Determination of sodium nifurstyrenate and nitrovin residues in edible food by liquid chromatography-tandem mass spectrometry after ultrasound-assisted extraction

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed for the determination of nitrovin and sodium nifurstyrenate residues in muscle and liver of swine and chicken and in muscle of fish. Sample preparation procedure includes ultrasound-assisted extraction with acetonitrile, defatting with n-hexane and final clean-up with solid phase extraction (SPE) on Oasis HLB cartridges. The analytes were detected in multiple reaction monitoring (MRM) under negative scan mode acquiring two diagnostic product ions for sodium nifurstyrenate and under positive mode for nitrovin. The averaged decision limits (CCα; α 1%) ranged 0.09-0.26 μg/kg while the detection capability (CCβ; β 5%) was 0.33-0.97 μg/kg in the tissues. Reasonable recoveries (71-110%) spiked in muscle and liver showed excellent relative standard deviation (RSD). The validated method was simple, rapid, sensitive, and complied with the regulations for the determination of nitrovin and sodium nifurstyrenate residues in food matrices.     

Detection of banned nitrofuran metabolites in animal plasma samples using UHPLC-MS/MS

The use of nitrofurans as veterinary drugs in food-producing animals has been banned in the EU since the 1990s. Monitoring programs in the EU are based on the detection of protein-bound metabolites after slaughter. An UHPLC-MS/MS method was developed and validated for pre slaughter determination of four nitrofuran metabolites (AHD, AOZ, SEM, AMOZ) in animal plasma (bovine, ovine, equine and porcine). This method is proposed as an alternative method for on-farm surveillance. Plasma samples were derivatised with 2-nitrobenzaldehyde and subsequently extracted with organic solvent. Extracts were concentrated and then analysed by UHPLC-MS/MS. The method was validated according to Commission Decision 2002/657/EC. Inter-species recovery for AHD, AOZ, SEM and AMOZ was 72, 74, 57 and 71%, respectively. Decision limits (CCα) were calculated from within laboratory reproducibility experiments to be 0.070, 0.059, 0.071 and 0.054 μg kg(-1), respectively. In addition, the assay was applied to incurred plasma samples taken from pigs treated with furazolidone.     

Matrix-assisted laser desorption/ionization mass spectrometry for quantitative determination of β-blocker drugs in one-drop of human serum sample

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been applied for the quantitative determination of β-blocker drugs in one-drop of human serum samples using drop-to-drop solvent microextraction (DDSME) as a preconcentrating probe. The optimum experimental conditions for β-blocker drugs were investigated and 1.8 μL volume of toluene for 10 min extraction time with the 5% addition of NaCl under pH 11.0 was found to be the best conditions for the separation and preconcentration of drugs from 30 μL of serum sample from a patient with high blood pressure. The optimized methodologies for DDSME/MALDI-MS analyses exhibited a good linearity with intra- and inter day precision value of 8.5-10.5% and 9.4-12.6%, respectively. The proposed DDSME/MALDI-MS offers a very simple, rapid and low-cost technique for the determination of β-blocker drugs in one drop of serum sample. The reported method has been successfully applied for the determination of propranolol and nadolol in small volume of serum sample from patient suffering from high blood pressure. In future, this technique could be applied for pharmacokinetic and clinical studies.     

除草剂扑草净和阿特拉津对海草与大型藻类的毒性比较

陆地径流等可引起海域中除草剂浓度升高, 从而威胁海洋大型植物——海草和大型藻类的生长。以叶绿素荧光为主要指标测定除草剂阿特拉津和扑草净的低、中、高(1、5和25 μg/L)浓度对4种常见海草:大叶藻(Zostera marina L.)、丛生大叶藻(Z. caespitosa M.)、矮大叶藻(Z. japonica Aschers. & Graebn.)、红须根虾形藻(Phyllospadix iwatensis M.)和2种常见大型藻类:孔石莼Ulva lactuca L.和海索面Nemalion helminthoides的光合抑制。结果显示, 低浓度1 μg/L的扑草净和5 μg/L的阿特拉津即对矮大叶藻、孔石莼和海索面产生了显著的光合抑制, 抑制率约而7.54%—12.97%; 大叶藻、丛生大叶藻和红须根虾形藻的扑草净和阿特拉津的显著作用浓度为5 μg/L, 在相同浓度下, 扑草净的光合抑制较阿特拉津更强, 同时, 矮大叶藻及两种大型藻类较其他3种海草成体对除草剂作用更为敏感。     

Development and validation of a liquid chromatography/tandem mass spectrometry method for quantitative determination of amoxicillin in bovine muscle

A simple, quick and economical liquid chromatographic/tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of amoxicillin in bovine muscle was developed and validated. The sample preparation procedure involved a liquid extraction with water, followed by a protein precipitation step with acetonitrile. The extract was purified by a liquid-liquid partition with dichloromethane and the upper aqueous layer was directly injected into the LC-MS/MS system. Chromatographic separation was achieved on a reversed phase column, using a mixture of acetonitrile, water and 0.005% formic acid in water as mobile phase. Gradient elution was performed at a flow rate of 0.2 mL min?1. Amoxicillin was detected using positive electrospray ionization in selected reaction monitoring (SRM) mode and was quantified using terbutaline as internal standard. The responses for standards prepared in solvent and in matrix were equivalent and additionally the absence of signal suppression was confirmed by the post column infusion technique. Amoxicillin stability in standard solution and in matrix was investigated at different times and storage conditions. Amoxicillin standards prepared in water were stable on storage up to 20 days at -20°C. Amoxicillin stability in matrix (spiked bovine muscle samples) was assessed up to 15 days at -20°C. The method was validated according to the parameters requested by European Commission Decision 2002/657/EC in terms of specificity, linearity, trueness, precision, decision limit (CCα) and detection capability (CCβ). All the trueness values fell within a range between 14.5% and 6.3%. Precision values for all levels of concentration tested were lower than the relative limit calculated by the Horwitz equation. The amoxicillin MRL is set at 50 μg kg?1 and the CCα and CCβ of the method were 61.2 μg kg?1 and 72.4 μg kg?1, respectively.     

Determination of clenbuterol in porcine tissues using solid-phase extraction combined with ultrasound-assisted dispersive liquid-liquid microextraction and HPLC-UV detection

A new pretreatment method, solid-phase extraction combined with dispersive liquid-liquid microextration (SPE-DLLME), was proposed in first time for the determination of clenbuterol (CLB) in porcine tissue samples. The tissue samples were firstly extracted by SPE, then its eluents were used as dispersant of the followed DLLME for further purification and enrichment of CLB. Various parameters (such as the type of SPE sorbent, the type and volume of elution solvent, the type and volume of extractant and dispersant, etc.) that affected the efficiency of the two steps were optimized. Good linearity of CLB was ranged from 0.19 μg/kg to 192 μg/kg with correlation coefficient (r2) of 0.9995. The limit of detection (LOD) was 0.07 μg/kg (S/N=3) and the recoveries at three spiked levels were ranged from 87.9% to 103.6% with the relative standard deviation (RSD) less than 3.9% (n=3). Under the optimized conditions, the enrichment factor (EF) for CLB could up to 62 folds. The presented method that combined the advantages of SPE and DLLME, had higher selectivity than SPE method and was successfully applied to the determination of CLB in tissue samples.     

Development and validation of a method for determination of corticosteroids in pig fat using liquid chromatography-tandem mass spectrometry

A new method was developed to determine five corticosteroids (prednisolone, methylprednisone, flumethasone, dexamethasone, and methylprednisolone) in pig fat samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) utilizing an optimized liquid-liquid extraction (LLE) and subsequent solid-phase extraction (SPE) for sample clean-up. In the sample preparation, a pig fat sample was dissolved in n-hexane and then extracted into the methanol-water (50/50, v/v) mixture that enabled extraction of only medium polar corticosteroids and not the non-polar components of matrices. This extract was cleaned-up and concentrated on polymeric Oasis HLB SPE cartridge. Separation involved isocratic solvent (methanol-acetate buffer, pH 5.4) and Ascentis Express Fused-Core type HLPC column; reduced the analysis time to 7.5 min, which is at least two times lower than time required for separation using conventional techniques. Other advantage of the developed method is the minimized ion suppression of LC-MS/MS analysis, which allowed detection of corticosteroids in sub μg/kg. Method was validated according to European Union (EU) Commission Decision 2002/657/EC. Measured parameters such as selectivity, linearity, recovery, within-laboratory reproducibility, decision limit, and detection capability satisfied the EU Directive. Ranges of mean recoveries and within-laboratory reproducibility were 81-100% and 8.0-20.5%, respectively. Decision limits were calculated in the range from 4.5 to 11.9 μg/kg for MRL compounds and varied from 0.1 to 0.2 μg/kg for banned substances. Limit of detections (LODs), calculated as three time signal-to-noise ratio, were in the range of 0.1-0.3 μg/kg.     

Determination of the new anthelmintic monepantel and its sulfone metabolite in milk and muscle using a UHPLC-MS/MS and QuEChERS method

This is the first paper to report a method for the detection of the new anthelmintic monepantel and its sulfone metabolite in goat's milk and ovine muscle. Samples were extracted and purified using a modified QuEChERS method. A concentration step was included when analyzing in the low μg kg(-1) range. Analysis was carried out by ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in a 13min run time using atmospheric pressure electrospray ionisation in the negative mode (ESI(-)) and multiple reaction monitoring (MRM) scanning. Monepantel (m/z 472) and monepantel-sulfone (m/z 504) both had product ions at m/z 186 and m/z 166. The method has been single-laboratory validated according to the 2002/657/EC guidelines. The mean recovery in milk was 108 and 106% for monepantel and monepantel-sulfone, respectively. The mean recovery in muscle was 109 and 108% for monepantel and monepantel-sulfone, respectively. The coefficients of variation for the within laboratory repeatability and reproducibility were ≤6.4% in milk and ≤14.2% in muscle. The decision limits (CCα) in milk were 2.20 and 2.08 μg kg(-1) for monepantel and monepantel-sulfone, respectively. The decision limits (CCα) in muscle were 771 and 746 μg kg(-1) for monepantel and monepantel-sulfone, respectively.     

Determination of antiepileptic drugs in biological material

Current analytical methodologies applied to the determination of antiepileptic drugs in biological material are reviewed. The role of chromatographic techniques is emphasized. Special attention is focused on new chemical entities as well as current trends such as high-speed liquid chromatographic techniques, hyphenated techniques and electrochromatography techniques. A review with 542 references.     

Nachweis von Zearalenon-4-β-D-Glucopyranosid in Weizen

Maize (Zea mays) cell cultures were used for the production of zearalenone-4-β-D-glucopyranoside as standard compound. Wheat samples were extracted with acetonitrile: water, applied to a florisil column and eluted with methanol:ethyl acetate. For determination and quantification of zearalenone-4-β-D-glucopyranoside and zearalenone a LC-MS method was developed. A concentration of 10 μg/kg zearalenone-4-β-D-glucopyranoside and zearalenone was detectable. The recovery rates were calculated to be 69% and 89% at a concentration level of 100 μg/kg for zearalenone-4-β-D-glucopyranoside and zearalenone, respectively.24 Bavarian wheat samples from harvest 1999 were analyzed. Zearalenone was present in 22 out of 24 field samples, the levels ranged from 11–860 μg/kg. Zearalenone-4-β-D-glucopyranoside was found in 10 out of the zearalenone positive samples (42%) at levels ranging from 17 to 104 μg/kg. The amounts of zearalenone-4-β-D-glucopyranoside were correlated to those of zearalenone (r2=0,86; b=0,10).     

Acute administration of the organochalcogen 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one induces biochemical and hematological disorders in male rats

Organochalcogens are extensively produced and employed by industry and agriculture, and the risk of occupational and environmental toxicity to them has been poorly understood. Here, we investigated the acute effect of a new organochalcogen 3-methyl-1-phenyl-2-(phenylseleno)oct-2-en-1-one on biochemical and hematological parameters in male Wistar rats. The animals were treated with a single intraperitoneal injection of the organochalcogen at doses of 125, 250 or 500 μg·kg(-1). After 60 min, the animals were sacrificed by decapitation, and the trunk blood was collected for determination of glucose, triglycerides, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase, lactate dehydrogenase, urea, creatinine, C-reactive protein, red blood cells, hematocrit, hemoglobin and white blood cells (WBC). Our results showed a reduction in cholesterol levels in all treated groups, an increase in ALT activity at doses of 250 and 500 μg·kg(-1), a decrease of hemoglobin and an increase in WBC in animals that received 250 and 500 μg·kg(-1) of the organoselenium. In addition, we observed an increase in neutrophil counts at 125 μg·kg(-1) dose and a decrease at 500 μg·kg(-1) dose. We also verified an increase in lymphocyte counts at the dose of 500 μg·kg(-1). Thus, the present study shows that the acute treatment with this new organochalcogen causes biochemical changes and hematological disorders in male rats.     

Simultaneous determination of thiamphenicol, florfenicol and florfenicol amine in eggs by reversed-phase high-performance liquid chromatography with fluorescence detection

A specific, sensitive and widely applicable reversed-phase high-performance liquid chromatography with fluorescence detection (RP-HPLC-FLD) method was developed for the simultaneous determination of thiamphenicol (TAP), florfenicol (FF) and florfenicol amine (FFA) in eggs. Samples were extracted with ethyl acetate-acetonitrile-ammonium hydroxide (49:49:2, v/v), defatted with hexane, followed by RP-HPLC-FLD determination. Liquid chromatography was performed on a 5 μm LiChrospher C(18) column using a mobile phase composed of acetonitrile (A), 0.01 M sodium dihydrogen phosphate containing 0.005 M sodium dodecyl sulfate and 0.1% triethylamine, adjusted to pH 4.8 by 85% phosphoric acid (B) (A:B, 35:65 v/v), at a flow rate of 1.0 mL/min. The fluorescence detector of HPLC was set at 224 nm for excitation wavelength and 290 nm for emission wavelength. Limits of detection (LODs) were 1.5 μg/kg for TAP and FF, 0.5 μg/kg for FFA in eggs; limits of quantitation (LOQs) were 5 μg/kg for TAP and FF, 2 μg/kg for FFA in eggs. Linear calibration curves were obtained over concentration ranges of 0.025-5.0 μg/mL for TAP with determination coefficients of 0.9997, 0.01-10.0 μg/mL for FF with determination coefficients of 0.9997 and 0.0025-2.50 μg/mL for FFA with determination coefficients of 0.9998, respectively. The recovery values ranged from 86.4% to 93.8% for TAP, 87.4% to 92.3% for FF and from 89.0% to 95.2% for FFA. The corresponding intra-day and inter-day variation (relative standard deviation, R.S.D.) found to be less than 6.7% and 10.8%, respectively.     

Determination of rifabutin by high-performance liquid chromatography using on-line concentration and column switching

A simple HPLC method has been developed that allows the sensitive determination of rifabutin (RBT) in human serum using on-line concentration and column switching. After pretreatment of the serum with acetonitrile and centrifugation, the samples were applied to a concentration column (CC) (Zorbax CN). Washing with phosphate buffer-methanol removed most of the contaminating substances. Via a six-port valve the CC was switched to the analytical mode. RBT was separated on a Chromspher RP 8 column (acetonitrile-phosphate buffer pH 7.4/sodium chloride) and determined photometrically at 278 nm. The lower limit of quantification for 200 μl serum precipitated with 200 μl acetonitrile and after injection of 2×150 μl was 33 μg/l and linearity was observed up to 27 mg/l. Different modes of sample application (single, repeated, and different injection volume portions), as well as washing time, cycle time and different CC materials were investigated.     

Bestimmung von Zearalenon in Speiseölen mit GPC und LC-ESI-MS/MS

A new method for the determination of zearalenone in edible oils with size exclusion chromatography (SEC) followed by LC-MS/MS as well as HPLC-FLD was developed and validated. By using the LC-MS/MS determination no further clean up step is necessary after the SEC. The correlation coefficient of 0.999 for the two detection systems is acceptable. In this research 77 edible oils were analyzed. The mean average value of 38 corn germ oils was 169 μg/kg, the maximum value amounted up to 921 μg/kg.     

Determination of caffeine and its metabolites by micellar electrokinetic capillary electrophoresis

The determination of caffeine and its analogues is important for a wide variety of analyses and is performed in an assortment of matrices ranging from food to clinical samples. While reversed-phase HPLC has become the standard analysis protocol in most laboratories, capillary electrophoresis has the advantages of higher separation efficiency and shorter separation time. The micellar capillary electrophoresis (MECC) separation of caffeine and its metabolites, theobromine, paraxanthine, theophylline and 1,3,7-trimethyluric acid was investigated using sodium dodecyl sulphate (SDS) as the micellar phase. The effects of pH, micelle concentration, buffer concentration, ionic strength, buffer salts, applied voltage and injection time were studied to select the optimum conditions for the determination of caffeine and its four analogues in drugs, foods and body fluids. Caffeine and its three analogues were resolved within 120 s with detection limits less than 1 μg/ml. Samples could be analyzed utilizing direct injection with satisfactory resolution and reproducibility.     

Quantification of multi-residue levels in peach juices, pulps and peels using dispersive liquid-liquid microextraction based on floating organic droplet coupled with gas chromatography-electron capture detection

In this paper, polychlorinated biphenyl (PCB), organochlorine pesticide (OCP) and pyrethroid pesticides in peach was investigated by comparing their residual level in peach juice, pulps and peels using dispersive liquid-liquid microextraction based on solidification of floating organic droplet (DLLME-SFO) combined with gas chromatography-electron capture detection (GC-ECD). Extraction conditions such as the type of extractant, volume of extractant and dispersant, salt effect and extraction time were optimized. For juice samples, the linearity of the method was obtained in the range of 10-2000 ng L(-1),with determination coefficients>0.99. The limits of detection (LOD) of the method were ranged between 2.8 and 18.5 ng L(-1). For pulp and peel samples, the developed method is linear over the range assayed, 1-20 μg kg(-1),with coefficients also >0.99. The relative recoveries of compounds analyzed from juice, pulp and peel samples were in the range of 73-106% with a relative standard deviation between 2.6 and 11.8%. The proposed method was applied to the simultaneous analysis of residues in real peach juice, pulp and peel samples. As a result, there were no target analytes found in peach juices and pulps while 3.3 μg kg(-1) cyhalothrin and 3.5 μg kg(-1) fenvalerate were found in peels. The experiment results revealed that the pyrethroid residues just deposited on the peels of the fruits, but did not move into pulps and juices.     

Determination of diclazuril, toltrazuril and its two metabolites in poultry tissues and eggs by gel permeation chromatography-liquid chromatography-tandem mass spectrometry

A new procedure has been described for the extraction of diclazuril (DIZ), toltrazuril (TOZ) and its two main metabolites toltrazuril sulphoxide (TZSO) and toltrazuril sulphone (TZS) from poultry tissues and eggs, using gel permeation chromatography (GPC). The analytes and the deuterated internal standard were extracted from the samples with ethyl acetate. The analytes were measured by LC coupled to an electrospray ionization tandem mass spectrometer operating in the negative ion mode. Excellent linear dynamic range was observed from 1 to 500 μg/L with the correlation coefficients (R(2)) better than 0.99 for all analytes. The method LOQ of the four analytes in real samples was 1.2 μg/kg for DIZ and TOZ, and 1.8 μg/kg for TZSO and TZS. These values are far lower than the maximum residue limits (MRLs) established by several control authorities. The developed method was accurate with overall recoveries in four matrices.     

Determination of isometamidium residues in animal-derived foods by liquid chromatography-tandem mass spectrometry

In this paper, a new determination method for isometamidium residues in animal-derived foods was developed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Isometamidium residues in bovine tissues and milk were extracted with the mixed solution of acetonitrile and 0.25 mol/L of ammonium formate-methanol (v/v, 1:1), concentrated and degreased, and determined by LC-MS/MS with quantification by external standard method. The results showed that the peak area of chromatogram was linearly related to the concentration of isometamidium in the range of 1-100 μg/L, and the limits of detection (LOD) and quantification (LOQ) were 0.05 μg/kg and 5 μg/kg, respectively. The average recoveries of spiked samples were in the range of 73.8-93.9% with relative standard deviations ranged from 2.3% to 7.5%. This method is simple, accurate and suitable for the identification and quantification for isometamidium in animal-derived foods.     

Determination of amdinocillin in plasma and urine by high-performance liquid chromatography

A rapid, sensitive and specific high-performance liquid chromatographic (HPLC) assay was developed for the determination of amdinocillin (formerly mecillinam) in human plasma and urine. The assay is performed by direct injection of a plasma protein-free supernatant or a dilution of urine. A 10-μm μBondapak phenyl column with an eluting solvent of water—methanol—1 M phosphate buffer, pH 7 (70:30:0.5) was used, with UV detection of the effluent at 220 nm. Azidocillin potassium salt [potassium-6-(d-(-)-α-azidophenyacetamido)-penicillanate] was used as the internal standard and quantitation was based on peak height ratio of amdinocillin to that of the internal standard. The assay has a recovery of 74.4 ± 6.3% (S.D.) in the concentration ranges of 0.1–20 μg per 0.2 ml of plasma with a limit of detection equivalent to 0.5 μg/ml plasma. The urine assay was validated over a concentration range of 0.025–5 mg/ml of urine, and has a limit of detection of 0.025 mg/ml (25 μg/ml) using a 0.1-ml urine specimen per assay.The assay was applied to the determination of plasma and urine concentrations of amdinocillin following intravenous administration of a 10 mg/kg dose of amdinocillin to two human subjects. The HPLC and microbiological assays were shown to correlate well for these samples.     

Toxicity and genotoxicity in Astyanax bimaculatus (Characidae) induced by microcystins from a bloom of Microcystis spp

Studies of genotoxicity in fish caused by cyanobacterial microcystins can be useful both in determining the sensitivity of native species, as well as comparing exposure routes. The genotoxicity caused by the microcystins LR and LA from a bloom collected in a eutrophic lake, was revealed in the fish Astyanaxbimaculatus, a native species from South America. LC50 (72 h) was determined as 242.81 μg L (-1) and LD50 (72 h) as 49.19 μg kg (-1) bw. There was a significant increase of DNA damage in peripheral erythrocytes, following intraperitoneal injection (ip) with tested concentrations of 24.58 μg kg (-1) bw and 36.88 μg kg (-1) bw, as well as through body exposure to a concentration of 103.72 μg L (-1) . Micronucleus (MN) induction was observed after ip injections of 24.58 μg kg (-1) bw and 36.88 μg kg (-1) bw for 72 h, as well as following body exposure for 72 at 103.72 μg L (-1) . Thus, both exposure routes resulted in MN induction and DNA damage. Apoptosis-necrosis testing was carried out only by ip injection with concentrations of 24.58 μg kg (-1) bw and 36.88 μg kg- 1 bw. Exposure to microcystins at lower concentrations induced more apoptosis than necrosis in peripheral erythrocytes, whereas exposure at higher concentrations gave rise to both conditions. Thus, Astyanax bimaculatus can be considered as a species sensitive to the genotoxic effects caused by microcystins.