Development of a two-dimensional liquid chromatography system with trapping and sample enrichment capabilities

A solid-phase microextraction (SPME) followed by a gas chromatographic-mass spectrometric (GC-MS) determination has been developed and validated for the determination of cyprodinil and fludioxonil in white wine samples. Extraction parameters such as the selection of SPME coating, the effect of the temperature, the effect of the headspace volume and the salt addition were studied and optimized, together with GC-MS analytical conditions. The divinylbenzene-Carboxen-polydimethylsiloxane (DVB-CAR-PDMS) fiber was the most appropriate for the determination of the two pesticides in wine. The quality parameters of the proposed method demonstrated a good precision (RSD about 5%), with detection limits of 0.1 and 0.2 microg/l for cyprodinil and fludioxonil, respectively. Fifteen commercial white wine samples produced in Rías Baixas area in Galicia (N.W. Spain) were analyzed with the SPME-GC-MS procedure. Some of the commercial wines (75%) presented the two pesticides in concentrations ranging from 0.9 to 28.6 microg/l. In conclusion, SPME-GC-MS has a great potential for fungicide determination in wines.     

葡萄酒中痕量木塞污染物的顶空固相微萃取-气相色谱串联质谱法检测

建立了顶空固相微萃取-气相色谱串联质谱检测葡萄酒中主要的痕量木塞污染物——2,4,6-三氯苯甲醚（TCA）的方法。通过优化萃取时间、温度、盐浓度、pH值等固相微萃取处理条件,采用2,4,6-三氯甲苯（TCT）为内标进行定量,气相色谱离子阱质谱法测定。选取TCA母离子和子离子分别为m/z210和m/z195,TCT的母离子和子离子为m/z195和m/z159。方法的定量下限（LOQ）为2.0 ng/L,回收率为71%-98%。该法操作简单、快速,适用于葡萄酒中痕量TCA残留的快速检测。     

Highly selective solid-phase extraction and large volume injection for the robust gas chromatography-mass spectrometric analysis of TCA and TBA in wines

A reliable solid-phase extraction (SPE) method for the simultaneous determination of 2,4,6-trichloroanisole (TCA) and 2,4,6-tribromoanisole (TBA) in wines has been developed. In the proposed procedure 50 mL of wine are extracted in a 1 mL cartridge filled with 50 mg of LiChrolut EN resins. Most wine volatiles are washed up with 12.5 mL of a water:methanol solution (70%, v/v) containing 1% of NaHCO3. Analytes are further eluted with 0.6 mL of dichloromethane. A 40 microL aliquot of this extract is directly injected into a PTV injector operated in the solvent split mode, and analysed by gas chromatography (GC)-ion trap mass spectrometry using the selected ion storage mode. The solid-phase extraction, including sample volume and rinsing and elution solvents, and the large volume GC injection have been carefully evaluated and optimized. The resulting method is precise (RSD (%) < 6% at 100 ng L(-1)), sensitive (LOD were 0.2 and 0.4 ng/L for TCA and TBA, respectively), robust (the absolute recoveries of both analytes are higher than 80% and consistent wine to wine) and friendly to the GC-MS system (the extract is clean, simple and free from non-volatiles).     

Development of a dispersive liquid-liquid microextraction method for the simultaneous determination of the main compounds causing cork taint and Brett character in wines using gas chromatography-tandem mass spectrometry

A novel dispersive liquid-liquid microextraction (DLLME) method, coupled to gas chromatography-tandem mass spectrometry (GC-MS/MS), was developed for simultaneously determining the main compounds responsible for cork taint (2,4,6-trichloranisole (TCA), 2,3,4,6-tetrachloroanisole (TeCA), 2,4,6-tribromoanisole (TBA) and pentachloranisole (PCA)) and Brett character (4-ethylguaiacol (EG), 4-ethylphenol (EP), 4-vinylguaiacol (VG) and 4-vinylphenol (VP)) in wines. Optimisation of DLLME procedure was performed by evaluating the type of disperser and extraction solvents and the temperature and salt addition effects. The volumes of disperser and extraction solvents were also optimised by means of a central composite design combined with desirability functions. Under optimum conditions, 5 mL of wine were extracted with an extraction mixture consisting of 1.43 mL of acetone, and 173 μL of chloroform at room temperature. The analytical characteristics of the method were evaluated. Satisfactory linearity (with correlation coefficients over 0.992), repeatability (below 11.6%) and between-days precision (below 11.0%) were obtained for all target analytes. Detection limits attained were at similar levels or even lower than the olfactory threshold of the studied compounds. Finally, the developed method was successfully applied to the analysis of wine samples. To our knowledge, this is the first time that DLLME has been applied to simultaneously determine the compounds responsible for cork taint and Brett character in wine.     

Analysis of corky off-flavour compounds at ultra trace level with multidimensional gas chromatography-electron capture detection

A robust method for routine quality control of corky off-flavour compounds in wine and cork soak matrices has been established. Based on an automated headspace solid phase microextraction (HS-SPME), the method needs only marginal sample preparation and achieves low (sub-ng L⁻¹) trace level detection limits (LODs) for the most relevant off-flavour compounds, such as 2,4,6-trichloroanisole (TCA), 2,3,4,6-tetrachloroanisole (TeCA) and 2,4,6-tribromoanisole (TBA). Particularly for wine matrix, reliable trace level quantification had only been achieved after applying heart-cutting multidimensional gas chromatography (MDGC). Using a halogen-sensitive electron capture detector (ECD) and quantification with a stable isotope dilution assay (SIDA), LODs of 0.1 ng L⁻¹ for TCA, TeCA and TBA could be obtained. Since a SIDA based quantification method is used with a non-mass spectrometric detector, the necessary chromatographic resolution of internal standard and target analyte peaks resulted from the use of highly deuterated [²H₅]-isotopologues.     

Determination of 2,4,6-trichloroanisole and 2,4,6-tribromoanisole on ng L-1 to pgL-1 levels in wine by solid-phase microextraction and gas chromatography-high-resolution mass spectrometry

A gas chromatography-high-resolution mass spectrometry (GC-HRMS) method using solid-phase microextraction (SPME) for the determination of 2,4,6-trichloroanisole (TCA) and 2,4,6-tribromoanisole (TBA) in wine at low ng L(-1) levels was developed. A robust SPME method was developed by optimizing several different parameters, including type of fiber, salt addition, sample volume, extraction and desorption time. The quantification limit for TCA and TBA in wine was lowered substantially using GC-HRMS in combination with the optimized SPME method and allowed the detection of low analyte concentrations (ng L(-1)) with good accuracy. Limits of quantification for red wine of 0.3 ng L(-1) for TCA and 0.2 ng L(-1) for TBA with gas chromatography-negative chemical ionization mass spectrometry and 0.03 ng L(-1) for TCA and TBA were achieved using GC-HRMS. The method was applied to 30 wines of which 4 wines were sensorically qualified as cork defected. TCA was found in three of these wines with concentrations in the range 2-25 ng L(-1). TBA was not detected in any of the samples.     

Determination of some phenolic compounds in red wine by RP-HPLC: method development and validation

A methodology employing reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and validated for simultaneous determination of five phenolic compounds in red wine. The chromatographic separation was carried out in a C(18) column with water acidify with acetic acid (pH 2.6) (solvent A) and 20% solvent A and 80% acetonitrile (solvent B) as the mobile phase. The validation parameters included: selectivity, linearity, range, limits of detection and quantitation, precision and accuracy, using an internal standard. All calibration curves were linear (R(2) > 0.999) within the range, and good precision (RSD < 2.6%) and recovery (80-120%) was obtained for all compounds. This method was applied to quantify phenolics in red wine samples from Santa Catarina State, Brazil, and good separation peaks for phenolic compounds in these wines were observed.     

Optimisation of a dispersive liquid-liquid microextraction method for the simultaneous determination of halophenols and haloanisoles in wines

A dispersive liquid-liquid microextraction (DLLME) method has been optimised for simultaneously extracting 2,4,6-trichloranisole (TCA), 2,3,4,6-tetrachloroanisole (TeCA), 2,4,6-tribromoanisole (TBA), pentachloroanisole (PCA), 2,4,6-trichlorophenol (TCP), 2,3,4,6-tetrachlorophenol (TeCP), 2,4,6-tribromophenol (TBP) and pentachlorophenol (PCP) from wine. The haloanisoles and halophenols were automatically determined using a gas chromatography-electron-capture detection (GC-ECD) system. Derivatisation of halophenols was performed at the same time as DLLME. Firstly, disperser and extraction solvents, salt addition and temperature conditions were selected. Then, the volume of disperser solvent, extraction solvent and derivatisation agent, and the percentage of base were optimised by means of a central composite design combined with desirability functions. The optimal extraction-derivatisation conditions found were 1.3 mL of acetone, 150 μL of carbon tetrachloride, 75 μL of acetic anhydride and a percentage of base of 0.7%; with no salt addition and at room temperature. Under these conditions, the proposed method showed satisfactory linearity (with correlation coefficients over 0.994), repeatability (below 9.7%) and reproducibility (below 9.9%). Moreover, detection limits were lower than the olfactory threshold of the compounds. The developed method was successfully applied to the analysis of red wine samples. To our knowledge, this is the first time that DLLME has been applied to determine cork taint responsible compounds in wine.     

In situ derivatization/solid-phase microextraction coupled with gas chromatography/negative chemical ionization mass spectrometry for the determination of trichloroethylene metabolites in rat blood

An in situ derivatization solid-phase microextraction (SPME) method has been developed for the determination of the trichloroethylene (TCE) metabolites, trichloroacetic acid (TCA), dichloroacetic acid (DCA) and trichloroethanol (TCOH), in rat blood. The analytical procedure involves derivatization of TCA and DCA to their ethyl esters with acidic ethanol, headspace sampling using SPME, and gas chromatography/negative chemical ionization mass spectrometry (GC/NCI-MS) determination. Parameters affecting both derivatization efficiency and the headspace SPME procedure, such as the concentration of sulfuric acid, amount of ethanol, derivatization-extraction temperature and time, sample preheating time, agitator speed and desorption conditions, were optimized. The method showed good linearity over the range of 1-1000 ng/mL in rat blood for each metabolite with correlation coefficients (R(2)) higher than 0.99. The intra-day and inter-day precision and accuracy were less than 10%. The relative recoveries for all analytes were greater than 84%. Validation results demonstrated that selected ion monitoring of the (35)Cl and (37)Cl isotopes using NCI resulted in reliable and sensitive quantitation of all three TCE metabolites. This validated method was successfully applied to study the toxicokinetic behavior of TCE metabolites following a 1 mg/kg oral dose of TCE.     

Application of fractional factorial experimental and Box-Behnken designs for optimization of single-drop microextraction of 2,4,6-trichloroanisole and 2,4,6-tribromoanisole from wine samples

In this paper a new method for the determination of 2,4,6-trichloroanisole (TCA) and 2,4,6-tribromoanisole (TBA) in wine samples is presented. Headspace single-drop microextraction (HS-SDME) was used for the extraction and preconcentration of the analytes, followed by analysis by gas chromatography and electron-capture detection (GC-ECD). The variables affecting extraction efficiency were optimized using fractional factorial experimental and Box-Behnken designs. The external calibration procedure was successfully carried out using a synthetic wine solution and diluted red wine samples. The method was also applied to white wine samples. Excellent detection limits of 8.1 and 6.1 ng L(-1) were achieved for TCA and TBA, respectively. Good precision and accuracy were obtained.     

Determination of biogenic amines in wines by pre-column derivatization and high-performance liquid chromatography coupled to mass spectrometry

A new HPLC method for determining biogenic amines in wines is developed. This method is based on pre-column amine derivatization, further separation of derivatives and on-line hyphenation of HPLC to atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Biogenic amines have been derivatized with 1,2-naphthoquinone-4-sulfonate at 65 °C and pH 9.2 for 5 min. The separation of derivatives has been accomplished in a C₁₈ analytical column using an elution gradient based on increasing the percentage of methanol. Derivatives have been ionized in positive mode and detected by selected ion monitoring. The operating conditions of the APCI-MS system (voltages, temperatures and gases) have been thoroughly optimized to obtain the maximum sensitivity for all analytes. In the selected conditions, APCI-MS spectra display little fragmentation and good signal-to-noise ratio. Depending on the amine characteristics, the main spectral peaks are due to mono- and di-derivative products. Figures of merit of the method have been established under the selected conditions using red wine samples. Recoveries ranging from 94% to 106% have been obtained which prove excellent accuracy of the method in the determination of histamine, putrescine, cadaverine, tryptamine, phenylethylamine, tyramine and serotonin in red wines. The proposed method has been applied to the analysis of commercial wines from different Spanish regions.     

Comparison of methods for the determination of ochratoxin A in wine

Different analytical methods for the determination of ochratoxin A (OTA) in wine have been compared. Sample clean-up was based on solid-phase extraction (SPE) with (i) immunoaffinity or (ii) RP-18 sorbent materials applying different experimental protocols. The detection of OTA was accomplished with high-performance liquid chromatography (HPLC) combined either with electrospray ionisation (ESI) tandem mass spectrometry (MS-MS) or fluorescence detection (FL). Comparative method evaluation was based on the investigation of 18 naturally contaminated red wine samples originating from different European countries. The analytical results are discussed in view of the respective method validation data and the corresponding experimental protocols. In general, analytical data obtained with RP-18 SPE combined with LC-MS-MS detection and immunoaffinity extraction combined with FL offered comparable good results in the sub-ppb concentration level indicating that high selectivity of either the sample clean-up or, alternatively the detection system are equally well-suited to guarantee an accurate OTA analysis in wine.     

Determination of oxadiazon residues by headspace solid-phase microextraction and gas chromatography-mass spectrometry

A method for the determination of trace amounts of the herbicide oxadiazon was developed using headspace solid-phase microextraction (HS-SPME), gas chromatography-mass spectrometry (GC-MS) and selected ion monitoring. It was applied to determine oxadiazon in ground water, agricultural soil, must, wine and human urine samples. To determine oxadiazon in liquid samples, a response surface methodology generated with a Doehlert design was applied to optimize the HS-SPME conditions using a 100 microm polydimethylsiloxane fibre. For the analysis of soil samples, they were mixed with water and the SPME fibre suspended in the headspace above the slurry. Ground water, human urine and must show linear concentration range of application of 0.5-50 ng ml(-1)' with detection limits < or =0.02 ng ml(-1). HS-SPME-GC-MS analysis yielded good reproducibility (RSD values between 6.5 and 13.5%). The method validation was completed with spiked matrix samples. The developed analytical procedure is solvent free, cost effective and fast.     

Critical aspects of the determination of pentafluorobenzyl derivatives of aldehydes by gas chromatography with electron-capture or mass spectrometric detection: Validation of an optimized strategy for the determination of oxygen-related odor-active aldehydes in wine

This work presents a thorough study of some aspects critical to the quantitative performance of methods for the determination of volatile aldehydes previously derivatized to pentafluorobenzyl hydroxylamine oximes. The conclusions of the study are further applied to the validation of an optimized procedure for the determination of oxidation-related aldehydes from wine. Aspects studied include the chromatographic injection, the analytical performance of electron-capture detection (ECD) or MS detection, and the way in which the derivatization is carried out. Different injection techniques have been optimized and compared (classical splitless-hot injection; cold splitless; and large volume solvent split injection). All of them were carried out in a programmed temperature vaporization (PTV) injector, with a 350 microL internal volume liner (3.4 mm internal diameter). Classical splitless injection of hexane extracts is troublesome and requires large carrier gas flows (>10 mL min(-1)). Cold splitless injection was clearly superior. Large volume solvent split injection has been also optimized. ECD has been found to lack the necessary selectivity for the determination of unsaturated aldehydes in wine, although the quantitation of several minor aldehydes is possible. MS detection has proven to be superior for the determination of these compounds in wine. The way in which the reagent is applied is also critical and for the case of wine is important to ensure that the reagent is applied after some of the major wine aldehydes have been eliminated. The finally proposed method is extremely sensitive. Method detection limits ranged from 0.002 microg L(-1) (for unsaturated aldehydes) to 0.73 microg L(-1) (for phenylacetaldehyde) and precision (measured as relative standard deviation) is < or =6% in all cases. The method makes it possible to determine quantitatively in a single run the wine aldehydes with sensory significance (isobutyraldehyde, 2-methylbutanal, isovaleraldehyde, (E)-2-hexenal, (E)-2-heptenal, (E)-2-octenal, (E)-2-nonenal, methional and phenylacetaldehyde).     

Determination of ethyl carbamate in alcoholic beverages: an interlaboratory study to compare HPLC-FLD with GC-MS methods

An international interlaboratory study on the determination of ethyl carbamate in alcoholic beverages by a new HPLC-FLD and by the official GC-MS methods is presented. The aim of this study was to improve the knowledge about precision and accuracy parameters of the new method and to compare the performance of both HPLC and GC methods. Five different samples representing table wines, fortified wines (red and white), distilled spirits, and wine spirits were available for analysis by each participant. Despite the low number of participants (6), the results obtained by the laboratories using the HPLC-FLD method are comparable to those obtained by GC-MS methods. The present study emphasizes the possibility to use, as routine, a much simpler analytical method than the current reference method by GC-MS for ethyl carbamate determination in alcoholic beverages.     

Analysis of organic acids and inorganic anions in beverage drinks by capillary electrophoresis

To meet the need for a new and validated analytical method for simultaneous determination of inorganic and organic acid anions in beverage drinks, a capillary zone electrophoresis (CZE) procedure had been developed based on a new background electrolyte (BGE) system containing 3 mmol/L 1,3,5-benzenetricarboxylic acid (BTA), 15 mmol/L tris(hydroxymethyl)aminomethane and 1.5 mmol/L tetraethylenepentamine (TEPA) at pH 8.4. Baseline separation of anions commonly found in beverage drinks could be achieved in less than 14 min with indirect UV detection at 240 nm. Comigration problems for hydroxycarboxylic acids could be solved using TEPA as BGE additive. The results indicate excellent repeatability for migration time (RSD, 0.27-0.67%, n = 5) and good precision for both peak height (RSD, 3.2-4.2%, n = 5) and peak area (RSD, 3.1-4.5%, n = 5). Under the optimized conditions and using corrected peak area for quantitation, an excellent linear dynamic range (with correlation coefficient > 0.997 in a concentration range from 0.005 to 2.0 mmol/L) and low detection limit (1-4 micro mol/L) were obtained for all the anions investigated. The applicability and reliability of the CE procedure developed were established by parallel method determination using established ion chromatography procedure for the analysis of inorganic and organic acid anions in orange juice and wine samples. Our CZE procedure provided a sensitive and economic technique for simultaneous determination of inorganic and organic acid anions in orange juice, red and white wine samples.     

Rapid headspace solid-phase microextraction-gas chromatographic-time-of-flight mass spectrometric method for qualitative profiling of ice wine volatile fraction. I. Method development and optimization

An analytical method for the determination of volatile and semi-volatile compounds representing various chemical groups in ice wines was developed and optimized in the presented study. A combination of the fully automated solid-phase microextraction (SPME) sample preparation technique and gas chromatographic-mass spectrometric (GC-MS) system to perform the final chromatographic separation and identification of the analytes of interest was utilized. A time-of-flight mass spectrometric (TOF-MS) analyzer provided very rapid analysis of this relatively complex matrix. Full spectral information in the range of m/z 35-450 was collected across the short GC run (less than 5 min). Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) 50/30 microm fiber performed best during the optimization experiments and it was used in the headspace SPME mode to isolate compounds from ice wine samples, consisting of 3 mL wine with 1g salt addition. After the sample incubation and extraction (both 5 min at 45 degrees C), analytes were thermally desorbed in the GC injector for 2 min (injector maintained at 260 degrees C) and transferred into the column. The MS data acquisition rate of 50 spectra/s was selected as optimal. The optimized analytical method did not exceed 20 min per sample, including both the isolation and pre-concentration of the analytes of interest, the final GC-TOF-MS analysis and the fiber bake-out. Both a linear temperature-programmed retention index (LTPRI) method using C(8)-C(20) alkanes loaded onto the fiber and a mass spectral library search were employed to identify the target compounds. The repeatability of the developed and optimized HS-SPME-GC-TOF-MS method for ice wine analysis, expressed as relative standard deviation (RSD, %, n=7), ranged from 3.2 to 9.0%.     

Dispersive liquid-liquid microextraction using non-chlorinated, lighter than water solvents for gas chromatography-mass spectrometry determination of fungicides in wine

A novel and low solvent consumption method for the sensitive determination of fungicide residues in wine samples is proposed. Analytes were extracted by dispersive liquid-liquid microextraction (DLLME) and further determined by gas chromatography-mass spectrometry (GC-MS). Under optimized conditions, a binary mixture of acetone and 1-undecanol (0.5 and 0.05 mL, respectively) was used to extract target compounds from diluted (1:1) wine samples. After centrifugation, the floating phase of 1-undecanol was solidified and separated from the liquid hydro-alcoholic matrix. Thereafter, it was allowed to melt at room temperature and injected in the GC-MS system. The method showed relative standard deviations (RSDs, %) below 13%, limits of quantification (LOQs) between 0.2 and 3.2 ng mL(-1) and linear responses for concentrations up to 300 ng mL(-1). The efficiency of the liquid-phase microextraction process was scarcely affected by the characteristics of wine samples, consequently pseudo-external standard calibration (using matrix matched standards of red and white wine) sufficed to achieve acceptable accuracy values: relative recoveries from 81 to 120%. The applicability of the method was demonstrated with commercial wine samples.     

Comparative study of two chromatographic methods for quantifying 2,4,6-trichloranisole in wines

Here we present the validation and the comparative study of two chromatographic methods for quantifying 2,4,6-trichloroanisole (TCA) in wines (red, rosé and white wines). The first method involves headspace solid-phase microextraction and gas chromatography with electron-capture detection (ECD). The evaluation of the performance parameters shows limit of detection of 0.3 ng l(-1), limit of quantification of 1.0 ng l(-1), recoveries around 100% and repeatability of 10%. The second one implies a headspace solid-phase microextraction and gas chromatography with mass spectrometric detection. The performance parameters of this second method are limit of detection of 0.2 ng l(-1), limit of quantification of 0.8 ng l(-1) and repeatability of 10.1%. From the comparative study we can state that both methods provide similar results and the differences between them are the better sensitivity of the GC-ECD method and the very shorter chromatogram running time of the GC-MS method. The two methods are able to quantify TCA below the sensorial threshold in red, rosé and white wines using just a calibration graph, thus they could be a very good tool for quality control in wineries.     

Determination of amphetamine in dog plasma by gas chromatography with mass selective detection

This paper describes the validation of an analytical method for the determination of amphetamine in beagle dog plasma by gas chromatography coupled to mass spectrometry (GC-MS). d-Amphetamine-d(6) was used as the internal standard. The method consisted of a rapid single-step liquid-liquid extraction and derivatization of amphetamine with 2,2,2-trichloroethyl chloroformate, followed by sensitive GC-MS detection. This is the first report utilizing the combination of trichloroethyl chloroformate as a derivatization reagent and a deuterated amphetamine analog as an IS for the quantification of amphetamine in plasma. The method was validated in terms of specificity, curve fit, precision, accuracy, recovery and stability, and was acceptable according to FDA draft guidelines for validation of bioanalytical methods. The limit of detection was 0.65 ng/mL. The calibration range was 5-150 ng/mL. The validated method was successfully employed for the quantitation of amphetamine in dog plasma samples for pharmacokinetic profiling.