Rapid and sensitive methodology for determination of ethyl carbamate in fortified wines using microextraction by packed sorbent and gas chromatography with mass spectrometric detection.

This work presents a new methodology to quantify ethyl carbamate (EC) in fortified wines. The presented approach combines the microextraction by packed sorbent (MEPS), using a hand-held automated analytical syringe, with one-dimensional gas chromatography coupled with mass spectrometry detection (GC-MS). The performance of different MEPS sorbent materials was tested, namely SIL, C2, C8, C18, and M1. Also, several extraction solvents and the matrix effect were evaluated. Experimental data showed that C8 and dichloromethane were the best sorbent/solvent pair to extract EC. Concerning solvent and sample volumes optimization used in MEPS extraction an experimental design (DoE) was carried out. The best extraction yield was achieved passing 300 µL of sample and 100 µL of dichloromethane. The method validation was performed using a matrix-matched calibration using both sweet and dry fortified wines, to minimize the matrix effect. The proposed methodology presented good linearity (R(2)=0.9999) and high sensitivity, with quite low limits of detection (LOD) and quantification (LOQ), 1.5 µg L(-1) and 4.5 µg L(-1), respectively. The recoveries varied between 97% and 106%, while the method precision (repeatability and reproducibility) was lower than 7%. The applicability of the methodology was confirmed through the analysis of 16 fortified wines, with values ranging between 7.3 and 206 µg L(-1). All chromatograms showed good peak resolution, confirming its selectivity. The developed MEPS/GC-MS methodology arises as an important tool to quantify EC in fortified wines, combining efficiency and effectiveness, with simpler, faster and affordable analytical procedures that provide great sensitivity without using sophisticated and expensive equipment.

Validation (in-house and collaboratory) of the quantification method for ethyl carbamate in alcoholic beverages and soy sauce by GC-MS.

A method for ethyl carbamate (EC) determination in alcoholic beverages and soy sauce was developed by GC-MS. We adopted the diatomaceous earth solid-phase extraction (SPE) column and elution solvent of ethyl acetate/diethyl ether (5:95 v/v) for sample cleaning. The in-house validation showed the limit of quantification (LOQ) was 5.0 µg/kg. In the accuracy assay, the total average recovery for was 96.7%. The relative standard deviations (RSDs) were <5%. Subsequently, a collaborative trial was organized for the further validation. The RSDs for repeatability and reproducibility were 1.2-7.8% and 2.3-9.6% respectively. It indicated that the present method performed well in different laboratories.

Multiple headspace solid-phase microextraction using a new fiber for avoiding matrix interferences in the quantitative determination of ethyl carbamate in pickles.

Multiple headspace solid-phase microextraction (HS-SPME) using a novel fiber coated with anilino-methyl triethoxy silicane-methacrylic acid/terminated silicone oil has been introduced as a useful pretreatment technique coupled to gas chromatography-flame ionization detector for the detection of ethyl carbamate in pickles. Anilino-methyl triethoxy silicane and methacrylic acid are put into use simultaneously with the aim to increase the hydrogen interaction strength between ethyl carbamate and the coating. In addition, the new fiber exhibits high thermal stability, good reproducibility, and long lifetime. Extraction temperature, extraction time, amount of desiccant, and amount of sample were well optimized to guarantee the suitability of multiple HS-SPME. Significant matrix interference was observed among various types of pickles and the multiple HS-SPME procedure was proved to be effective in avoiding the matrix effect by a complete recovery of the analyte. The method showed satisfactory linearity (0.1-100 mg kg(-1)), precision (4.25%, n = 5), and detection limit (0.038 mg kg(-1)). The accuracy of the method was evaluated by comparison with standard addition method and the results were statistically equivalent. The study indicates that the multiple HS-SPME procedure is simple, convenient, accurate, and low-cost, and most of all, can be used for quantitative analysis in complex matrix without matrix effect.

Derivatization followed by gas chromatography-mass spectrometry for quantification of ethyl carbamate in alcoholic beverages.

A sensitive and rapid analytical methodology based on derivatization followed by gas chromatography-mass spectrometry (GC-MS) was developed for the quantitative determination of the toxic contaminant ethyl carbamate (EC, urethane, C(2)H(5)OCONH(2)) in alcoholic samples. EC was extracted using liquid-liquid extraction technique, and then silylated with bis-(trimethylsilyl)trifluoroacetamide, analysed finally by GC-MS. The isopropyl carbamate was used as the internal standard for quantitative analysis of EC in alcoholic samples. In this work, the sample extraction and derivatization reaction conditions were investigated, and the optimal extraction conditions obtained were: pH 9 and solvent of ethyl acetate, and the derivatization conditions were: derivatization reaction temperature of 80°C and time duration of 30 min. With the optimal conditions, the method validations were also studied. In the validation studies, EC exhibited good linearity with a regression coefficient of 0.9999. The limit of detection and limit of quantification were 0.30 and 5.0 µg/kg, respectively. The precision was less than 8.4%. Finally, the proposed technique was successfully applied to the analysis of EC in 35 kinds of alcoholic samples. The experimental results have demonstrated that the proposed technique is a fast, reliable and low-cost method for determination of EC in alcoholic samples.

Multiple headspace solid-phase microextraction after matrix modification for avoiding matrix effect in the determination of ethyl carbamate in bread.

This study presents the potential of multiple headspace solid-phase microextraction (multiple HS-SPME) for the quantification of analytes in solid samples. Multiple HS-SPME shares the same advantages as SPME. It also enables a complete recovery of the target compound and therefore the matrix effect, which commonly appears in SPME-based analysis, is avoided. A method based on multiple HS-SPME for the determination of the toxic contaminant ethyl carbamate (EC) in bread samples has been developed and validated, using gas chromatography with flame ionization detector. A novel polyethylene glycol/hydroxy-terminated silicone oil fiber was prepared for the first time and subsequently used instead of commercial ones because of its high extraction ability and good operational stability. An important problem still remained in multiple HS-SPME of EC in fresh bread samples. The adsorption of EC by water in the samples caused low transport of analyte to the headspace, which made multiple HS-SPME invalidated. Mixing with anhydrous sodium sulphate, the sensitivity of the method was improved and the problem was solved. The proposed method showed satisfactory linearity (0.15-1500 µg g(-1)), precision (1.6%, n=5) and limit of detection (0.041 µg g(-1)). Good recoveries, from 92.5 to 103.4%, were observed at three spiking levels. The method was applied to 14 bread samples. The multiple HS-SPME technique offers several advantages including reducing the manipulation time and cost, and avoiding analyte losses, especially in the analysis of a large number of samples in different matrices.

Multiple headspace solid-phase microextraction of ethyl carbamate from different alcoholic beverages employing drying agent based matrix modification.

Multiple headspace solid-phase microextraction (MHS-SPME) combined with gas chromatography-nitrogen phosphorus detector is proposed to determine the toxic contaminant ethyl carbamate (EC) in various alcoholic beverages after matrix modification. The remarkable feature of this method is that matrix effect, which commonly appears in SPME-based analysis, is avoided by determining the total amount of the analyte in the sample. To increase the sensitivity of the method, a novel polyethylene glycol/hydroxy-terminated silicone oil fiber was developed by sol-gel technique and applied for the analysis. Owing to the high polarity and hydrophilia of EC, an important problem still remains because the adsorption by sample matrix causes low transport of EC to the headspace and thus invalidates MHS-SPME for quantification. Mixing with anhydrous sodium sulphate, the sensitivity of the method can be improved. A Taguchi's L(16) (4(5)) orthogonal array design was employed to evaluate potentially significant factors and screen the optimum conditions for MHS-SPME of EC. Under the optimized conditions, limit of detection of 0.034 mg L(-1) was obtained. Relative standard deviation of replicate samples (n=6) was 2.19%. The proposed method was linear in the range of 0.04-100 mg L(-1), and the coefficient of determination was 0.9997. The method was used to determine EC in various alcoholic beverages. The concentrations obtained were compared with those obtained by standard addition method and no statistically significant differences were observed.

Optimization of headspace solid-phase microextraction for analysis of ethyl carbamate in alcoholic beverages using a face-centered cube central composite design.

The headspace solid-phase microextraction (HS-SPME) of ethyl carbamate from alcoholic beverages was optimized for the first time using a face-centered cube central composite design (CCD). The factors expected to influence the extraction process are discussed. Firstly, some of factors are fixed based on the opinion of expert and previous experiments, which reduce the number of factors and then avoid very complex response models and large variability. Secondly, for three remaining inexplicit factors, sample temperature, pH and %NaCl, a face-centered cube central composite design was performed and a response surface equation was derived. The statistical parameters of the derived model were r=0.974 and F=20.183. The optimum conditions were obtained using a grid method. Next, the method was analytically evaluated using the optimum conditions. The detection limit, relative standard deviation, linear range and recovery were 3microgL(-1), 4.3-8.6%, 10-160microgL(-1), and 92.8-97.5%, respectively. Finally, the method was applied to a variety of alcoholic beverages.

Application of positive ion chemical ionisation and tandem mass spectrometry combined with gas chromatography to the trace level analysis of ethyl carbamate in bread.

A rapid, sensitive and selective method has been developed and validated for the analysis of the contaminant ethyl carbamate (EC) in bread products at the part-per-billion level. The new procedure uses positive ion chemical ionisation (PICI) and tandem mass spectrometry (MS/MS), combined with gas chromatography (GC), on a 'bench-top' triple-quadrupole mass spectrometer. Ammonia was the PICI reagent gas of choice because of its ability to produce abundant [M+H]+ and [M+NH4]+ ions from EC and deuterium-labelled EC (LEC) used as an internal standard. For identification and quantification, selected reaction monitoring (SRM) was used to follow the precursor-to-product ion transitions of m/z 107 --> 90, m/z 107 --> 62 and m/z 90 --> 62 for EC, as well as m/z 112 --> 63 for the LEC internal standard. The limits of detection and quantification were 0.6 and 1.2 microg kg(-1), respectively, and the recovery of the method was 101 +/- 10% at 10 microg kg(-1) and 98 +/- 5% at 100 microg kg(-1). The precision of the method, established under conditions of intermediate reproducibility, did not exceed a relative standard deviation of 7%. The quantitative performance of the new GC/PICI-SRM procedure compared favourably with that of a reference method based on GC/MS and selected ion monitoring (correlation coefficient, r = 0.997). However, the new method had the advantages of reduced sample preparation time, improved sensitivity and unambiguous identification of EC at all concentrations. Application of the new method to the analysis of 50 UK breads showed that levels of EC ranged from 0.6 to 2.3 microg kg(-1) in retail products and from 3.1 to 12.2 microg kg(-1) for breads prepared using domestic breadmaking machines (dry weight basis). Toasting bread in a domestic toaster led to increases of between two- and three-fold in mean EC concentrations.

Analysis of ethyl carbamate in wines using solid-phase extraction and multidimensional gas chromatography/mass spectrometry.

The method describes a rapid and accurate procedure for the analysis of ethyl carbamate in wines. The separation of the ethyl carbamate (EC), the target analyte, from alcohol and the sample matrix is a challenge to many analytical chemists. After alcohol removal from the sample, EC was extracted and concentrated by solid-phase extraction. For analysis of EC, large-volume injection on a programmable temperature vaporization (PTV) inlet was used followed by multidimensional gas chromatography/mass spectrometry (MDGC/MS) using electron-impact ionization (EI). For quantitation, the ratio of ions produced during EI at m/z 62 (EC) and 64 (isotopically labeled EC) was monitored. The use of solid-phase extraction and MDGC/MS removes the majority of the matrix interference encountered in other methods. A linear dynamic range was established from 0.387 to 1160 ng/mL, with a limit of detection at 0.1 ng/mL and limit of quantitation at 1 ng/mL.

An improved method for determination of ethyl carbamate in Korean traditional rice wine.

An improved extraction method for ethyl carbamate, a genotoxic and carcinogenic compound found in various fermented foods and beverages, was investigated for its determination in the two most typical Korean traditional rice wines, takju and yakju. When the rice wines were extracted twice with chloroform at 30 degrees C for 60 min, the recovery of ethyl carbamate was less than 16%. When they were saturated with NaCl before extraction, the recovery of ethyl carbamate increased to 24.4% in takju and 67.2% in yakju. Adjustment of pH to 9.0 after NaCl saturation in takju resulted in a dramatic increase of recovery to 81.2%, but not in yakju. When the contents of ethyl carbamate and its precursor, urea, in various Korean traditional rice wines were determined, there was no correlation between the two contents. This is due to the fact that storage time is more important than urea content in the formation of ethyl carbamate in rice wine. In addition, its storage at high temperature resulted in a dramatic increase in ethyl carbamate content according to the prolonged storage time, suggesting that storage time and temperature play a key role in the formation of ethyl carbamate in Korean traditional rice wine.

Tabaquismo: alteraciones producidas por el uretano en los receptores de macrófagos y el sistema del complemento / Smoking: alterations by urethano in macrophages, complement system

Se comunican los resultados de un análisis de la acción del uretano sobre las células inmunocompetentes (macrófagos), el sistema del complemento y también en los enterocitos. El estudio de los receptores de macrófagos por medio de la formación de rosetas puso de manifiesto que concentraciones altas de uretano destruyen a los macrófagos y disminuyen la unión con IgG y el tercer componente del complemento. Los resultados obtenidos experimentalmente in vitro sugieren que de acuerdo con el grado y tiempo de la exposición al humo de tabaco se puede romper el equilibrio del sistema inmunitario y de otras poblaciones celulares que desembocan en los procesos patológicos que se relacionan con tabaquismo