6.18%、5.11%和7.28%、5.79%;加标样品平均回收率为76.4%、79.9%;OA与DTX1和DTX2的交叉反应率分别为53%和79%。结果表明,该方法可用于贝类样品中DTX1和DTX2的残留检测。     

利用蛋白磷酸酶活力抑制法检测牡蛎体内的腹泻性贝毒

基于腹泻性贝毒(Diarrhetic Shellfish Poison,DSP)中大田软海绵酸(Okadaic acid,OA)和鳍藻毒素(Dinophysis toxins,DTXs)能够抑制蛋白磷酸酶活力的特点,人们建立了一种利用碱性蛋白磷酸酶活力变化检测贝类中大田软海绵酸毒性当量的生物化学测试方法。本实验利用该方法对威海出入境检验检疫局采集的3个牡蛎样品进行分析,结果表明:3个牡蛎样品中不含有OA和DTXs毒素,但水解后可检出OA毒性,其中两个牡蛎水解样品的毒性当量分别为1.81和1.21μg OA eq./kg贝组织(湿重)。     

大田软海绵酸和鳍藻毒素-1纯化样品的分析

采用高效液相色谱-荧光检测、高效液相色谱-紫外检测、流动注射进样-质谱检测和高效液相色谱-质谱联用分析等方法对从有毒利玛原甲藻(Prorocentrum lima Dodge)中分离纯化的大田软海绵酸(OA)和鳍藻毒素-1(DTX1)毒素样品进行定性鉴定和纯度分析,为毒素标准品的制备提供材料和方法依据.通过柱前衍生、H...     

海产贝类脂溶性贝毒素的高效液相色谱-串联质谱分析方法及其食用安全性评价

针对海产贝类存在多种脂溶性贝毒素复合污染的现状,采用高效液相色谱-串联质谱联用技术(HPLC-MS/MS)对海产贝类中的常见脂溶性贝毒素进行同步检测,结合多种毒素复合污染的风险评估方法,用于市售海产贝类的食用安全风险评价。结果表明,在选定的实验条件下,8种典型脂溶性贝毒素加标回收率在63.2%~88.8%之间,方法的精密度(相对标准偏差(RSD)≤14.5%)和灵敏度(检出限为0.5~2.7 ng/g)良好,能满足海产贝类样品的检测要求。在采集的105个市售海产贝类样品中,42.86%的样品中至少检出了一种脂溶性贝毒素,其中鳍藻毒素-1(DTX1)的含量均值最高,为47.6μg/kg,对海产贝类污染最严重。根据每日人均贝类摄入量(TDI)和各种脂溶性贝毒素的急性中毒参考剂量(ARf D),通过计算综合风险指数∑ERI进行市售海产贝类食用安全性评价,结果表明,在所检测的样品中,存在食用安全隐患和高风险的市售海产贝类比率为19.05%,其中扇贝的食用安全风险最大。本研究建立的基于海产贝类中脂溶性贝毒素物质组复合污染的风险评价方法,与欧盟的海产品贝毒素限量标准评价方法(单指标法)相比更加严格,可以使贝类食用者更好地规避中毒风险。     

Liquid chromatography with mass spectrometry--detection of lipophilic shellfish toxins

A rapid multiple toxin method based on liquid chromatography with mass spectrometry (LC/MS) was developed for the detection of okadaic acid (OA), dinophysistoxin-1 (DTX-1), DTX-2, yessotoxin (YTX), homoYTX, 45-hydroxy-YTX, 45-hydroxyhomo-YTX, pectenotoxin-1 (PTX-1), PTX-2, azaspiracid-1 (AZA-1), AZA-2, and AZA-3. Toxins were extracted from shellfish using methanol-water (80%, v/v) and were analyzed using a C8 reversed-phase column with a 5 mM ammonium acetate-acetonitrile mobile phase under gradient conditions. The method was validated for the quantitative detection of OA, YTX, PTX-2, and AZA-1 in 4 species (mussels, Mytilus edulis; cockles, Cerastoderma edule; oysters, Crassostrea gigas; king scallop, Pecten maximus) of shellfish obtained from United Kingdom (UK) waters. Matrix interferences in the determination of the toxins in these species were investigated. The validated linear range of the method was 13-250 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. Recovery and precision ranged between 72-120 and 1-22%, respectively, over a fortification range of 40-160 microg/kg for OA, PTX-2, and AZA-1 and 100-400 microg/kg for YTX. The limit of detection, reproducibility, and repeatability of analysis showed acceptable performance characteristics. A further LC/MS method using an alkaline hydrolysis step was assessed for the detection of OA, DTX-1, and DTX-2 in their esterified forms. In combination with the LC/MS multiple toxin method, this allows detection of all toxin groups described in Commission Decision 2002/225/EC.     

Survey of the distribution of red tide toxins (okadaic acid and dinophytoxin-1) in the Dalian Bay sea area of China by micellar electrokinetic capillary chromatography.

Two kinds of diarrhoetic shellfish toxins, okadaic acid (OA) and dinophytoxin-1 (DTX-1) were determined by micellar electrokinetic capillary chromatography (MEKC) with ultraviolet detection. A detection limit of 3.25 microg/mL for both of them was achieved. The UV absorbance of these toxins measured at 200 nm showed good linearity in the range of 6.25-200 microg/mL with R = 0.992 for OA and 0.997 for DTX-1. Three kinds of shellfish (Chlamys farreri, Mytilus edulis and Ruditaps philippinarum) collected from eight locations (sampling in the intertidal zone) along the Dalian Bay sea area of China were surveyed in February and May of 2000. Results indicated that three kinds of shellfish were contaminated by OA and DTX-1. Based on per gram of hepatopancreas in February, the contamination contents ranged from 0 to 1.26 microg for OA and from 0 to 1.82 microg for DTX-1, and in May, the contents ranged from 0 to 1.45 microg for OA and 0 to 2.56 microg for DTX-1. Among the eight locations, Hei Shi Jiao and Long Wang Tang were the most contaminated areas. Of the three kinds of shellfish, Mytilus edulis was the most significant species in accumulating OA and DTX-1.     

Liquid chromatography with electrospray ion-trap mass spectrometry for the determination of yessotoxins in shellfish

Yessotoxins are a group of large polyether toxins, produced by marine dinoflagellates, which cause widespread contamination of filter-feeding shellfish. A new, sensitive liquid chromatography-mass spectrometry (LC-MS) method has been developed for the determination of yessotoxin (YTX) and 45-hydroxy-yessotoxin (45-OHYTX), a major metabolite in shellfish. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap MS in negative mode. The molecular related ion species at m/z 1141 [M-2Na+H]- was used as the parent ion for multiple MS experiments. MS-MS and MS3 gave major fragment ions at m/z 1061 [1141-SO3H]- and m/z 945 [1061-C9H12O]-. Predominant ions, that are due to the fragmentation of the backbone structure of YTXs, were observed at the MS4 stage. Reversed-phase LC using a C16 amide column was preferable to C18 phases for the separation of YTX and 45-OHYTX. Optimum calibration and reproducibility data were obtained for YTX using LC-MS-MS; r 2=0.9960, RSD < or = 6.3% at 0.25 microg YTX/g (n=5). The detection limit (S/N=3) was 30 pg YTX on-column which corresponded to 3 ng/g shellfish tissue.     

Multiresidue method for determination of algal toxins in shellfish: single-laboratory validation and interlaboratory study

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).     

Liquid chromatographic methods for the isolation and identification of new pectenotoxin-2 analogues from marine phytoplankton and shellfish.

Two acidic analogues of the polyether marine toxin, pectenotoxin-2 (PTX-2), responsible for diarrhetic shellfish poisoning (DSP), have been isolated from the toxic marine phytoplankton (Dinophysis acuta), collected in Irish waters. Liquid chromatography with fluorimetric detection (LC-FLD) analyses of the extracts of bulk phytoplankton samples, following derivatisation with 9-anthryldiazomethane (ADAM) or 1-bromoacetylpyrene (BAP), showed a complex toxin profile with peaks corresponding to okadaic acid (OA) and its isomers, dinophysistoxin-2 (DTX-2) and DTX-2C, as well as other unidentified lipophilic acids. LC-UV analysis showed the presence of a diene moiety in these new compounds and two acids have been isolated. LC coupled with mass spectrometry (MS) and tandem mass spectrometry (LC-MS-MS) were used to gain structural information. Through flow injection analysis (FIA)-MS, both in positive and negative ion modes, the molecular weight of 876 for both compounds was determined. Collision Induced Dissociation (CID) from each parent ion, as performed both in positive and negative ion mode, produced mass spectra which were very similar to those obtained for authentic PTX-2 (mw 858). These new compounds have been confirmed to be pectenotoxin-2 seco acids (PTX-2SAs) and they are closely related to PTX-2 except that they contain an open chain carboxylic acid rather than a lactone ring. Toxic mussels also contained these pectenotoxin-2 analogues.     

Confirmation of okadaic acid, dinophysistoxin-1 and dinophysistoxin-2 in shellfish as their anthrylmethyl derivatives using UV radiation

A rapid and simple method for confirmation of the diarrhetic shellfish poisons (DSP): okadaic acid (OA), dinophysistoxin-1 (DTX-1) and dinophysistoxin-2 (DTX-2) using fluorescence detection following derivatization with 9-chloromethylanthracene, has been established as an alternate to LC/MS. Exposure of the anthrylmethyl derivatives of OA, DTX-1 and DTX-2 to near UV light (300-400 nm) resulted in the loss of these compounds to below detection limits within 30 min, with a concurrent appearance of two additional compounds. Based on the mass spectral evidence, we propose that these newly formed compounds are the decarboxylation products of the derivatized diarrhetic shellfish poisons. UV radiation is, therefore, proposed as a rapid and simple confirmation technique for these DSP in mussel samples.     

Determination of lipophilic toxins by LC/MS/MS: single-laboratory validation

An LC/MS/MS method has been developed, assessed, and intralaboratory-validated for the analysis of the lipophilic toxins currently regulated by European Union legislation: okadaic acid (OA) and dinophysistoxins 1 and 2, including their ester forms; azaspiracids 1, 2, and 3; pectenotoxins 1 and 2; yessotoxin (YTX), and the analogs 45 OH-YTX, Homo YTX, and 45 OH-Homo YTX; as well as for the analysis of 13-desmetil-spirolide C. The method consists of duplicate sample extraction with methanol and direct analysis of the crude extract without further cleanup or concentration. Ester forms of OA and dinophysistoxins are detected as the parent ions after alkaline hydrolysis of the extract. The validation process of this method was performed using both fortified and naturally contaminated samples, and experiments were designed according to International Organization for Standardization, International Union of Pure and Applied Chemistry, and AOAC guidelines. With the exception of YTX in fortified samples, RSDr below 15% and RSDR were below 25%. Recovery values were between 77 and 95%, and LOQs were below 60 microg/kg. These data together with validation experiments for recovery, selectivity, robustness, traceability, and linearity, as well as uncertainty calculations, are presented in this paper.     

Validation of a confirmatory method for lipophilic marine toxins in shellfish using UHPLC-HR-Orbitrap MS

Lipophilic marine toxins are produced by harmful microalgae and can accumulate in edible filter feeders such as shellfish, leading to an introduction of toxins into the human food chain, causing different poisoning effects. During the last years, analytical methods, based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been consolidated by interlaboratory validations. However, the main drawback of LC-MS/MS methods remains the limited number of compounds that can be analyzed in a single run. Due to the targeted nature of these methods, only known toxins, previously considered during method optimization, will be detected. Therefore in this study, a method based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS) was developed. Its quantitative performance was evaluated for confirmatory analysis of regulated lipophilic marine toxins in shellfish flesh according to Commission Decision 2002/657/EC. Okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX), and 13-desmethyl spirolide C (SPX-1) were quantified using matrix-matched calibration curves (MMS). For all compounds, the reproducibility ranged from 2.9 to 4.9 %, repeatability from 2.9 to 4.9 %, and recoveries from 82.9 to 113 % at the three different spiked levels. In addition, confirmatory identification of the compounds was effectively performed by the presence of a second diagnostic ion (¹³C). In conclusion, UHPLC-HR-Orbitrap MS permitted more accurate and faster detection of the target toxins than previously described LC-MS/MS methods. Furthermore, HRMS allows to retrospectively screen for many analogues and metabolites using its full-scan capabilities but also untargeted screening through the use of metabolomics software. Figure

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超高效液相色谱-串联质谱法同时测定血浆与尿液中12种脂溶性贝类毒素

生物样品中脂溶性贝类毒素的检测,可为食物中毒等突发公共卫生事件的流行病学调查以及中毒者的临床救治提供技术支持.目前的研究存在目标化合物少,以及方法前处理复杂、灵敏度低等问题.该研究通过优化前处理和色谱分离技术,建立了超高效液相色谱-串联质谱法测定血浆、尿液中12种脂溶性贝类毒素的方法.实验对提取试剂以及流动相的选择进行...     

In-house validation of a liquid chromatography tandem mass spectrometry method for the analysis of lipophilic marine toxins in shellfish using matrix-matched calibration

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of lipophilic marine toxins in shellfish extracts (mussel, oyster, cockle and clam) was validated in-house using European Union (EU) Commission Decision 2002/657/EC as a guideline. The validation included the toxins okadaic acid (OA), yessotoxin (YTX), azaspiracid-1 (AZA1), pectenotoxin-2 (PTX2) and 13-desmethyl spirolide-C (SPX1). Validation was performed at 0.5, 1 and 1.5 times the current EU permitted levels, which are 160 μg kg^-1 for OA, AZA1 and PTX2 and 1,000 μg kg^-1 for YTX. For SPX1, 400 μg kg^-1 was chosen as the target level as no legislation has been established yet for this compound. The method was validated for determination in crude methanolic shellfish extracts and for extracts purified by solid-phase extraction (SPE). Extracts were also subjected to hydrolysis conditions to determine the performance of the method for OA and dinophysistoxin esters. The toxins were quantified against a set of matrix-matched standards instead of standard solutions in methanol. To save valuable standard, methanolic extract instead of the homogenate was spiked with the toxin standard. This was justified by the fact that the extraction efficiency is high for all relevant toxins (above 90%). The method performed very well with respect to accuracy, intraday precision (repeatability), interday precision (within-laboratory reproducibility), linearity, decision limit, specificity and ruggedness. At the permitted level the accuracy ranged from 102 to 111%, the repeatability from 2.6 to 6.7% and the reproducibility from 4.7 to 14.2% in crude methanolic extracts. The crude extracts performed less satisfactorily with respect to the linearity (less than 0.990) and the change in LC-MS/MS sensitivity during the series (more than 25%). SPE purification resulted in greatly improved linearity and signal stability during the series. Recently the European Food Safety Authority (EFSA) has suggested that to not exceed the acute reference dose the levels should be below 45 μg kg^-1 OA equivalents and 30 μg kg^-1 AZA1 equivalents. A single-day validation was successfully conducted at these levels. If the regulatory levels are lowered towards the EFSA suggested values, the official methods prescribed in legislation (mouse and rat bioassay) will no longer be sensitive enough. The validated LC-MS/MS method presented has the potential to replace these animal tests.