Time-resolved fluoroimmunoassay for 19-nortestosterone residues in aquaculture tissues.

A competitive time-resolved fluoroimmunoassay (TR-FIA) was developed for the determination of 19-nortestosterone (17beta-NT) residues in aquaculture tissues. The limit of detection (LOD) was determined to be 0.08 ng g-1 and the limit of quantification (LOQ) was less than 0.8 ng g-1. The results obtained by the TR-FIA and ELISA showed a good correlation. The established TR-FIA was validated for the determination of incurred aquaculture tissues and confirmed by liquid chromatography tandem mass spectrometry (LC/MS/MS). This proposed technique could be applied to routine residue analysis.     

Assessment of commutability for candidate certified reference material ERM-BB130 “chloramphenicol in pork”

Chloramphenicol (CAP), an effective antibiotic against many microorganisms, is meanwhile banned in the EU for treatment of food-producing animals due to adverse health effects. The Institute for Reference Materials and Measurements (IRMM) is currently developing a certified reference material (CRM) for CAP in pork, intended for validation and method performance verifications of analytical methods. The material will be certified using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and gas chromatography–mass spectrometry (GC–MS) methods and has a target CAP level around the minimum required performance limit (MRPL) of 0.3 μg/kg. To prove that the material can be applied as a quality control tool for screening methods, a commutability study was conducted, involving five commercially available enzyme-linked immunosorbent assay kits and one biosensor assay (BiaCore kit). Meat homogenates (cryo-milled wet tissue) with CAP concentrations around the MRPL and the candidate CRM (lyophilised powder) were measured by LC–MS/MS and GC–MS as well as the six screening methods. Pairwise method comparisons of results obtained for the two sample types showed that the CRM can successfully be applied as quality control (QC) sample to all six screening methods. The study suggests that ERM-BB130 is sufficiently commutable with the investigated assays and that laboratories applying one of the investigated kits therefore benefit from using ERM-BB130 to demonstrate the correctness of their results. However, differences among the assays were observed, either in the abundance of bias between screening and confirmatory LC and GC methods, the repeatability of test results, or goodness of fit between the methods.     

改良的QuEChERS与HPLC-HESI/MS/MS同时测定中华鳖组织中氯硝柳胺和酰胺醇类药物及其代谢物的残留量

建立了中华鳖(Trionyx sinensis)组织(血浆、肌肉、裙边、肝脏和肾脏)中氯硝柳胺、氯霉素、甲砜霉素、氟苯尼考和氟苯尼考胺同时测定的高效液相色谱-加热电喷雾电离源串联质谱法(HPLC-HESI/MS/MS)。样品经改进的QuEChERS方法提取净化,以氨化乙腈为提取剂,十八烷基硅烷键合硅胶(C18)粉为净化剂,甲醇-水为流动相,流速为0.3 mL/min,以Waters Symmetry~ C_(18)(2.1 mm×100 mm,3.5μm)为色谱分离柱,采用正负离子分段扫描和多反应监测模式(MRM)检测。氯霉素、甲砜霉素、氟苯尼考和氟苯尼考胺采用内标标准曲线法定量,氯硝柳胺采用基质匹配标准曲线外标法定量。结果表明,在0.3~100μg/L范围内,5种待测物均呈良好的线性关系,相关系数(r2)均不小于0.998 7。在1~20μg/kg加标水平下,中华鳖空白血浆、肌肉、裙边、肝脏和肾脏的加标回收率为77.9%~105.3%(n=6),相对标准偏差为2.7%~10.5%(n=6),方法的检出限分别为0.5、0.1、0.5、0.5、0.5μg/kg,定量下限分别为1.0、0.3、1.0、1.0、1.0μg/kg。该方法操作简便、准确、灵敏度高,适用于中华鳖组织中氯硝柳胺、氯霉素、甲砜霉素、氟苯尼考和氟苯尼考胺残留量的同时测定。     

Determination of Chloramphenicol Residues in Foods by ELISA and LC-MS/MS Coupled with Molecularly Imprinted Solid Phase Extraction

The combination of molecularly imprinted solid-phase extraction (MISPE) with ELISA and LC-MS/MS was developed for the detection of chloramphenicol (CAP) in honey samples. Significant recoveries of 99.1 ± 7.1 and 98.8 ± 8.2% were obtained for intra- and inter-assay determination by ELISA determination, respectively. The limit of detection of CAP was 0.034 μg kg^?1 and the limit of quantification was 0.046 μg kg^?1. Determination and validation of CAP by using LC-MS/MS were performed following the same extraction and purification process as for the ELISA. The results demonstrated that the CAP samples purified by using MISPE were simultaneously applicable to analysis by ELISA and LC-MS/MS.     

Determination of Nitrofuran Residues in Tilapia Tissue by Enzyme‐Linked Immunosorbent Assay and Confirmation by Liquid Chromatography Tandem Mass Spectrometric Detection

Furazolidone is a broad‐spectrum antibiotic that is frequently used in aquaculture on account of its excellent antibacterial properties. In this study, both the enzyme‐linked immunosorbent assay (ELISA) and high‐performance liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) methods were used to analyze the content of residual 3‐amino‐2‐oxazolidinone (AOZ), a metabolite of furazolidone in Tilapia tissue. Homogenized fish samples were spiked with various amounts of AOZ, and following combined acid‐hydrolysis and derivatization of the homogenized tissue with 2‐NBA (2‐nitrobenzaldehyde), sample clean‐up was performed and the derived 2‐nitrophenylmethylene‐3‐amino‐2‐oxazolidinone (NPAOZ) was analyzed. Using the LC‐MS/MS method, a linear correlation between measured concentration Y and spiked concentration × was observed: Y = 0.4518X ? 0.0166, R² = 0.9972. The linear equation for the ELISA method was Y = 0.9322X + 0.5168, R² = 0.9066. These results demonstrated that the ELISA method might overestimate the residual AOZ content at low concentrations. The detection limit and recovery of the known addition were 0.05 μg kg^?1a and 108% for the LC‐MS/MS method and 0.31 μg kg^?1 and 305% for the ELISA method, respectively.     

Discrimination of eight chloramphenicol isomers by liquid chromatography tandem mass spectrometry in order to investigate the natural occurrence of chloramphenicol

This paper describes the discrimination of eight different isomers of chloramphenicol (CAP), an antibiotic banned for use in food producing animals, by reversed phase and chiral liquid chromatography in combination with tandem mass spectrometric detection. Previously, by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) the presence of CAP was confirmed in some grass and herb samples collected on Mongolian pastures up to concentrations of 450 μg kg(-1). It was not possible to establish the cause of CAP residues which has initiated research on the natural occurrence of this drug. CAP occurs in the para-configuration and in the meta-configuration and contains two chiral centers thus eight different isomeric configurations exist, namely four (RR, SS, RS, SR) meta-stereoisomers and four para-stereoisomers. It is known that only RR-p-CAP has antimicrobial properties. To find out if the CAP detected in the plant material samples is the active configuration, a high resolution reversed phase LC-MS/MS system was tested for its ability to separate the different isomers. This system proved to be able to discriminate between some isomers, but not between RR-p-CAP and SS-p-CAP, also called dextramycin. Despite a detailed elucidation of the product ions and the fragmentation patterns of all isomers, MS/MS did not add sufficient specificity for full discrimination of the isomers. Therefore a chiral liquid chromatographic separation with MS/MS detection that is able to distinguish all isomers was developed and finally the isomeric ratio of non-compliant plant material samples and some CAP formulations was determined using this system. This showed that Mongolian grass and herb samples only contain the biological active isomer of CAP as do the obtained formulations. Therefore the CAP present in the plant material might origin from the production by soil organisms or from a manufactured source.     

Fast and selective extraction of chloramphenicol from soil by matrix solid‐phase dispersion using molecularly imprinted polymer as dispersant

The molecularly imprinted polymer (MIP) was synthesized and used as dispersant of matrix solid‐phase dispersion (MSPD) for the extraction of chloramphenicol (CAP) in soil samples. The satisfactory recovery of CAP was obtained by the optimized extraction conditions: 1:2 as the ratio of sample to MIPs; 5 min as the dispersion time; 30% aqueous methanol as washing solvent and methanol as elution solvent. The CAP extracted from soil was determined by LC‐MS/MS. The slight ion suppression phenomenon was observed for the CAP when the sample was cleaned up by MSPD with MIP as dispersant, when compared with C18 as MSPD dispersant, which caused significant ion suppression. LOD of CAP is 4.1 ng/g. RSDs of intra‐ and inter‐day tests ranging from 3.1 to 6.2% and from 3.9 to 8.3% are obtained. At all three fortified levels (20, 100 and 500 ng/g), recoveries of CAP are in the range of 86.9–92.6%. The effect of ageing time of spiked soil sample on the CAP recovery was examined. The CAP recovery decreased from 91.0 to 36.9% when the ageing time changed from 1 day to 4 wk.     

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。     

Screening for chloramphenicol residues in the tissues and fluids of treated cattle by the four plate test, Charm II radioimmunoassay and Ridascreen CAP-Glucuronid enzyme immunoassay.

The administration of chloramphenicol (CAP) is banned in food animals in the European Union (EU). It is, therefore, important to have adequate screening methods to determine if residues of CAP and its major metabolite, chloramphenicol-glucuronide (CAP-Gluc), are present in samples taken for monitoring purposes. Six castrated male cattle were treated with a single intramuscular injection of 10 mg kg-1 CAP. Animals were sampled once daily for urine and were slaughtered at 3 and 6 d post-injection. Samples of bile, kidney, liver and diaphragmatic muscle were removed at slaughter. All matrices were analysed using the four plate test (FPT) bioassay, the Charm II radioimmunoassay and a Ridascreen CAP-Glucuronid competitive enzyme immunoassay (EIA). The FPT detected CAP residues in urine samples taken up to 2 d post-treatment. The Charm assay detected CAP in the urine for up to 4 d post-treatment. The EIA detected CAP throughout the 6 d sampling period. Samples of bile were positive by both the EIA and the Charm assay at day 3 and day 6. No zones of inhibition were obtained using the FPT in bile or diaphragm either with or without sample pre-treatment with beta-glucuronidase. However, the kidney and the liver from one animal killed at day 6 gave larger zones of inhibition after treatment with beta-glucuronidase, indicating the presence of CAP. The kidneys of all treated animals slaughtered at day 3 were positive by both the EIA and the Charm assay but none of the kidneys at day 6 tested positive by either method. Owing to technical difficulties, the Charm assay was not suitable for the analysis of liver. The EIA failed to detect CAP in the liver of any treated animal. It is concluded that urine appears to be the best matrix for screening purposes. The sensitivity of the FPT is inadequate for the determination of CAP residues were minimal withdrawal periods have been observed. The Charm assay and the EIA were suitable for the detection of both CAP and CAP-Gluc in tissues and body fluids for longer periods post-administration. The EIA was more sensitive for the determination of low concentrations of CAP and its metabolite.     

Quantitative liquid chromatography/tandem mass spectrometry determination of chloramphenicol residues in food using sub-2 microm particulate high-performance liquid chromatography columns for sensitivity and speed

The use of chloramphenicol (CAP)--a highly effective broad-spectrum antibiotic used in animal husbandry--is banned in many countries. Therefore, a very low minimum required performance limit (MRPL) of 0.3 microg/kg CAP in meat for human consumption has been defined. Analytical methods capable of quantifying and confirming such low residue levels require sophisticated instrumentation. Preferably sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) or gas chromatography/mass spectrometry (GC/MS) methods have been used. This paper suggests the use of sub-2 microm particulate high-performance liquid chromatography (HPLC) columns to gain additional sensitivity and improve resolution as well as speed. Depending on the operating conditions, higher chromatographic resolution and speed can be obtained at the price of a significantly increased operating pressure, requiring dedicated LC equipment. A 3-4-fold overall improvement of the signal-to-noise ratio for CAP was obtained compared to more classical 5 microm particulate HPLC columns. The proposed analytical methodology includes an enzymatic digestion, which liberates glucuronide-bound CAP from kidney tissue. The extracts obtained after an Extrelut clean-up are sufficiently pure to permit routine injection of biological samples into the sub-2 microm particulate HPLC column, without observing rapid deterioration of peak shape or column clogging problems. The time for one chromatographic run was 4.2 min. The described method was validated for two particularly difficult matrices (kidney and honey). Decision limits (CC alpha) were 0.007 microg/kg (honey) und 0.011 microg/kg (kidney), which are significantly below the current MRPL.     

Determination of chloramphenicol in aquatic products by graphene‐based SPE coupled with HPLC‐MS/MS

An effective analytical protocol using graphene‐based SPE coupled with HPLC‐MS/MS for determination of chloramphenicol (CAP) in aquatic products has been developed. In the present work, graphene was evaluated as SPE sorbents for the analytes enrichment and clean up. The target analytes were quantified by a triple‐quadrupole linear ion trap MS in multiple‐reaction monitoring mode. In addition, the proposed method was validated according to Commission Decision 2002/657/EC. The calibration curve was linear over the range of 0.5–100 ng/mL. The mean values of RSD of intra‐ and interday ranging from 1.48 to 4.29% and from 3.25 to 7.42% were obtained, respectively. In the three fortified levels, the recoveries of CAP ranging from 92.3 to 103.4% with RSDs ≤ 5.58% were obtained. The proposed method has been successfully applied to the analysis of CAP in several aquatic product samples, indicating that graphene was a potential SPE sorbent for the enrichment of trace residues in food samples.     

Determination of chloramphenicol residues in bee pollen by liquid chromatography/tandem mass spectrometry

A novel liquid chromatographic/tandem mass spectrometric (LC/MS/MS) method was developed for the trace residue determination of chloramphenicol (CAP) in bee pollen. CAP was extracted from bee pollen with a mixture of methanol and 1% metaphosphoric acid solution, followed by a 2-stage solid-phase extraction enrichment and cleanup. The first stage involved a polymeric cartridge, and the second stage involved an alumina neutral cartridge. The LC separation was performed on a C18 column with 10 mM ammonium formate-acetonitrile (7 + 3) as the mobile phase and MS detection with negative-ion electrospray ionization. CAP-d5 was used as the internal standard. The method was validated according to Commission Decision 2002/657/EC. The calibration curves were linear between 0.1 and 5.0 ng/mL, and overall recoveries ranged from 98 to 113%. Decision limits (CCalpha) ranged from 0.05 to 0.07 microg/kg, and detection capabilities (CCbeta) ranged from 0.08 to 0.12 microg/kg. The developed method was applied to 11 samples.     

Quantitative determination of trigonelline in mouse serum by means of hydrophilic interaction liquid chromatography–MS/MS analysis: Application to a pharmacokinetic study

Trigonelline is a pyridine alkaloid found in fenugreek seeds and coffee beans. Most of the previous studies are concerned with the quantification of trigonelline along with other constituents in coffee herbs or beverages. Only a few have focused on its determination in animal or human tissues by applying different modes of HPLC with UV or MS detection. The aim of the study was to develop and validate a fast and simple method for trigonelline determination in serum by the use of hydrophilic interaction liquid chromatography (HILIC) with ESI‐MS/MS detection. Separation of trigonelline was achieved on a Kinetex HILIC column operated at 35°C with acetonitrile–ammonium formate (10 mm , pH = 3) buffer mixture (55:45, v/v) as the mobile phase. The developed method was successfully applied to determine trigonelline concentration in mouse serum after intravenous administration of 10 mg/kg. The developed assay is sensitive (limit of detection = 1.5 ng/mL, limit of quantification = 5.0 ng/mL) and linear in a concentration range from 5.0 to 250.0 ng/mL. Sample preparation is limited to deproteinization, centrifugation and filtration. The application of the HILIC mode of chromatography with MS detection and selection of deuterated trigonelline as internal standard allowed a rapid and precise method of trigonelline quantification to be to developed.     

Gas chromatography/negative ion chemical ionization mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry quantitative profiling of N-acetylcysteine conjugates of valproic acid in urine: application in drug metabolism studies in humans

We report a GC/NICI-MS assay and a LC/ESI-MS/MS assay for the analysis of N-acetylcysteine (NAC) conjugates of (E)-2,4-diene VPA (NAC I and NAC II) identified in humans. The assay also includes the analysis of the NAC conjugate of 4,5-epoxy VPA (NAC III), an identified metabolite in rats treated with 4-ene VPA for its use in metabolic studies in animals. The highly sensitive GC/MS assay was designed to monitor selectively the diagnostic and most abundant [M - 181](-) fragment anion of the di-PFB derivatives of NAC I, NAC II, and NAC IV, the internal standard (IS) and the PFB derivative of NAC III. The higher selectivity of LC/MS/MS methodology was the basis for an assay which could identify and quantitate the underivatized conjugates simultaneously using MRM of the diagnostic ions m/z 130 and 123 arising from the CID of their protonated molecular ions [MH](+). The GC/MS assay employed liquid-liquid extraction whereas the LC/MS/MS assay used a solid-phase extraction procedure. Linearity ranges of the calibration curves were 0.10-5.0microg ml(-1) by GC/MS and 0.10-1.0microg ml(-1) by LC/MS/MS for NAC I, NAC II and NAC III (r(2) = 0.999 or better). Both assays were validated for NAC I and NAC II and provided good inter- and intra-assay precision and accuracy for NAC I and NAC II. The LOQ by LC/MS/MS was 0.1microg ml(-1), representing 1 ng of NAC I and NAC II. The same LOQ (0.1microg ml(-1)) was observed by GC/MS and was equivalent to 100 pg of each metabolite. NAC III was detected at concentrations as low as 0.01 microg ml(-1) by both methods. The total urinary excretion of the NAC conjugates in four patients on VPA therapy was determined to be 0.004-0.088% of a VPA dose by GC/MS and 0.004-0. 109% of a VPA dose by LC/MS/MS.     

Determination of chloramphenicol residues in milk, eggs, and tissues by liquid chromatography/mass spectrometry

A new liquid chromatography/mass spectrometry (LC/MS) method is presented for the determination of chloramphenicol (CAP) residues in milk, eggs, chicken muscle and liver, and beef muscle and kidney. CAP is extracted from the samples with acetonitrile and defatted with hexane. The acetonitrile extracts are then evaporated, and residues are reconstituted in 10mM ammonium acetate--acetonitrile mobile phase and injected into the LC system. CAP is determined by reversed-phase chromatography using an Inertsil ODS-2 column and MS detection with negative ion electrospray ionization. Calibration curves were linear between 0.5-5.0 ng/g for all matrixes studied. The relative standard deviations for measurements by this method were generally <12%, and average recoveries ranged from 80 to 120%, depending on the matrix involved. The method detection limits of CAP ranged from 0.2 to 0.6 ng/g, which are comparable to previously reported results. The proposed method is rapid, simple, and specific, allowing a single analyst to easily prepare over 40 samples in a regular working day.     

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.     

基于磁性金属有机框架化合物-适配体探针的氯霉素仿生比色传感器研究

构建了一种基于磁性金属有机框架化合物( MOF)-适配体探针的仿生比色传感器,用于食品中氯霉素残留分析。首先将氯霉素( CAP)的适配体标记到Fe3 O4磁珠上获得捕获探针,进而采用该适配体的互补链标记到铁基MOF( Fe-MOF)上作为纳米示踪剂( MOF-cDNA),将捕获探针和示踪剂杂交结合后,可获得铁磁性仿生复合探针。当氯霉素和此类探针孵育后,其与捕获探针上的适配体结合,将纳米示踪剂释放到溶液中,并经过磁分离后进入上清液。由于Fe-MOF具有过氧化物酶的性质,可以催化TMB-H2 O2系统显色,由此构建了一种高选择性的氯霉素比色传感器。在最佳反应条件下,本法对氯霉素的检测范围在0.001~10 ng/mL之间,最低检出限为0.3 pg/mL(S/N=3),实际样品的加标回收率为86.9%~93.5%,且不受其它抗生素干扰。用此方法检测牛奶样品中氯霉素的结果与商业化ELISA方法一致。此类无酶标记仿生探针具有高催化活性且成本较酶标探针大大降低;该分析方法利用磁分离简化了前处理步骤,可用于奶制品中氯霉素的快速灵敏分析。     

Determination and confirmation of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment using high-performance liquid chromatography/tandem mass spectrometry

An accurate, reliable, and reproducible analytical method using HPLC/MS/MS for the determination of tulathromycin residues in bovine liver and porcine kidney via their common hydrolytic fragment (CP-60,300) was developed and validated. Briefly, the method involved an initial acid treatment of intact tissues, which yielded the common fragment (CP-60,300). A portion of the acid hydrolyzate was purified by SPE using a strong cation exchange cartridge. Evaporation of the purified extract was followed by reconstitution in aqueous buffer and analysis by HPLC/MS/MS under isocratic conditions. The developed method provided acceptable sensitivity for determinative surveillance of tulathromycin in porcine kidney and bovine liver with an LOQ of 7.50 and 2.75 microg/g, respectively. The overall recovery and precision of 45 determinations of each tissue were 97.8% (5.3%) for porcine kidney and 96.9% (7.9%) for bovine liver. Accuracy, precision, linearity, specificity, and ruggedness were demonstrated. An HPLC/MS/MS method was also developed for use in these tissues as a confirmatory assay following modifications to the MS detection parameters. The confirmatory method demonstrated acceptable sensitivity for confirmatory evaluation of tulathromycin in porcine kidney and bovine liver at tolerances of 15 and 5.5 microg/g, respectively.     

Rapid method for the confirmatory analysis of chrysoidine in aquaculture products by ultra‐performance liquid chromatography–tandem mass spectrometry

A sensitive and fast method for the quantification of the illegal dye chrysoidine in aquaculture products with ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC‐MS/MS) is presented. Muscle tissues were made alkaline with sodium hydroxide and extracted with ethyl acetate. After evaporation and subsequent defatting with n‐hexane, extracts were directly injected onto the UPLC‐column. Chromatography was performed on a C₁₈ column using 0.1% formic acid in water and an acetonitrile gradient within 6 min. Mass spectrometric analysis was performed in the positive electrospray MS/MS mode. The limit of quantification was 0.25 ng/g, which was 30 times lower than the only previously published method with gas chromatographic detection. A complete validation according to the scientific literature and as defined by the European Union was performed. The applicability of the method was shown in the analysis of more than 50 unknown samples in the framework of a monitoring program. Copyright © 2010 John Wiley & Sons, Ltd.