Capillary electrophoresis coupled to electrospray mass spectrometry for the determination of organic and inorganic arsenic compounds in water samples.

Some compounds of arsenic are extremely toxic to the human body even at low levels. Long-term exposure from drinking contaminated water causes adverse health effects. This has led the European Union and other global organizations to reduce the maximum residue limit for the total arsenic up to 10 µg L-1 in water for human consumption. The toxicity of arsenic depends on the oxidation state, chemical structure and solubility in the biological environment. Main analytical procedures report on the determination of arsenic species in water samples which have been developed based on atomic spectroscopy -speciation performed in the sample treatment step- and inductively coupled plasma-mass spectrometry (ICP-MS). In such cases, speciation is performed by coupling to high-performance liquid chromatography (HPLC). As far as we know, the determination of arsenic species in water samples at low concentration levels by capillary electrophoresis (CE) coupled to electrospray mass spectrometry (ESI-MS) has not been described up to now. In this research CE-ESI(-)-MS is proposed for the identification and simultaneous quantification of organic and inorganic arsenic species in water samples for human consumption. The target compounds were dimethylarsinate (DMA), mono-methylarsonate (MMA), arsenite (i-As(III)) and arsenate (i-As(V)). Optimization of the composition and nature of both the electrophoretic separation medium -using hexafluoro-2-propanol (HFIP) as an additive- and the sheath liquid was performed and aimed at achieving the best sensitivity and separation efficiency for the CE-ESI(-)-MS coupling. Two simple sample treatments were proposed: one based on dispersive liquid-liquid micro-extraction (DLLME) intended for a rapid determination of the total content of inorganic arsenic in the water sample; and the second based on partial evaporation of the water sample to detect each of the four main arsenic species. For the second, limits of detection between 0.02 and 0.04 µg L-1 were found, which are similar results as the ones achieved with methods based on HPLC-ICP-MS. The DLLME + CE-ESI(-)-MS method was applied to the determination of total inorganic arsenic in eleven water samples in the province of Salamanca (Spain). Five of the analyzed samples had values close to, or superior to the maximum residue limit for the total of arsenic in water intended for human consumption (10 µg L-1). The validated method CE-ESI(-)-MS for arsenic speciation was applied with success to the analysis of the weakly mineralized water sample (dry residue < 50 mg L-1) of both groundwater and bottled water.

Analysis of Isoflavones in Foods.

In recent years the nutritional and bioactive properties of foods are being intensively investigated with a view to control, in addition to food quality, their possible influence on human health. Because of this, there is a growing demand for rapid, selective, sensitive, and validated methods for analysis and quantification. Bioactive plant compounds include those with weak estrogenic activity (phytoestrogens), among which are the isoflavones. Some of the beneficial activities that have been attributed to isoflavones are anticarcinogenic activity, the prevention of cardiovascular disease, the improvement of bone health, and antioxidant activity. The objective of this work is to provide an updated review of the methods used in sample preparation and subsequent analysis for the determination of isoflavones in food samples, including both soybean and soy products, as well as other foods with low isoflavone contents. The review focuses on the most common sample preparation techniques used during the last 10 years, including both conventional solvent extraction and other more recent extraction techniques. Separation and detection methods, including current trends in liquid chromatography analysis, such as the use of monolithic columns or ultra-high-pressure liquid chromatography, are also discussed.

Capillary electrophoresis coupled to mass spectrometry employing hexafluoro-2-propanol for the determination of nucleosides and nucleotide mono-, di- and tri-phosphates in baby foods.

The present work describes a method for the simultaneous determination of unmodified nucleosides and nucleotide mono-, di- and tri-phosphates by capillary electrophoresis coupled to mass spectrometry (CE-MS). The use of hexafluoro-2-propanol (HFIP) in the separation medium, and as an additive to the sheath liquid of the electrospray interface (ESI), generated a highly efficient and sensitive method. Instrumental limits of detection in the range of 14-53ngmL-1 for nucleosides and 7-23, 20-49 and 64-124ngmL-1 for nucleotide mono-, di-, and tri-phosphates, respectively, were found. Sample treatment involved diluting an aliquot of baby food with ultra-high quality water and applying centrifugation-assisted ultrafiltration (CUF). The proposed method was validated and used to analyse a variety of baby food samples (16 in total) such as fish, meat, fruits, and baby dairy desserts that may endogenously contain these analytes.

Determination of nucleosides and nucleotides in baby foods by hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of hydrophilic ion-pairing reagents.

In this work we propose a rapid and efficient method for the joint determination of nucleosides and nucleotides in dairy and non-dairy baby foods based on hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of diethylammonium (DEA) as a hydrophilic ion-pairing reagent (IP-HILIC-MS/MS). Sample treatment of the baby food included dilution with water and centrifugal ultrafiltration (CUF) with an additional washing step that notably improved the global performance of the process. Later dilution of the extract with acetonitrile allowed adequate separation in the HILIC system. With the proposed treatment, we obtained extraction recoveries higher than 80% and, additionally, no matrix effects were observed. The CUF-IP-HILIC-MS/MS method was validated according to the 2002/657/EC decision and was used for the quantification of nucleotides and nucleosides in sixteen samples of commercial baby foods.

Analysis of free nucleotide monophosphates in human milk and effect of pasteurisation or high-pressure processing on their contents by capillary electrophoresis coupled to mass spectrometry.

A simple, efficient and green analytical method for the determination of free nucleotide monophosphates in human milk is proposed. It involves centrifugal ultrafiltration (CUF) as sample treatment and capillary electrophoresis-electrospray mass spectrometry (CE-ESI-MS) for separation and simultaneous quantification. The optimised method, applied to the analysis of human milk samples, included their dilution (1:5) with water followed by CUF treatment. No matrix effects were found. The method provided limits of detection between 0.08 and 0.13 µg mL(-1) and limits of quantification between 0.26 and 0.43 µg mL(-1). The intralaboratory repeatability and reproducibility afforded relative standard deviation values lower than 10%. The method was applied to the study of the effects of Holder pasteurisation and high-pressure processing on the nucleotide contents in samples from a human milk bank. The results showed concentration values between 0.5 and 10 µg mL(-1), with higher concentrations for the samples treated by pasteurisation. The effect of freezing time on the content of nucleotides was also assessed.

A validated method for the determination of nucleotides in infant formulas by capillary electrophoresis coupled to mass spectrometry.

In this work CE-ESI-MS is proposed for the identification and simultaneous quantification of several ribonucleotide 5'-monophosphates in infant formula (IF) samples. The target compounds were adenosine 5'-monophosphate, cytidine 5'-monophosphate, guanosine 5'-monophosphate, uridine 5'-monophosphate, and inosine 5'-monophosphate. To our knowledge, the application of CE for the determination of these bioactive compounds in IFs has not yet been described. Optimization of the composition of the electrophoretic separation buffer and -mainly- the injection medium was carried out with a view to obtaining the best sensitivity and separation efficiency for the CE-MS coupling. Different sample treatments were assayed and one based on centrifugal ultrafiltration proved to be the simplest and most compatible with CE separation of the analytes and their ionization by the electrospray source. The whole optimized method (centrifugal ultrafiltration treatment prior to CE-MS) was validated according to the 2002/657/EC decision, obtaining a reliable and robust CE-MS method to determine these compounds in IF samples, with LODs between 0.8 and 1.8 µg/g (S/N = 3) and recoveries in the 90-106% range.

Capillary electrophoresis coupled to mass spectrometry for the determination of anthelmintic benzimidazoles in eggs using a QuEChERS with preconcentration as sample treatment.

Benzimidazoles (BZDs) are anthelmintic agents widely used in veterinary medicine. Their use in food-producing animals increases the possibility of residues appearing in animal tissues and products. Most analytical procedures reported for the determination of BZDs have been developed based on liquid chromatography (LC) because of their polar nature - zwitterionic - and thermal lability. To our knowledge, the determination of these compounds by capillary electrophoresis coupled to mass spectrometry (CE-MS) has not yet been described. In this work CE-MS is proposed for the identification and simultaneous quantification of several benzimidazoles in egg samples. The target compounds were 2-aminobenzimidazole, carbendazim, albendazole-2-aminosulphone, 5-hydroxy-thiabendazole, oxibendazole, albendazole, fenbendazole, oxfendazole, albendazole-sulphone, fenbendazole-sulphone. Optimization of the composition and nature - organic/aqueous - of both the electrophoretic separation buffer and the injection medium was carried out with a view to obtaining the best sensitivity and separation efficiency for the CE-MS coupling. A comparative study was carried out on different sample treatments for analyte extraction from egg samples. Two of them comprised a solvent extraction step followed by clean-up using a new commercial polymeric sorbent (Evolute ABN(©)), and the third was a particularization of the general extractive method so called Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS). Different modifications of the QuEChERS method were assayed, which included a later preconcentration step based on either SPE with MCX(©) sorbents or evaporation. The whole optimized method (QuEChERS with preconcentration prior to CE-MS) was validated according to the 2002/657/EC decision obtaining a CE-MS method sufficiently reliable and robust to determine residues of these compounds in egg samples of different origins with limits of detection between 3 and 51 µgL(-1) (S/N=3) and recoveries in the 74-112% range.

Programed nebulizing-gas pressure mode for quantitative capillary electrophoresis-mass spectrometry analysis of endocrine disruptors in honey.

The application of programed nebulizing-gas pressure (PNP) has been previously described to be a simple strategy for the separation of anions by capillary electrophoresis-electrospray-mass spectrometry (CE-ESI-MS). The PNP mode provided high resolution and stable analyses and also had the advantage of allowing the use of capillaries wider than the 50-75 µm conventional ones. Here, the application of the PNP approach to the quantitative analysis of pollutants in real samples by CE-ESI-MS is described for the first time; in particular, for the determination several endocrine disruptors (2,4-dichlorophenol, 2,4,5-trichlorophenol, pentachlorophenol, bisphenol-A, 4-tert-butyl-phenol, and 4-tert-butyl benzoic acid) in honey. For sample pretreatment, different liquid-liquid extraction (LLE) procedures were assayed and compared to the QuEChERS(©) methodology prior to electrophoretic analysis. With the application of the PNP approach to CE-ESI-MS, the limits of detection achieved were in the 1-4 ng/g range with a simple liquid-liquid procedure without any further clean-up step; relative standard deviation values in the 2-9% range were found. The analytical characteristics allow the proposed method to be used in the control analysis of these compounds in honey.

Programmed nebulizing gas pressure for efficient and stable capillary electrophoresis-mass spectrometry analysis of anionic compounds in positive separation mode.

Here we have developed a method for the analysis of anionic compounds by capillary electrophoresis coupled to electrospray ionization-mass spectrometry (CE-ESI-MS). The strategy proposed is based on the application of programmed nebulizing gas pressure (PNP), which is applied during the different steps of analysis: sample injection, electrophoretic separation, and detection by the mass spectrometer. The proposed procedure prevents the frequent drops in current observed in the analysis of anions and allows high separation efficiency by capillary electrophoresis to be maintained because it is not necessary to employ pressured-assisted electrophoresis. Additionally, for the first time we describe the use of 100 µm capillaries in capillary electrophoresis-mass spectrometry, allowing the loading of larger samples and hence greater sensitivity, with no loss of the efficiency achieved with the 50 µm capillaries usually used.

Determination of endocrine disruptors in honey by CZE-MS using restricted access materials for matrix cleanup.

An analytical method based on CZE coupled to ESI-MS is proposed for the identification and simultaneous quantification of several endocrine-disrupting chemicals in honey. The target compounds were the chlorophenols: 2,4-dichlorophenol, 2,4,5-trichlorophenol and pentachlorophenol, and bisphenol-A, 4-tert-butylphenol, and 4-tert-butylbenzoic acid. A two-step optimization of the ESI-MS detection was carried out. First, the organic solvent present in the sheath liquid was selected and its effect on the analytical signal was studied. The best results in terms of the intensity of the MS signals were obtained with methanol. Thus, an experimental design technique (Doehlert type) was used for the optimization of the other parameters: the NH(3) concentration in the sheath liquid, the flow of the sheath liquid, the nebulizer pressure in ESI, and the drying gas temperature and flow. Here, we developed a new sample treatment based on the combined use of a restricted access material and a polymeric sorbent for SPE. The LOD achieved were in the range of 5-31 ng/g. The intraday precision of the proposed method was determined from replicate analyses (n=4) at a concentration level of 50 ng/g, with RSD values in the range of 15-23%. The results revealed that the proposed method is suitable for the reliable quantification of endocrine-disrupting chemicals in honey at nanograms per gram levels.

Ultrasonic solvent extraction and nonaqueous CE for the determination of herbicide residues in potatoes.

In the present work we address the development of a simple and effective method for the determination of triazine herbicide residues in horticultural products by CE in nonaqueous media (NACE). Potato samples were selected as a representative matrix of such foods with a nonfatty content. Isolation of the analytes from the sample matrix was accomplished by extraction with organic solvents, assisted by ultrasound; a clean-up step of the organic extracts was carried out with SPE, using an Oasis MCX(R) sorbent to retain the analytes directly from the organic medium. The detection limits achieved in spiked potatoes (1.7-4.0 mug/kg) were lower than the default value of maximum residue level (MRL) established by current EU legislation for pesticide residues in foodstuffs. The results obtained were compared with HPLC in order to evaluate the performance of the NACE procedure.

In-capillary preconcentration of pirimicarb and carbendazim with a monolithic polymeric sorbent prior to separation by CZE.

CZE was assayed for the separation of carbamate pesticides susceptible to protonation (Pirimicarb, Carbendazim). Different electrophoretic media with high organic contents were explored, adequate separation and resolution being achieved when a BGE based on ACN with acetic acid in the presence of SDS as an ionic additive was used. With a view to increasing the sensitivity of the method, an in-capillary SPE step prior to the electrophoretic separation was developed. We employed a monolithic polymer formed in situ within the capillary as a medium for analyte retention. The synthesized monolithic bed exhibited high porosity and allowed samples to be loaded at flow rates of about 65 microL/min by applying a pressure of 12 bar. A 5-cm length of monolithic sorbent was used to preconcentrate the target analytes from aqueous samples. The analytes retained were eluted from the polymeric phase directly in the separation capillary with the same electrophoretic medium used for their further separation by CZE. For a 15-min preconcentration time, the in-line SPE-CZE approach proposed here permitted the determination of these pesticides in drinking water at a concentration level of 0.1 microg/L, as demanded by current EU legislation.

Sensitive determination of herbicides in food samples by nonaqueous CE using pressurized liquid extraction.

We have developed a method involving extraction with mixtures of solvents under pressure (pressurized liquid extraction (PLE)) for the determination of triazine herbicides in a series of samples from the food industry. The organic extracts obtained were subjected to a clean-up step with SPE, using Oasis MCX sorbents, after which they were analyzed by NACE. Potato was chosen as a representative matrix of horticultural products since it has a high water content. Spiked potato samples were used to optimize extraction conditions. In order to compare the results obtained with NACE, different studies were also conducted using HPLC. The detection limits in NACE were similar to those found with HPLC and were of the order of 10-15 microg/kg, depending on the analyte. Satisfactory results were obtained on applying the method proposed for the potato matrix (PLE with separation by electrophoresis) to other food matrices such as other tubercles, fruits, vegetables and cereals.

Ion-pair association and acid-base equilibria in nonaqueous capillary electrophoresis of weakly basic compounds.

CE in nonaqueous media was used to study the migrating behavior of two weakly basic s-triazine pesticides and one of their metabolites. The target pesticides were selected to be representative for each of the two main groups: propazine and deethylatrazine for the chloro-s-triazines group and ametryn for the methylthio-s-triazines group. To elucidate the phenomena involved, systematic studies were carried out in the different organic media studied. Absolute mobilities were determined in 50% v/v methanol (MeOH)/ACN by extrapolation of the effective mobilities to zero ionic strength in the presence of different concentrations of perchloric acid. Conductivity measurements performed in MeOH and 50 and 20% v/v methanol/ACN permitted the evaluation of the associations of the components of the BGE. The effects of ionic strength on the actual mobilities of the compounds were determined in the presence of perchloric acid and SDS in different organic media. Two different ion-pair equilibria were considered: one due to the presence of perchlorate anions present in the BGE and second that from the added dodecyl sulfate anions. Bearing in mind that these weakly basic compounds can exhibit ion-pair and acid-base equilibria, the acid-base and ion-pair parasite reaction coefficients were determined. Finally, the effects of ionic strength, ion-pair interactions and acid-base properties on the effective electrophoretic mobilities of the analytes are discussed.

Comparative study of separation and determination of triazines by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis: application to residue analysis in natural waters.

The separation and determination of a mixture of chloro- and methylthiotriazines in water samples by both micellar electrokinetic capillary chromatography (MEKC) and nonaqueous capillary zone electrophoresis (NA-CZE) were compared. The characteristics of both methods proved to be very similar in terms of separation efficiency and analysis times, but application of these methods for the analysis of triazines in natural waters, with a prior preconcentration step, revealed significant differences. A preconcentration step by solid-phase extraction (SPE) with Oasis HLB cartridges was accomplished for the determination of triazines at sub-ppb levels in drinking and river waters; when NA-CZE was used after this SPE step, electropherograms with fewer interferences and more stable baselines were obtained than when separation was carried out using MEKC. Another aspect related to the application to real samples was the lack of precision encountered upon evaluating the electrophoretic signals generated when using SPE coupled with NA-CZE. Here, we demonstrate the importance of choosing an appropriate internal standard for analyte quantification. It is recommended that a triazine belonging to the same family as that of the triazine to be determined should be used as internal standard.