Simultaneous determination of benzimidazoles and their metabolites in plasma using high-performance liquid chromatography/tandem mass spectrometry: application to pharmacokinetic studies in rabbits

An HPLC/MS/MS method was developed for the simultaneous determination of the following benzimidazole anthelmintics and metabolites in plasma: flubendazole, albendazole, fenbendazole, mebendazole, thiabendazole, hydrolyzed flubendazole, albendazole sulfoxide, albendazole sulfone, albendazole aminosulfone, oxfendazole, fenbendazole sulfone, aminomebendazole, hydroxymebendazole, and 5-hydroxythiabendazole. The sample preparation process involved a pH-dependent extraction of the analytes. Chromatographic separation was performed on a C18 column with a mobile phase gradient starting with methanol-water (20 + 80, v/v) containing 0.1% formic acid. The overall average recoveries of the analytes based on a matrix-matched calibration ranged from 75.0 to 120.0%, with RSD values of <20.0%. The LODs ranged from 0.08 to 2.0 microg/kg and the LOQs from 0.3 to 5.0 microg/kg. The validated method was used in pharmacokinetic studies of benzimidazole compounds in rabbits, and the elimination of the metabolites was measured quantitatively.     

Development and validation of a liquid chromatographic-electrospray tandem mass spectrometric multiresidue method for anthelmintics in milk

A liquid chromatographic-tandem mass spectrometric multiresidue method for the simultaneous quantitative determination of the tetrahydroimidazole, levamisole and the benzimidazoles thiabendazole, oxfendazole, oxibendazole, albendazole, fenbendazole, febantel and triclabendazole in milk has been developed and validated. The anthelmintic residues were extracted with ethyl acetate. The liquid chromatographic separation was performed on a reversed-phase C18 column with gradient elution. The analytes were detected by tandem quadrupole mass spectrometry after positive electrospray ionisation by multiple reaction monitoring. The confirmatory method is very sensitive and each component can be detected at a residue level lower than 1 microgram/l. The method is validated according to the revised European Union requirements and all parameters were found conform the criteria. The evaluated parameters were linearity, specificity, stability, recovery, precision (repeatability and within-laboratory reproducibility) and analytical limits (detection limit, decision limit and detection capability). This analytical method is applied in the Belgian monitoring programme for classical anthelmintic veterinary drugs in raw farm cow's milk.     

Do proficiency tests always verify laboratories’ performance? The case of FAPAS PT 0270

In this paper, the case of FAPAS PT 0270 “Doramectin and Oxfendazole in Sheep Liver” is discussed. During evaluation of the data received from participants (determination of total, oxidised oxfendazole residue and calculation of the sum of oxfendazole and oxfendazole sulfone residues), significant differences were observed between the results obtained by use of two analytical approaches. This phenomenon can be explained by the route of oxfendazole metabolism, which results in the presence of fenbendazole in the sample. This was not predicted by the provider; consequently, not all the necessary tests on the material were conducted. Due to the high uncertainty of the z-scores in this test, the results of the PT cannot be used for purposes of evaluation, and the benefits of participation in PT 0270 are questionable.     

Analysis of veterinary drug residues in cheese by ultra‐high‐performance LC coupled to triple quadrupole MS/MS

A simple, reliable, and fast multiresidue method has been developed for the determination of 17 veterinary drugs belonging to several families (macrolides, sulfonamides, and anthelmintics) in cheese at trace levels. Ultra‐high‐performance LC coupled to MS/MS has been used for the analysis of these compounds in less than 9 min. Veterinary drug residues have been extracted from cheese samples using a QuEChERS (quick, easy, cheap, effective, rugged, and safe)‐based extraction procedure without applying any further clean‐up step. Matrix‐matched calibration was used for quantification and recoveries were calculated at three concentration levels (10, 50, and 100 μg/kg). The obtained values ranged from 70 to 110% for the selected compounds except for tylosin and josamycin at 100 μg/kg (111.7 and 112.7%, respectively). Intra‐ and interday precision were also evaluated and RSDs were lower than 25% in all the cases. LOQs ranged from 0.3 μg/kg (for thiabendazole, oxfendazole, mebendazole, josamycin, and fenbendazole) to 10.5 μg/kg (abamectin), whereas decision limit and detection capability ranged from 2.3 (thiabendazole) to 11.3 (abamectin) and 4.2 (thiabendazole) to 14.3 μg/kg (abamectin), respectively. Finally, 13 samples were analyzed and traces of thiabendazole were detected in two different cheeses.     

液相色谱串联质谱法同时测定饲料中8种苯并咪唑类药物

建立了同时测定饲料中8种苯并咪唑类药物(噻苯咪唑、丙硫咪唑、硫苯咪唑、苯硫氧咪唑、氟苯咪唑、甲苯咪唑、丙氧苯唑和三氯苯唑)的液相色谱串联质谱分析方法。饲料样品用酸化乙腈直接提取,提取液用甲酸溶液稀释后进行分析。分析时用XBridgeTMC18色谱柱,以甲酸溶液-乙腈体系进行梯度洗脱,MRM方式测定,基质外标法定量。8种苯并咪唑类药物均在0.02~10.0 mg.L-1范围内呈良好的线性关系,相关系数(r2)均不低于0.990,在饲料样品中的检出限为2.1~63.0μg.kg-1。饲料中苯并咪唑类药物在0.50、30、200 mg.kg-13种加标水平下的回收率为84%~104%,相对标准偏差均小于10.0%。方法分析单个样品约需30 min,该方法适合饲料中8种苯并咪唑类药物的同时分析。     

Simultaneous determination of praziquantel,pyrantel embonate,febantel and its active metabolites,oxfendazole and fenbendazole,in dog plasma by liquid chromatography/mass spectrometry

A liquid chromatography–electrospray–mass spectrometry method (LC/MS) has been developed and validated for determination of praziquantel (PZQ), pyrantel (PYR), febantel (FBT), and the active metabolites fenbendazole (FEN) and oxfendazole (OXF), in dog plasma, using mebendazole as internal standard (IS). The method consists of solid‐phase extractions on Strata‐X polymeric cartridges. Chromatographic separation was carried out on a Phenomenex Gemini C₆‐Phenyl column using binary gradient elution containing methanol and 50 mm ammonium–formate (pH 3). The method was linear (r² ≥ 0.990) over concentration ranges of 3–250 ng/mL for PYR andFEB, 5–250 ng/mL for OXF and FEN, and 24–1000 ng/mL for PZQ. The mean precisions were 1.3–10.6% (within‐run) and 2.5–9.1% (between‐run), and mean accuracies were 90.7–109.4% (within‐run) and 91.6–108.2% (between‐run). The relative standard deviations (RSD) were <9.1%. The mean recoveries of five targeted compounds from dog plasma ranged from 77 to 94%.The new LC/MS method described herein was fully validated and successfully applied to the bioequivalence studies of different anthelmintic formulations such as tablets containing PZQ, PYR embonate and FBT in dogs after oral administration. Copyright © 2015 John Wiley & Sons, Ltd.     

Comparative study of sample preparation methods; supported liquid membrane and solid phase extraction in the determination of benzimidazole anthelmintics in biological matrices by liquid chromatography-electrospray-mass spectrometry

Supported liquid membrane (SLM) and solid phase extraction (SPE) have been applied as clean-up and/or enrichment techniques for a mixture of five benzimidazole anthelmintics compounds, namely albendazole, fenbendazole, mebendazole, oxibendazole, and thiabendazole. Two biological matrices, mainly urine and milk, and ultra high purity (UHP) water were spiked with a mixture of these five compounds. Waters Oasis^® MCX and International Sorbent Technology (IST) HCX SPE sorbents were used. The liquid membrane used for clean-up and/or enrichment of these compounds was 5% tri-n-octylphosphine oxide (TOPO) dissolved in n-undecane/di-n-hexyl ether (1:1). The SLM extraction efficiencies and SPE percentage recoveries ranged between 60 and 100%. The detection limits (DLs) for different benzimidazole compounds by SPE/LC-ES-MS for thiabendazole, oxibendazole, and albendazole was 0.1 ng/L, for fenbendazole and mebendazole was 1 and 10 ng/L, respectively. Similarly, the detection limits of SLM/LC-ES-MS for thiabendazole, oxibendazole, and albendazole was 0.1 ng/L and for fenbendazole and mebendazole was 1 ng/L. The results of optimization of various parameters of the SLM method are reported.     

Multiresidue analysis of insecticides in soil by gas chromatography with electron-capture detection and confirmation by gas chromatography-mass spectrometry.

A rapid multiresidue method has been developed for the analysis of nine insecticides (organochlorines, pyrethroids and organophosphorus) in soil. The method is based on the sonication extraction of residues from a certain amount of soil placed in a small column, using ethyl acetate. The effect of the residence time of insecticides in soil, the material of the columns used (glass or plastic columns) and the soil moisture content on the recovery of these compounds was also studied. Residues were determined by gas chromatography with electron-capture detection. The average recovery through the method obtained for these compounds varied from 90 to 108% with a relative standard deviation between 1 and 11%. The results of this study pointed out that the recoveries of insecticide residues obtained with plastic or glass columns at different soil moisture content were similar and that the residence of these compounds in soil during several days did not affect their recovery from soil. Confirmation of residue identity was performed by gas chromatography coupled with mass spectrometry.     

Bioassays for the detection of growth-promoting agents, veterinary drugs and environmental contaminants in food

Residues of growth-promoting agents, veterinary drugs and environmental contaminants in food products are routinely analyzed with chemical-analytical methods, using physical and spectrometric properties of a compound. Since residue limits are in general based on biological properties of compounds, bioassays offer in theory a good alternative. As a consequence, these assays are much more suitable for the detection of mixtures of compounds with common biological properties, including possibly unknown agonists. Using modern molecular biological techniques, a new generation of bioassays has been developed, showing in general a higher sensitivity and specificity for the target compounds. The CALUX (chemical activated luciferase expression) assay was developed for the detection of polyhalogenated compounds, based on their affinity for the aryl hydrocarbon (Ah) receptor. This paper focuses on the specificity of the assay. The benzimidazole compounds oxfendazole, fenbendazole, febantel, thiabendazole, mebendazole, omeprazole, lanzoprazole and benomyl were shown to give a positive response in the assay. Similar results were obtained with dexamethasone, corticosterone and cortisol, which in addition were able to enhance the response obtained with TCDD. Similarly to the flavonoids alpha- and beta-naphtoflavone, the reported Ah receptor antagonist 4-amino-3-methoxyflavone showed a strong positive response at a concentration of 400 microM, but failed to inhibit the response obtained with TCDD. It is concluded that the chances of false-negative results appear to be minimal and can be recognized. False-positive or, better, unwanted results are in theory more likely to occur. Possible solutions to avoid or detect these type of results are discussed. In general, these kinds of assays offer great possibilities for screening of food samples. In addition to the further optimization of these assays, future work should be focused on the development of rapid, sample and selective extraction procedures.     

Determination of bithionol, bromophen, nitroxynil, oxyclozanide, and tribromsalan in milk with liquid chromatography coupled with tandem mass spectrometry

LC/MS/MS was developed to determine the residues of bithionol (BTN), bromofen (BMF), nitroxynil (NTX), oxyclozanide (OCZ), and tribromsalan (TBS) in milk. Samples were extracted with ethyl acetate and cleaned up by liquid-liquid separation with acetonitrile and n-hexane. The compounds were determined by RP-LC using a C18 column with 0.1% formic acid-methanol. Mass spectral acquisition was performed in the negative mode by applying selected-reaction monitoring. The method was validated in milk spiked with these compounds at 5-600 microg/kg; average recoveries were in the range 83.8-97.1%, with RSD values of 1.4-8.0%. The interassay RSDs were less than 11%. The LODs of these compounds in milk were 0.1 microg/kg. The method was applied to 24 raw milk samples. The concentration of these compounds in all samples was lower than the Japanese maximum residue limits. The method is rapid, sensitive, and specific for monitoring residues of BTN, BMF, NTX, OCZ, and TBS in milk.     

高效液相色谱-串联质谱法同时测定肉类罐头中双酚-二缩水甘油醚

建立了同时测定肉类罐头中双酚A-二缩水甘油醚(BADGE)、双酚F-二缩水甘油醚(BFDGE)及其衍生物双酚A-(2,3-二羟丙基)甘油醚(BADGE•H2O)、双酚A-双(2,3-二羟丙基)醚(BADGE•2H2O)、双酚A-(3-氯-2-羟丙基)(2,3-二羟丙基)醚(BADGE•H2O•HCl)、双酚A-(3-氯-2-羟丙基)甘油醚(BADGE•HCl)、双酚A-双(3-氯-2-羟丙基)醚(BADGE•2HCl)、双酚F-双(2,3-二羟丙基)醚(BFDGE•2H2O)、双酚F-双(3-氯-2-羟丙基)醚(BFDGE•2HCl)9种环境激素的高效液相色谱-串联质谱分析方法。样品经叔丁基甲醚提取,HLB固相萃取小柱净化,C18色谱柱分离,用5 mmol/L醋酸铵溶液(含0.1%甲酸)与甲醇为流动相梯度洗脱,质谱多反应监测(MRM)模式检测,基质标准校正,外标法定量。结果表明,这9种化合物在10.0～2000.0 μg/L范围内线性关系良好;定量限(以信噪比≥10计)为10.0 μg/kg;在高、中、低3个加标水平下9种化合物的平均添加回收率为79.6%～100.9%,相对标准偏差为6.3%～12.1%。该方法具有较高的灵敏度和准确度,能满足法规要求的对肉类罐头中双酚A-二缩水甘油醚、双酚F-二缩水甘油醚及其衍生物残留量的快速检测及准确定量。     

Capillary high-performance liquid chromatography with mass spectrometry for simultaneous determination of major flavonoids, iridoid glucosides and saponins in Flos Lonicerae

Flos Lonicerae, a traditional herbal medicine, has been used in China to treat some inflammatory disease. Several different classes of compounds have been separated from the herb to assess their pharmacological activities. Among these classes, flavonoids, iridoid glycosides and saponins have been well studied and may be responsible for its clinical application. Therefore, quality control of Flos Lonicerae is an important issue for drug safety and validity evaluations. A quantitative method consisting of solid phase extraction followed by capillary high-performance liquid chromatography-diode array detection/electrospray ionization mass spectrometry (capillary HPLC-DAD/ESI-MS) was developed for simultaneously assay of 24 compounds in Flos Lonicerae. Under optimized capillary HPLC-DAD/ESI-MS conditions, these compounds, including nine flavonoids, eight iridoid glucosides and seven saponins, were separated with high efficiency in the selected-ion monitoring (SIM) mode. Linearity of the method was good with correlation coefficients (r(2)) in the range of 0.9935-0.9998 and detection limits were lower than 2.57 ng/mL for most of analytes. The obtained recoveries varied between 91.0 and 108.7% with the relative standard deviations (RSDs) within 8.74% (n=3). The capillary HPLC-DAD/ESI-MS method was also successfully applied to the analysis of these compounds in five species of Flos Lonicerae. It was demonstrated to be a powerful tool for comprehensive analysis of herbal medicines, owing to its exclusive selectivity and excellent sensitivity.     

Determination of fenbendazole and its metabolites in trout by a high-performance liquid chromatographic method.

A high-performance liquid chromatographic (HPLC) method based on solid phase extraction was developed for the simultaneous determination of fenbendazole (FBZ), fenbendazole sulfoxide (FBZ-SO) and fenbendazole sulfone (FBZ-SO2) in trout muscle and skin tissues. The compounds were extracted with acetonitrile and the extract was concentrated and cleaned up by solid phase extraction on C18 and CN cartridges. The extract was analysed by reversed-phase gradient HPLC on a C18 column followed by ultraviolet detection at 290 nm. The method's detection limits were 4.0 micrograms kg-1 for FBZ, 4.5 micrograms kg-1 for FBZ-SO and 3.8 micrograms kg-1 for FBZ-SO2. The mean recovery in muscle was 88% for FBZ, 94% for FBZ-SO and 92% for FBZ-SO2 at a level of 5-150 micrograms kg-1. The corresponding mean recoveries in skin tissue were 88, 81 and 86% at a level of 10-100 micrograms kg-1. The mean relative repeatability standard deviation was 9.2% at a level of 5 micrograms kg-1, 5.9% at a level of 10-100 micrograms kg-1 and 2.3% at a level of 150 micrograms kg-1. The method was applied to trout given feed containing FBZ in an aquaculture pilot plant. The three compounds FBZ, FBZ-SO and FBZ-SO2 were all detected in muscle and skin tissues shortly after administration. The concentrations were generally highest in skin tissue.     

奥芬达唑质控标准中关键杂质成分的合成

苯并咪唑氨基甲酸酯类化合物(Benzimidazolecarbamate,BZC)是一类广谱抗寄生虫抗菌剂,其广泛使用的典型代表药物为奥芬达唑。 但该药物中的杂质化合物不易获得,导致生产质控不足。 本文发现了奥芬达唑中4种主要杂质成分,包括芬苯达唑、5-苯磺酰基-1H-2-甲氧甲酰氨基苯并咪唑、5-苯亚磺酰基-1H-2-氨基苯并咪唑和2-二(5-苯亚磺酰基-1H-2-苯并咪唑基)-碳酰二胺的简单高效化学合成方法。 特别是通过两种不同的关环策略实现了关键吡唑环的有效合成,以及控制氧化条件得到硫醚的亚砜和砜的氧化产物。 该方法不仅可为奥芬达唑的质控提供标准品,而且为基于奥芬达唑类似物的新型杀虫、抗菌及抗肿瘤等药物的研发提供了一条简单高效的合成线路。     

Sulphonamide Residues in Italian Surface and Drinking Waters: A Small Scale Reconnaissance

The occurrence of antibiotics in the aquatic environment is an important emerging issue due to potential adverse effect of these compounds on ecosystem and human health. For a correct environmental risk assessment there is a need for appropriate analytical methods for monitoring antibiotic residues in a variety of water matrices. This paper describes a method for the determination of eleven sulphonamide compounds in surface and drinking waters using solid-phase extraction and liquid chromatography-tandem mass spectrometry. Recoveries of the analytes in both surface and drinking water matrices at different fortification levels, always exceed 87%; the limits of quantification in surface water samples are between 0.005 and 0.021 μg L⁻¹ depending on the compound, and the interday method precision is less than 12%. Matrix effects were evaluated in drinking and surface water samples. The method has been applied to a small scale reconnaissance of river, lake, mineral and municipal water samples; results indicate the occurrence of sulphonamides in some surface and mineral waters analyzed.     

Determination of endosulfan isomers and endosulfan sulfate in tomato juice by matrix solid-phase dispersion and gas chromatography

A rapid method based on matrix solid-phase dispersion was developed for the determination of endosulfan isomers and endosulfan sulfate in commercial tomato juice. After the optimisation of different parameters such as the type of adsorbent, the extraction solvent, and the extraction assistance by sonication, the recoveries obtained ranged from 81 to 100% with relative standard deviations equal to or lower than 10%. The analysis of samples was accomplished using gas chromatography with electron-capture detection and the identity of endosulfan residues was confirmed by gas chromatography-mass spectrometry with selected ion monitoring. The detection limit for these compounds, calculated as three times the background noise, was 1 microg/kg. The proposed method was applied to the analysis of these compounds in commercial juice samples and levels of endosulfan between 1 and 5 microg/kg were detected in some samples.     

固相萃取-气相色谱-质谱法同时测定水中9种药品及个人护理用品

采用固相萃取-气相色谱-质谱联用技术,建立了水体中9种药品及个人护理用品（水杨酸、萘普生、布洛芬、对乙酰氨基酚、降固醇酸、三氯生、双氯酚酸、酮洛芬和双酚A）的定量分析方法。水样品经稀盐酸调节至pH=3后,采用Oasis HLB固相萃取小柱进行富集净化;选用三甲基氢氧化硫（TMSH）为衍生化试剂,在常温条件下对目标物进行快速甲基化;以气相色谱-质谱法选择离子监测模式（GC-MS-SIM）进行检测,由2,4,5-涕丙酸为内标进行定量分析。本实验分别对固相萃取和衍生化等前处理条件进行了系统的研究。在优化的实验条件下,方法的回收率为50.7~115.4%,相对标准偏差均不高于10%,检出限为0.03~0.30 μg/L,定量限为0.15~1.50 μg/L。利用该方法对东莞市某农田灌溉水进行了分析,4种目标物在样品中有检出,最大质量浓度范围为0.176~0.998 μg/L。结果表明该方法操作简便,灵敏可靠。     

Application of capillary zone electrophoresis with large-volume sample stacking to the sensitive determination of sulfonamides in meat and ground water

A CZE method with UV-Vis detection has been established and validated for the determination of nine sulfonamides: sulfapyridine, sulfamethazine, sulfamerazine, sulfamether, sulfadiazine, sulfadimethoxine, sulfamethoxazole, sulfachlorpyridazine, and sulfamethizole. Optimum separation was obtained on a 64.5 cm x 75 microm bubble cell capillary using a buffer containing 45 mM sodium phosphate and 10% methanol at pH 7.3, with temperature and voltage of 27 degrees C and 25 kV, respectively. p-Aminobenzoic acid was used as an internal standard . Taking into account the lack of sensitivity of the UV-Vis detection, the application of an on-line preconcentration methodology, such as large-volume sample stacking with polarity switching has been proposed. This procedure combined with a solvent extraction/SPE method applied for off-line preconcentration and cleanup provides a significant improvement in the LODs, ranging from 2.59 to 22.95 mug/L for the studied compounds; the quantification of these residues being possible below the levels established by EU legislation in animal food products, such as meat. Satisfactory recoveries were also obtained in the analysis of these compounds in ground water.     

Simultaneous determination of nine types of phthalate residues in commercial milk products using HPLC-ESI-MS-MS

A multi-residue method was developed for the confirmation and quantitation of nine types of phthalates in milk using high-performance liquid chromatography electrospray ionization tandem mass spectrometry. The samples were extracted with acetonitrile. The analytes were separated using a 0.1% formic acid-methanol system as the mobile phase, and a linear gradient elution program. Mass spectral acquisition was achieved by selectively monitoring the ions in electro-spray ionization mode. Qualitative analysis was based on the retention time and the mass spectrum results, and the quantity was carried out by comparison with the external standard. The mean recoveries for each analyte ranged from 65.2% to 98.3%, with relative standard deviations below 11.2%. The limits of detection were 5～25 μg/kg, and the limits of quantitation were 17～83 μg/kg, depending on the compounds. This method has the merits of convenient operation, high sensitivity, and good repeatability, making it an effective method for analysis of phthalates in milk. And the proposed analytical method has been applied to the analysis of phthalates presented in four commercial milk products. The main phthalate residues were DBP and DMP. And the amount of DBP was found to be more than 100 μg/kg in all this milk products.     

Organochlorine residue analysis of commercial milks by capillary gas chromatography

The determination of organochlorine pesticides and polychlorinated biphenyls in milks requires the use of efficient extraction methods. A rapid procedure has been developed, based on extraction of organochlorine residues from milk on to octadecylsilica solid phase extraction cartridges and elution with hexane. The addition of different organic solvents to the milk before solid phase extraction has been studied. The use of methanol to disrupt the fat globules enables almost complete recovery of the residues with minimum extraction of fatty substances. Recovery experiments were performed for eighteen compounds present at ppb levels in whole, two per cent, and skimmed milks. The average recoveries of the compounds from two per cent and skimmed milks were 73–84%; values for whole milk were lower. The residues were determined by gas chromatography using two kinds of capillary column (non-polar and semi-polar) and electron capture detection. The procedure shows low lipid carry over, and extraneous interferences are minimal. The method has been applied to the detection of organochlorine pesticides and nine individual polychlorinated biphenyls in commercial milks. The results obtained demonstrated the presence of very low levels of organochlorine residues in the commercial milks analyzed.