Peer validation of a method to confirm chloramphenicol in honey by liquid chromatography-tandem mass spectrometry

Chloramphenicol (CAP) is extracted from an aqueous dilution of honey using ethyl acetate. The extracts are evaporated and redissolved in water. CAP is then extracted from the aqueous solutions using reversed-phase solid-phase extraction cartridges. CAP is eluted from the reversed-phase cartridges with acetonitrile-water and re-extracted into ethyl acetate. The ethyl acetate is evaporated, and the residue is reconstituted in an aqueous solution. Extracts are chromatographed using a reversed-phase column and analyzed by electrospray negative mode tandem mass spectrometry. Four product ions of precursors m/z 321 or 323 are monitored. The method meets confirmation criteria recommended by the U.S. Food and Drug Administration and 4-point identification criteria established by the European Union. With slight modifications to accommodate different equipment, the method was validated in 2 laboratories.     

Quantitative determination of chloramphenicol in milk powders by isotope dilution liquid chromatography coupled to tandem mass spectrometry

A method is described for the determination of residues of the illegal antibiotic chloramphenicol (CAP) in milk powders. The analyte is quantified by liquid chromatography coupled to electrospray ionisation tandem mass spectrometry (LC-ESI-MS-MS) operating in negative ion multiple reaction monitoring mode (MRM) after a liquid-liquid extraction followed by a clean-up step on solid phase extraction (SPE) cartridge. Because of the presence of two chlorine atoms in the CAP molecule, four specific transition reactions of CAP were monitored by MS-MS in selecting m/z 321 --> 257, 321 --> 152 (35Cl2) and m/z 323 --> 257, 323 --> 152 (37Cl35Cl). Two calibration curves were constructed by plotting the area ratio of m/z 321 --> 152 versus 326 --> 157 and m/z 321 --> 257 versus 326 --> 262 against their corresponding amount ratio. Indeed, even if m/z 321 --> 152 was found to give a higher MS-MS response (calibration curve used by default), an interfering chemical substance was sometimes observed for some milk extracts and not for the transition m/z 321 --> 257. The quantitation method was validated according to the European Union (EU) criteria for the analysis of veterinary drug residues at 0.1, 0.2 and 0.5 microg/kg concentration levels using d5-CAP as internal standard. The decision limit (CCalpha) and detection capability (CCbeta) of CAP in milk were calculated for m/z 321 --> 152 at 0.02 microg/kg and 0.03 microg/kg, respectively, and for m/z 321 --> 257 at 0.02 microg/kg and 0.04 microg/kg, respectively. At the lowest fortification level (i.e. 0.1 microg/kg), repeatability and within-laboratory reproducibility were calculated for m/z 321 --> 257 both at 0.02 microg/kg and for m/z 321 --> 152 at 0.03 and 0.05 microg/kg, respectively. Moreover, the measurement of uncertainty of the analytical method was calculated at the same spiking levels and falls within the precision values of the within-laboratory reproducibility. This method can be applied to several types of milk powders (e.g. full cream, skim) and can serve as a monitoring tool to avoid that unacceptable levels of residues of CAP enter the food chain.     

Rapid determination of chloramphenicol residues in milk powder by liquid chromatography-elektrospray ionization tandem mass spectrometry

A simple and rapid liquid chromatography tandem mass spectrometry (LC-ESI-MS-MS) confirmation method for the analysis chloramphenicol (CAP) in milk powder has been developed. Samples were extracted by using liquid-liquid extraction steps with ethyl acetate. Lipids were removed using hexsan. LC separation was achieved by using a Phenomenex Luna C-18 column and acetonitryle-water as a mobile phase. The mass spectrometer was operated in multiple reaction monitoring mode (MRM) with negative electro-spray interface (ESI-). The four transitions were monitored m/z 321-->257, 321-->194, 321-->152, 326-->157 (IS) and for quantification, the transition m/z 321-->152 was chosen. Validation of the method was done according to criteria of Decision Commission No 2002/657 EC. Validation includes the determination of specification, linearity, precision (within- and between-day), accuracy, decision limit (CC alpha) and detection capability (CC beta). Samples were fortified at CAP levels 0.30, 0.45 and 0.60 microg/kg with CAP-5d as internal standard. The precision within-day (RSD%) was lower than 12% and accuracy (RE%) ranged from -9.8 to -3.7%. The precision between-day (RSD%) was less than 15%. The limit of decision (CC alpha) and detection capability (CC beta) for milk powder 0.09 and 0.11 microg/kg. Value CC alpha and CC beta were calculated for the 321-->152 ion transition. This method has been successfully used for routine analysis.     

Determination of the antibiotic chloramphenicol in meat and seafood products by liquid chromatography-electrospray ionization tandem mass spectrometry

A confirmatory method based on isotope dilution liquid chromatography-electrospray ionization tandem mass spectrometry is described for the determination of the antibiotic chloramphenicol (CAP) in foods. The method is quantitative and entails liquid-liquid extraction followed by a clean-up step on a silica gel solid-phase extraction cartridge. Mass spectral acquisition is done in the negative ion mode applying multiple reaction monitoring of two diagnostic transition reactions for CAP (m/z 321 --> 257 and m/z 321--> 152). In addition, the presence of two chlorine atoms in the CAP molecule provides further analyte certainty by assessing the 37Cl/35Cl ratio using the transition reactions m/z 323 --> 257 and m/z 323 --> 152. Validation of the method in chicken meat is conducted according to the latest European Union criteria for the analysis of veterinary drug residues at levels of 0.05, 0.10, and 0.20 microg/kg, employing [2H5]-chloramphenicol as internal standard. The decision limit and the detection capability were calculated at 0.01 microg/kg and 0.02 microg/kg, respectively. At the lowest fortification level (i.e. 0.05 microg/kg), precision values below 14 and 17% were achieved under repeatability and within-laboratory reproducibility conditions, respectively. The accuracy of the method was within 20, 15, and 5% of the target values at the 0.05, 0.10, and 0.20 microg/kg fortification levels, respectively. The applicability of this procedure was demonstrated by the analysis of other meat (turkey, pork, beef) and seafood (fish, shrimps) products. The method is robust and suitable for routine quality control operations, and more than 200 sample injections were performed without excessive pollution of the mass spectrometer or loss of LC column performance.     

In-house validation of a liquid chromatography/electrospray tandem mass spectrometry method for confirmation of chloramphenicol residues in muscle according to Decision 2002/657/EC

In this work we present an in-house validation study for the confirmatory analysis of chloramphenicol (CAP) in muscle according to the Commission Decision 2002/657/EC requirements. CAP is extracted in acetonitrile and after liquid-liquid partitioning with n-hexane is identified and quantitatively determined by ion trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) analysis in the negative ion mode. CAP was identified using the precursor ion and at least two product ions, meeting the qualitative and quantitative criteria set by the European Commission in the Decision 2002/657/EC for confirmation of prohibited veterinary drug residues. We calculated mean drug recoveries, CCalpha and CCbeta of the method, and reported data on specificity, ruggedness and within-laboratory reproducibility. Finally, we point out and discuss some problems and questions arising from controversy about the application of Decision 2002/657/EC.     

气相色谱-质谱联用法测定动物组织中氯霉素、氟甲砜霉素和甲砜霉素的残留量

建立了气相色谱-负离子化学电离源质谱同时测定动物组织中氯霉素(CAP)、甲砜霉素(TAP)和氟甲砜霉素(FF)残留量的方法。样品用乙酸乙酯提取,正己烷分配去脂肪,再用Florisil柱进一步净化,甲苯作为反应介质,用N,O-双(三甲基硅基)三氟乙酰胺(BSTFA)-三甲基氯硅烷(TMCS)(体积比为99∶1)进行硅烷化处理,用间硝基氯霉素(m-CAP)作为内标进行测定。CAP的检测限可达到0.03 μg/kg,TAP和FF的检测限可达到0.2 μg/kg;上述3种药物的标准曲线的线性相关系数均大于0.99。CAP,FF和TAP的批内测定的精密度(以相对标准偏差表示)依次为5.5%,10.4%和8.8%;批间测定的精密度依次为7.4%,20.7%和19.1%。回收率为80.0%~111.5%,相对标准偏差为1.2%~15.4%。该方法前处理步骤简单,处理后杂质干扰少,灵敏度高,适用性强,可用于猪肉及禽类、水产品等多种动物组织中氯霉素类药物残留的检测。     

Determination of residues of tricaine in fish using liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination of residues of the anaesthetic tricaine mesilate (MS222) in fish tissues is described. Residues were extracted from homogenized tissues with McIllvaine buffer/methanol and purified over a C18 solid-phase extraction column followed by LC-MS/MS analysis. In the multiple-reaction monitoring mode of the mass spectrometer, chromatograms were recorded by monitoring the m/z 166-->m/z 138 and m/z 166-->m/z 94 transitions for quantification and confirmation of the residues in the finfish matrix, respectively. Recoveries were in the range of 67%+/-10% (n=6) for tilapia at 2 microg kg(-1), 95%+/-7% (n=6) at 2 microg kg(-1) in salmon and 92%+/-3% (n=5) for trout at 2.5 microg kg(-1). The limits of detection were 0.5, 0.6 and 0.6 microg kg(-1) in trout, salmon and tilapia, respectively. No residues of tricaine were found in eight sampled aquacultured fish (salmon and trout) bought from the local market.     

Liquid chromatography/tandem mass spectrometry analysis of chloramphenicol in cooked crab meat

A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method is described for the extraction, cleanup, determination, and confirmation of chloramphenicol (CAP) in cooked crab meat. The method involves pulverization of cooked crab meat with dry ice; extraction of the CAP into ethyl acetate (EtOAc); evaporation (by N2) of the EtOAc; addition of methanol, aqueous NaCl, and heptane; extraction of the lipids into the heptane, followed by extraction of the aqueous phase with EtOAc; evaporation (by N2) of the EtOAc; dissolution into methanol-water; filtration; and separation/detection/confirmation using LC/MS/MS. Crab meat was fortified at 0.25, 0.50, and 1.0 ng/g (ppb) chloramphenicol. Average absolute recoveries were 67, 84, and 86%, respectively, with relative standard deviation values all less than 1%. Four daughter ions (m/z 152, 176, 194, and 257) were monitored off the m/z 321 precursor ion. Determination was based on a standard curve using the peak areas of the m/z 152 daughter ion (the base peak) for standard solutions equivalent to 0.10, 0.20, 0.50, and 1.0 ppb in tissue (made with control crab extract). A set of 6 matrix controls (unfortified crab meat) was also analyzed, in which no chloramphenicol was detected. For identification purposes, the ion ratios (of each daughter ion versus the base daughter ion) of the fortified crab versus those of the chloramphenicol standards agreed within 10% (relative) at fortified chloramphenicol concentrations of 0.25-1.0 ppb.     

气相色谱-质谱法测定乳制品中光引发剂异丙基硫杂蒽酮的残留量

建立了采用气相色谱（GC）-质谱(MS)检测由包装材料迁移到乳制品中的光引发剂异丙基硫杂蒽酮残留量的方法。使用氘代蒽为内标,样品经Carrez试剂除蛋白质后用丙酮-正己烷(体积比为1∶1)提取,上层提取液用氟罗里硅土固相萃取小柱净化。采用单四极杆质谱进行样品筛选和定量,选取的监测离子为m/z 184,m/z 224,m/z 239,m/z 254(异丙基硫杂蒽酮)和m/z 80,m/z 94,m/z 188,m/z 160(氘代蒽)。疑似样品采用离子阱串联质谱法进行确证,选取的母离子和子离子分别为m/z 254,m/z 239(异丙基硫杂蒽酮)和m/z 188,m/z 160(氘代蒽)。本方法的测定低限(LOQ)分别为7.0 μg/L(GC-MS)和5.0 μg/L(GC-MS/MS),回收率为74.9%~89.6%。采用该方法对11种不同类型的乳制品进行了检测,发现了两例阳性样品。     

Liquid chromatography/electrospray tandem mass spectrometric analysis of incurred ractopamine residues in livestock tissues

Ractopamine HCl is a beta-adrenergic agonist (beta-agonist) recently approved by the U.S. Food and Drug Administration, but not other governmental agencies, for use in finishing swine. For these reasons, it was important to develop and validate mass spectrometric methods for the detection and confirmation of ractopamine residues in livestock marker tissues. Incurred tissues in cattle, sheep, turkeys, and ducks were generated during 7-day ractopamine feeding (20 ppm in diets) periods. Disposition of ractopamine residues in liver and pigmented retinal epithelium was determined in animals slaughtered with withdrawal periods of 0, 3, and 7 days. Ractopamine residues, purified using solid-phase extraction, were measured using liquid chromatography (LC) and electrospray with detection by tandem mass spectrometry (MS/MS) in the multiple reaction-monitoring (MRM) mode. Total ractopamine residues (parent ractopamine + hydrolyzed conjugates) in liver were detected in all species on withdrawal day 0 (2-97 ppb) and were greatly diminished in all species by withdrawal day 7 (<1 ppb). Bovine and ovine retina had lower levels of ractopamine (0.5-3 ppb) than liver, and occular residues increased with withdrawal time, suggesting redistribution into this tissue. Lower limits of quantification were found to be approximately 0.1 ppb in liver and retina. Incurred ractopamine residues were confirmed by the precise and accurate agreement of MRM intensity ratios of diagnostic fragment ions (m/z 284, 164, and 121) from the protonated molecule between ractopamine residues in incurred samples and an authentic ractopamine standard. The limits of confirmation in liver and retina using recognized acceptance criteria were below 1 ppb. The high sensitivity and specificity for measurement and confirmation of ractopamine residues suggests this method will be applicable for regulatory residue surveillance programs.     

纺织品、皮革及其制品中五氯苯酚残留量的气相色谱-质谱法测定

介绍应用气相色谱-质谱法测定纺织品、皮革及其制品中防霉剂五氯苯酚的残留量,试样经硫酸溶液酸化后用正己烷提取,用气相色谱-质谱选择离子测定。方法简便、快速、灵敏,检出限20μg／kg,添加回收率86．7％～93．1％,变异系数4．1％～5．9％。     

Development of a liquid chromatography/electrospray tandem mass spectrometry method for confirmation of chloramphenicol residues in milk after alfa-1-acid glycoprotein affinity chromatography

In this work we present a method for confirmatory analysis of chloramphenicol (CAP) in bovine and buffalo raw milk. CAP is extracted in acetonitrile and purified by affinity chromatography on an alpha-1-acid glycoprotein (AAG) column, then is identified and determined by ion-trap liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI-MS/MS) analysis in the negative ion mode. CAP was identified at the minimum required performance limit (MRPL) of 0.30 ppb, by monitoring the [M-H]- ion and at least two product ions, meeting the qualitative and quantitative criteria set by the European Commission in Decision 2002/657/EC for confirmation of prohibited veterinary drugs. The trueness and within-day and between-day repeatability data are also reported. Moreover, the loading capacity of affinity columns towards CAP was tested. This method, based on the molecular recognition between drug and AAG during the purification step to improve sample cleanup, represents a quantitative and repeatable procedure for confirmatory analysis, and fits the requirements for the routine official control of CAP residues in raw milk.     

Confirmatory analysis of residues of stanozolol and its major metabolite in bovine urine by liquid chromatography-tandem mass spectrometry

A reliable method for the confirmation of the synthetic hormone stanozolol and its major metabolite, 16beta-hydroxystanozolol, in bovine urine by liquid chromatography coupled with tandem mass spectrometry has been developed. [2H3]Stanozolol was used as internal standard. Sample preparation involved enzymatic hydrolysis, liquid-liquid extraction and purification on an amino solid-phase extraction column. The analytes were ionized using atmospheric pressure chemical ionization with a heated nebulizer interface operating in the positive ion mode, where only the protonated molecules, [M+H]+, at m/z 329 and m/z 345, for stanozolol and 16beta-hydroxystanozolol, respectively, were generated. These served as precursor ions for collision-induced dissociation and three diagnostic product ions for each analyte were identified for the unambiguous hormone confirmation by selected reaction monitoring liquid chromatography-tandem mass spectrometry. The accuracy ranged from 19.7 to 14.9% and from 18.9 to 13.2% for stanozolol and 16beta-hydroxystanozolol, respectively. The precision ranged from 12.4 to 2.4% and from 13.1 to 1.8% for stanozolol and 16beta-hydroxystanozolol, respectively. The limit of quantification of the method was 1 ng/ml in the bovine urine for both stanozolol and 16beta-hydroxystanozolol. The developed method fulfils the European Union requirements for confirmatory methods.     

Pre-electrospray ionisation manifold methylation and post-electrospray ionisation manifold cleavage/ion cluster formation observed during electrospray ionisation of chloramphenicol in solutions of methanol and acetonitrile for liquid chromatography-mass spectrometry employing a commercial quadrupole ion trap mass analyser

We have observed unusual mass spectra of chloramphenicol (CAP) in solutions of methanol or acetonitrile showing intense ions at m/z 297, m/z 311, m/z 325 and m/z 339. The observed ions were different from those which are traditionally observed in the full scan ESI mass spectra of CAP with ions of m/z 321, m/z 323 and m/z 325. We have evidence to show that this process starts with offline methylation of CAP in solutions of methanol or acetonitrile to give m/z 339. Investigations using nuclear magnetic resonance (NMR) spectroscopy showed that there is a methylene group somewhere within the CAP molecule but not attached to any of the carbon atoms when the CAP is dissolved in methanol or acetonitrile before infusion into the mass spectrometer. The possible locations of attachment were speculated to be the electronegative atoms apart from the chlorine atoms due to valence considerations. The methylene group is attached to the nitrogen atom and forms a bond as observed in the MS/MS spectra of m/z 297, m/z 311, m/z 325 and m/z 339 which give m/z 183 as the base peak in all cases. Further experiments showed that there is cleavage of the methylated CAP molecule followed by cluster ion formation involving addition of methylene groups to the CAP fragment with m/z 183 to produce ions of m/z including m/z 297, m/z 311, m/z 325 and m/z 339. This process occurs in the mass spectrometer in the region housing the tube lens and is triggered when the ions are accelerated through this region by application of a negative tube lens offset voltage. This region affords collision of the charged droplets with a collision gas in this case nitrogen to strip the droplets of their solvent molecules. Experiments to follow the intensities of m/z 183, m/z 311, m/z 321, m/z 323, m/z 325 and m/z 339 as the tube lens offset voltage was varied were done in which the intensities of m/z 311, m/z 325 and m/z 339 were observed to be at their peak when the tube lens offset voltage was set at -40 V. When the tube lens offset voltage is swung to +40 V, thus decelerating the ions through the capillary skimmer region via the tube lens, the traditionally observed spectra with m/z 321, m/z 323 and m/z 325 were observed.     

Determination of M+4 stable isotope labeled cortisone and cortisol in human plasma by microElution solid-phase extraction and liquid chromatography/tandem mass spectrometry

A sensitive microElution solid-phase extraction (SPE) liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed and validated for the determination of M+4 stable isotope labeled cortisone and cortisol in human plasma. In this method, M+4 cortisone and M+4 cortisol were extracted from 0.3 mL of human plasma samples using a Waters Oasis HLB 96-well microElution SPE plate using 70 microL methanol as the elution solvent, and chromatographed on a Waters Symmetry C18 column (4.6 x 50 mm, 3.5 microm). M+9 cortisone and M+9 cortisol were used as the internal standards. A PE Sciex API 4000 tandem mass spectrometer interfaced with the liquid chromatograph via a turboionspray source was used for mass analysis and detection. The selected reaction monitoring (SRM) of precursor --> product ion transitions were monitored at m/z 365.2 [M+H](+) --> 167.0 and at m/z 367.3 [M+H](+) --> 125.1 for M+4 cortisone and M+4 cortisol, respectively. The lower limit of quantitation was 0.1 ng mL(-1) and the linear calibration range was from 0.1 to 100 ng mL(-1) for both analytes. This method demonstrated to be very reproducible and reliable.     

Tetrahydrogestrinone: discovery, synthesis, and detection in urine

Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one or THG) was identified in the residue of a spent syringe that had allegedly contained an anabolic steroid undetectable by sport doping control urine tests. THG was synthesized by hydrogenation of gestrinone and characterized by mass spectrometry and NMR spectroscopy. We developed and evaluated sensitive and specific methods for rapid screening of urine samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of underivatized THG (using transitions m/z 313 to 241 and 313 to 159) and gas chromatography/high-resolution mass spectrometry (GC/HRMS) analysis of the combination trimethylsilyl ether-oxime derivative of THG (using fragments m/z 240.14, 254.15, 267.16, and 294.19). A baboon administration study showed that THG is excreted in urine.     

超声萃取/液相色谱-串联质谱法测定染整助剂中的二硬脂基二甲基氯化铵

建立了超声辅助萃取/液相色谱-串联质谱( LC - MS/MS)测定染整助剂中二硬脂基二甲基氯化铵(DSDMAC)的方法.将样品以甲醇为萃取溶剂进行超声萃取,萃取液经过滤后采用LC-MS/MS测定.采用电喷雾离子源,定性离子对为m/z 550.5/298.3、522.5/298.3、522.5/270.3、494.5/...     

气相色谱/质谱/质谱法分析石油基引燃物

应用Ｖａｒｉａｎ ３８００ ＧＣ／Ｓａｔｕｒｎ ２０００ ＭＳ 气相色谱／质谱联用分析仪（简称ＧＣ／ＭＳ／ＭＳ）对以汽油、煤渍和紫油为引燃物，燃烧聚丙稀化纤地毯和土壤时的模拟现场物品进行了引燃物的检出。发现采用ＧＣ／ＭＳ／ＭＳ方法比用ＧＣ／ＭＳ方法可以较彻底地排队由于柱注失、地毯燃物分解产物、土壤背景产物和地毯增塑剂等引起的干扰，而且可以提高检测灵敏度。详细考察了以不同质荷比的离子作母离子时对ＧＣ     

Determination of the metabolites of gestrinone in human urine by high performance liquid chromatography, liquid chromatography/mass spectrometry and gas chromatography/mass spectrometry

Gestrinone was studied by high performance liquid chromatography (HPLC) for screening and by gas chromatography/mass spectrometry (GC/MS) for confirmation. When the chromatograms of blank, spiked urine and dosed urine were compared by HPLC, two unknown metabolites were found and these were excreted as the conjugated forms. Metabolites 1 and 2 were tested by LC/MS and LC/MS/MS and both had parent ions at m/z 325. The fragment ion of metabolite 1 was at m/z 263 and ions for metabolite 2 were m/z 307 [MH - H(2)O](+), 289, 279 and 241. LC/MS/MS of m/z 263 as the parent ion of metabolite 1 gave fragment ions at m/z 245 and 217, which were assumed to be [263 - H(2)O](+) and [235 - H(2)O](+), respectively. The trimethylsilyl (TMS)-enol-TMS ether derivative of gestrinone displayed three peaks in its GC/MS chromatogram, formed by tautomerism.     

Analysis of Chlormequat residues in grain using liquid chromatography-mass spectrometry (LC-MS/MS)

A fast, sensitive and specific method for routine determination of residues from Chlormequat (CAS no. 7003-89-6) is described. The method is based on a simple clean-up using an SPE-C₁₈ cartridge, high-performance liquid chromatography on a standard C₁₈ column (Spherisorb S5 ODS1) and specific detection and quantification by electrospray mass spectrometry (LC-MS/MS). ¹³C-Chlormequat was synthesised for use as internal standard. Samples were extracted with methanol – water – acetic acid. Internal standard and ammonium acetate were added before C₁₈-cartridge clean up and residues eluted with methanol – water – acetic acid, containing 50 mM ammonium acetate. Chromatographic separation was achieved using a solvent composed of acetonitrile – methanol – water – acetic acid (53:21:25:1 by volume), containing 50 mM ammonium acetate. Electrospray ionisation mass spectrometry was employed using m/z 58 (daughter ion of the Chlormequat quaternary ammonium ion, m/z 122) and m/z 61 (daughter ion of the ¹³C-Chlormequat quaternary ammonium ion, m/z 125) for quantification. The LC analysis time was 15 min and the limit of detection of the analytical method was 9 μg/kg. The performance of the method was demonstrated analysing grain material from an inter-comparison study. In Denmark the primary use of Chlormequat chloride (CCC, cycocel, or chlorocholin chloride, CAS no. 999-81-5) is for winter cereals and 11 such winter wheat samples from the Danish National Pesticide Survey were analysed. Residue contents were from below 0.01 up to 0.45 mg/kg, and thus below the EU maximum residue level of 2.0 mg/kg for wheat.