Online sample concentration and analysis of drugs of abuse in human urine by micelle to solvent stacking in capillary zone electrophoresis.

A sensitive and rapid CZE-UV method was developed to determine drugs and their metabolites' presence in human urine. Ten drugs of abuse were analyzed including four amphetamines, cocaine, cocaethylene, heroin, morphine, 6-monoacetylmorphine, and 4-methylmethcathinone. An MSS (micelle to solvent stacking) approach was evaluated to enhance method sensitivity. This method considers composition of the micellar sample solution matrix and the injection time. Several analytical conditions influencing the resolution of the drugs mixture as pH and buffer concentration, organic solvent content, were also investigated. The base-line separation of all studied analytes in the same run was achieved within 18 min in an uncoated fused silica capillary (50 µm id × 60 cm) using a background solution containing 50 mM phosphate buffer pH 2.5 and 30% ACN v/v. Other experimental parameters such as applied voltage and capillary temperature were set up at 20 kV and 20°C, respectively. LOD values ranging between 15 and 75 ng/mL for all studied compounds were obtained. From a comparison with conventional CZE, the proposed method provides an increase of sensitivity (39- to 55-fold enhancement factor). Under optimal MSS-CZE conditions, good linearity was achieved (R2 ≤ 0.9998). The method was finally applied to the analysis of urine samples spiked with a standard mixture after a sample pretreatment, reaching satisfactory recovery values.

Capillary electrochromatography-mass spectrometry for the determination of 5-nitroimidazole antibiotics in urine samples.

The separation of eight antibiotics belonging to 5-nitroimidazole family was carried out by means of CEC coupled with MS. Preliminary experiments were carried out with ultraviolet detection in order to select the proper stationary and mobile phase. Among the different stationary phases studied (namely Lichrospher C18, 5 µm particle size; Cogent(TM) Bidentate C18, 4.2 µm; Pinnacle II™ Phenyl, 3 µm; Pinnacle II™ Cyano, 3 µm), Cogent™ Bidentate C18 (4.2 µm) gave the best performance. For CEC-MS coupling, a laboratory assembled liquid-junction-nano-spray interface was used. In order to achieve a good sensitivity, special attention was paid to both optimization of the sheath liquid composition as well as selection of the injection mode. Under optimized CEC-ESI-MS conditions, the separation was accomplished within 22 min by using a column packed with a mixture of Bidentate C18:Lichrospher Silica-60 (5 µm) 3:1 w/w, an inlet pressure of 11 bar, a voltage of 15 kV, and a mobile phase composed by 45:10:45 v/v/v ACN/MeOH/water containing ammonium acetate (5 mM pH 5). A combined hydrodynamic and electrokinetic injection of 8 bar, 15 kV, and 96 s was adopted. The method was validated in terms of repeatability and intermediate precision of retention times and peak areas, linearity, and LODs and LOQs. RSDs values were <2.9% for retention times and <16.1% for peak areas in both intraday and interday experiments. LOQ values were between 0.09 and 0.42 µg/mL for all compounds. Finally, the method was applied to the determination of three most employed 5-nitroimidazole antibiotics (metronidazole, secnidazole, and ternidazole) in spiked urine samples, subjected to a SPE procedure. Recovery values in the 67-103% range were obtained. Furthermore, for the selected antibiotics, CEC-MS(2) spectra were obtained providing the unambiguous confirmation of these drugs in urine samples.

Determination of key flavonoid aglycones by means of nano-LC for the analysis of dietary supplements and food matrices.

A method for the analysis of flavonoids (myricetin, quercetin, naringenin, hesperitin, and kaempferol), with interesting bioactivity, has been developed and validated utilizing nano-LC technique. In order to find optimal conditions, capillary columns (75 µm id × 10 cm) packed with different types of stationary phases, Kinetex® C18 core-shell (2.6 µm particle size), Hydride-based RP-C18 (sub-2 µm particle size), and LiChrospher® 100 RP-18 endcapped (5 µm particle size) were evaluated. The method was validated using Hydride-based RP-C18 stationary phase, with sub-2 µm particle size. A good chromatographic performance, expressed in terms of repeatability (RSD, in the range 1.63-4.68% for peak area), column-to-column reproducibility (RSD not higher than 8.01% for peak area), good linearity and sensitivity was obtained. In particular limit of detection values between 0.07 and 0.31 µg/mL were achieved with on column focusing technique. The method was applied to the determination of studied flavonoids in dietary supplements as well as in food matrices. The amount of quercetin found in the first analyzed dietary supplement, was in agreement to the labeled content. In the other samples, where the content of flavonoids was not labeled, most of the studied flavonoids were determined in amounts somewhere comparable to those reported in literature.

Enantiomers separation by nano-liquid chromatography: use of a novel sub-2 µm vancomycin silica hydride stationary phase.

A novel sub-2 µm chiral stationary phase (CSP) was prepared immobilizing vancomycin onto 1.8 µm diol hydride-based silica particles. The CSP was packed into fused silica capillaries of 75 µm i.d. with a length of 11 cm and evaluated by means of nano-liquid chromatography (nano-LC) using model compounds of both pharmaceutical and environmental interest (some non-steroidal anti-inflammatory drugs, ß-blockers and herbicides). The study of the effect of the linear velocity of the mobile phase on chromatographic efficiency showed good enantioresolutions up to a value of 5.11 at the optimal linear velocity with efficiencies in terms of number of plates per meter in the range 51,650-68,330. The results were compared with the ones obtained employing 5 µm vancomycin modified diol-silica particles packed in capillaries of the same i.d. For the acidic analytes the sub-2 µm CSP showed better performances, the baseline chiral separation of several studied compounds occurred in an analysis time of less than 3 min. Column-to-column packing reproducibility (n=3) expressed as relative standard deviation was in the range 2.2-5.8% and 0.5-7.7% for retention times and peak areas, respectively.

Current applications of miniaturized chromatographic and electrophoretic techniques in drug analysis.

In the last decade, miniaturized separation techniques have become greatly popular in pharmaceutical analysis. Miniaturized separation methods are increasingly utilized in all processes of drug discovery as well as quality control of pharmaceutical preparation. The great advantages presented by the analytical miniaturized techniques, including high separation efficiency and resolution, rapid analysis and minimal consumption of reagents and samples, make them an attractive alternative to the conventional chromatographic methods for drug analysis. The purpose of this review is to give a general overview of the applicability of capillary electrophoresis (CE), capillary electrochromatography (CEC) and micro/capillary/nano-liquid chromatography (micro-LC/CLC/nano-LC) for the analysis of pharmaceutical formulations, active pharmaceutical ingredients (API), drug impurity testing, chiral drug separation, determination of drugs and metabolites in biological fluids. The results concerning the use of CEC, micro-LC, CLC, and nano-LC in the period 2009-2013, while for CE, those from 2012 up to the review draft are here summarized and some specific examples are discussed.

A nano-LC/UV method for the analysis of principal phenolic compounds in commercial citrus juices and evaluation of antioxidant potential.

In this study, a simple and rapid methodology to analyze and quantify principal flavanones in citrus fruit juices through the use of a nano-LC/UV-Vis apparatus, employing a 75 µm id capillary column packed with sub-2 µm particles C18 stationary phase for 10 cm, was developed. All compounds were baseline resolved working with a step gradient elution mode in 10 min. The developed analytical method was validated and the resulting RSD% for intra- and interday repeatability, related to retention time and peak area, were <4.7 and 5.5%, respectively. LOD and LOQ values corresponded to 0.40 and 1.56 µg/mL for didymin, hesperitin, and narinegenin, while for the other flavanones were 0.78 and 3 µg/mL, respectively. Good linearity with acceptable determination coefficients R(2) was obtained in the range between LOQ concentration and 200 µg/mL (500 µg/mL naringin and hesperidin). Good recovery values were also obtained. Then, the method was applied to the analysis of selected hand-squeezed and commercial citrus juices. Further, the nano-LC system was coupled to a mass spectrometer to confirm analyte identification. Antioxidant capacity of selected samples was also evaluated measured by Folin-Ciocalteu assay and Trolox equivalent antioxidant capacity assay. Results were compared to determine total flavanones concentrations.

Nano-liquid chromatography in nutraceutical analysis: determination of polyphenols in bee pollen.

In this study, a nano-liquid chromatography based method for the simultaneous separation of 16 polyphenols employing UV-vis detection has been developed. A 100 µm I.D. capillary column packed with C18 core-shell particles (2.6 µm particle size, 100 Å) for 10 cm was employed. The separation of analytes was performed with a step gradient in less than 20 min, using 0.5% formic acid aqueous solution and acetonitrile as eluents. The optimized analytical method was validated and the resulting RSD% for intra-day and inter-day repeatability, related to retention time, retention factor and peak area, were below 4.68 and 5.57%, respectively. LOD and LOQ values were as low as 0.78 and 3.12 µg/mL, while linearity, assessed in the concentration range of interest for all analytes, gave R(2)≥0.990. The method was finally applied to the analysis of polyphenols extracted from a collected bee pollen. Nine polyphenols, namely o-, p-coumaric acid, ferulic acid, myricetin, cinnamic acid, quercetin, naringenin, hesperitin and kaempferol, were identified. All analytes, with the exception of p-coumaric acid and myricetin, which partially co-eluted with other pollen components, were also quantified in the sample.

Enantiomeric separations by means of nano-LC.

Enantiomers represent a class of compounds extensively investigated since they can show totally different behaviors when they interact with a chiral environment. Because of their identical chemical structure (they differ only in the spatial arrangement of the atoms in the molecule), the separation of optical isomers is a challenging task of analytical chemistry. So far employed methods for the separation of enantiomers are mainly based on chromatography. CE as well was considered as an analytical technique suitable for chiral separations, characterized by high efficiency and low consumption of reagent. Recently, miniaturization was introduced in LC to answer the needs to perform analyses in the minimum time, to use the smallest amount of samples and to reduce environmental pollution. Nano-LC represents nowadays a valid alternative to the abovementioned conventional analytical techniques, and can be advantageously exploited for enantiomeric separation especially because it needs minute amounts of the chiral material necessary to carry out enantiomeric separations. This review describes the development and applications of nano-LC in the field of chiral separations. The data reported in literature show its relevance for the study enantiomers-chiral selectors interaction, as well as for application in pharmaceutical and clinical research.

Cyclodextrins as a chiral mobile phase additive in nano-liquid chromatography: comparison of reversed-phase silica monolithic and particulate capillary columns.

Enantioseparations of racemic nonsteroidal anti-inflammatory drugs (naproxen, ibuprofen, ketoprofen, flurbiprofen, suprofen, indoprofen, cicloprofen, and carprofen) were performed by nano-liquid chromatography, employing achiral capillary columns and heptakis(2,3,6-tri-O-methyl)-ß-cyclodextrin (TM-ß-CD) or hydroxylpropyl-ß-cyclodextrin (HP-ß-CD) as a chiral mobile phase additive (CMPA). Working under the same experimental conditions (in terms of mobile phase and linear velocity), the performance of a RP-C18 monolithic column was compared with that of a RP-C18 packed column of the same dimensions (100 µm i.d. × 10 cm). Utilizing a mobile phase composed of 30% ACN (v/v) buffered with 50 mM sodium acetate at pH 3, and containing 30 mM TM-ß-CD, the monolithic column provided faster analysis but lower resolution than the packed column. This behavior was ascribed to the high permeability of the monolithic column, as well as to its minor selectivity. HP-ß-CD was chosen as an alternative to TM-ß-CD. Employing the monolithic column, the effects of different parameters such as HP-ß-CD concentration, mobile phase composition, and pH on the retention factor and the chiral resolution of the analytes were studied. For the most of the analytes, enantioresolution (which ranged from R(s) = 1.80 for naproxen to R(s) = 0.86 for flurbiprofen) was obtained with a mobile phase consisting of sodium acetate buffer (25 mM, pH 3), 10% MeOH, and 15 mM HP-ß-CD. When the same experimental conditions were used with the packed column, no compound eluted within 1 h. Upon increasing the percentage of organic modifier to favor analyte elution, only suprofen eluted within 30 min, with an R(s) value of 1.14 (20% MeOH). Replacing MeOH with ACN resulted in a loss of enantioresolution, except for naproxen (R(s) = 0.89).

Analysis of polyphenols and methylxantines in tea samples by means of nano-liquid chromatography utilizing capillary columns packed with core-shell particles.

In this study, a rapid separation of eleven polyphenols and three methylxanthines was obtained by means of nano-liquid chromatography (nano-LC), employing a 100 µm I.D. capillary column packed with C18 core-shell particles (2.7 µm) for 10 cm. All compounds were baseline resolved with a step gradient elution in less than 15 min. The developed analytical method was validated and the resulting RSD% for intra-day and inter-day repeatability, related to retention time, retention factor and peak area, were below 5.1 and 5.7%. LOD and LOQ values corresponded to 0.300 and 0.625 µg/mL, while linearity range assessed gave R² no lower than 0.990. Then, the method was used to determine studied compounds in tea extracts. Further, the nano-LC system was coupled with a mass spectrometer to confirm the components present in real samples. Finally the results were compared with those obtained using a capillary column (100 µm I.D. × 10 cm) packed with C18 sub-2 µm particles, applying the same nano-LC experimental conditions.

Fast-liquid chromatography using columns of different internal diameters packed with sub-2 µm silica particles.

Capillary columns (50, 75 and 100 µm I.D.) were packed with silica C18 sub-2 µm particles for 50mm and were employed in nano-liquid chromatography (nano-LC) for fast chromatographic separations. Mixtures of nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids were used as models. Separations no longer than 3 min were obtained with the three capillary columns employing a mobile phase of 35/65 (v/v) and 45/55 (v/v) ACN/H(2)O in 0.1% formic acid (HFo) for steroids and NSAIDs, respectively. Among the capillary columns used, best results, in terms of retention factor and selectivity, were achieved with the 50 µm I.D. column. The same sample mixtures were analyzed by micro-liquid chromatography (µ-LC), employing a commercial microbore column Hypersil GOLD™ (50 mm × 1.0 mm I.D.) packed with C18 1.9 µm particles, and working at the same linear velocity. The results were compared with those obtained with the 50 µm I.D. column in terms of analysis time, efficiency and selectivity. An evaluation of Van Deemter curves was also done for both columns. Finally both nano-LC and µ-LC methods, developed for the separation of the steroid mixture, were validated and applied to the determination of dexamethasone in commercial tablets. Relative standard deviation (RSD%), intra and inter days, of retention time were in the 0.1-1.0% and 2.3-3.5% ranges respectively, for nano-LC, while for µ-LC they were in the 0.6-1.4% and 0.9-1.6% ranges. Peak areas RSD% were also satisfactory (not higher than 6.3% for inter days values). LOD were between 0.010 and 0.040 µg/mL for nano-LC and between 0.1 and 0.5 µg/mL for µ-LC. To assess the linearity of both methods, six concentration levels were injected for three times in two different concentration ranges. Correlation coefficients (r²) of 0.998 and 0.997 were obtained for µ-LC and nano-LC respectively. Good recovery data were also found from analysis of real samples, utilizing 11α-hydroxyprogesterone as internal standard.

Advances in the enantioseparation of ß-blocker drugs by capillary electromigration techniques.

ß-Blocker drugs or ß-adrenergic blocking agents are an important class of drugs, prescribed with great frequency. They are used for various diseases, particularly for the treatment of cardiac arrhythmias, cardioprotection after myocardial infarction (heart attack), and hypertension. Almost all ß-blocker drugs possess one or more stereogenic centers; however; only some of them are administered as single enantiomers. Since both enantiomers can differ in their pharmacological and toxicological properties, enantioselective analytical methods are required not only for pharmacodynamic and pharmacokinetic studies but also for quality control of pharmaceutical preparations with the determination of enantiomeric purity. In addition to the chromatographic tools, in recent years, capillary electromigration techniques (CE, CEC, and MEKC) have been widely used for enantioselective purposes employing a variety of chiral selectors, e.g. CDs, polysaccharides, macrocyclic antibiotics, proteins, chiral ion-paring agents, etc. The high separation efficiency, rapid analysi,s and low consumption of reagents of electromigration methods make them a very attractive alternative to the conventional chromatographic methods. In this review, the development and applications of electrodriven methods for the enantioseparation of ß-blocker drugs are reported. The papers concerning this topic, published from January 2000 until December 2010, are summarised here. Particular attention is given to the coupling of chiral CE and CEC methods to MS, as this detector provides high sensitivity and selectivity.

Multi-walled carbon nanotubes-dispersive solid-phase extraction combined with nano-liquid chromatography for the analysis of pesticides in water samples.

In this work, the simultaneous separation of a group of 12 pesticides (carbaryl, fensulfothion, mecoprop, fenamiphos, haloxyfop, diclofop, fipronil, profenofos, fonofos, disulfoton, nitrofen, and terbufos) by nano-liquid chromatography with UV detection is described. For the analyses, a 100 µm internal diameter capillary column packed with silica modified with phenyl groups was used. Experimental parameters, including the use of a trapping column for increasing the sensitivity, were optimized and validated. A preliminary study of the applicability of a rapid and practical dispersive solid-phase extraction (DSPE) procedure was developed for the extraction of some of these pesticides (carbaryl, fensulfothion, fenamiphos, fipronil, profenofos, fonofos, disulfoton, nitrofen, and terbufos) from Milli-Q water samples using multi-walled carbon nanotubes (MWCNTs). The method was validated through a recovery study at three different levels of concentration, obtaining limits of detection in the range 0.016-0.067 µg/L (below European Union maximum residue limits) for the majority of the pesticides. In this work, MWCNTs were reused up to five times, representing an important reduction of the waste of stationary phase. Furthermore, DSPE permitted a clear diminution of the total sample treatment time with respect to conventional SPE.

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode.

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100 µm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50°C. Linear correlation of lnk vs. 1/T was observed for all the columns; ΔH° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation. To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.

Capillary electrochromatography as a new tool to assess drug affinity for membrane phospholipids.

This work proposes a new capillary electrochromatography (CEC) method for determination of drug partition in membrane phospholipids. CEC experiments were carried out in a 100 µm (ID) fused-silica capillary, partially packed with a chromatographic phospholipid stationary phase, so-called Immobilized Artificial Membrane, IAM.PC.DD2. The observed retention values were corrected by both the electro-osmotic and electrophoretic mobility values, measured by capillary electrophoresis (CE) experiments, assuming the values of the logarithms of "chromatographic" affinity factors, log k(CEC) as indexes of affinity for phospholipids. Analogously to biochromatography, all the values were determined with a totally aqueous mobile phase, or extrapolated to 100% aqueous buffer. The analytes were 16 structurally unrelated compounds, of basic, neutral, and acidic nature. To evaluate the effectiveness of CEC data to describe partition in phospholipids, log k(CEC) were related to both log P and log k(w)(IAM) values. log P are the lipophilicity values expressed as the logarithms of n-octanol/water partition coefficients and log k(w)(IAM) are the retention data measured by High Performance Liquid Chromatography (HPLC) on an IAM.PC.DD2 column, assumed as the reference values for phospholipid affinity. Phospholipid affinity scale by CEC related to that achieved by HPLC, but only if two different subclasses were considered separately, i.e. protonated and unprotonated analytes; indeed, all the compounds protonated at the experimental pH value (7.0) were retained stronger in CEC than in HPLC. This discrepancy may be due to the use of different buffers in CEC and HPLC since, to avoid the occurrence of a high current, the eluent in CEC experiments was of different composition and lower ionic strength than in HPLC. CEC analyses were faster and required lower amounts of both solvent and stationary phase than HPLC; moreover, with the exception of only three analytes, all analyses were performed with 100% aqueous eluents avoiding time-consuming and tedious extrapolation procedures.

Analysis of Aloe-based phytotherapeutic products by using nano-LC-MS.

This article proposes a chromatographic method for the analysis of extracts of Aloe plants. The method was developed with a laboratory assembled nano-LC system coupled with a UV detector, followed by an IT-mass spectrometer. With a step gradient mode of ACN/H(2)O mixtures and employing a capillary column packed with C(18) (100 µm id), a complete separation of the following anthrones was achieved: aloin (in its two isomeric forms A and B), 5-hydroxyaloin and 7-hydroxyaloin (in its two isomeric forms A and B). The optimized nano-LC-MS method was validated for the quantification of aloin, the main component of Aloe with known pharmacological activities. RSD values obtained for retention time and peak areas were 1.3 and 12.1%, respectively. LOD and LOQ values of 0.4 and 1.5 µg/mL were obtained for each aloin isomer. The method was applied to the analysis of Aloe vera and A. ferox extracts in order to acquire a fingerprint, characteristic for each plant. Several phenolic compounds were detected by UV and identified by MS. A. vera and A. ferox showed different profiles and it was possible to discriminate them. Several commercial formulations, declared to contain Aloe extracts, were analyzed. Comparing their chromatograms with those obtained from A. vera and A. ferox, it was possible to recognize the Aloe species and to determine aloin.

CEC-ESI ion trap MS of multiple drugs of abuse.

This article describes a method for the separation and determination of nine drugs of abuse in human urine, including amphetamines, cocaine, codeine, heroin and morphine. This method was based on SPE on a strong cation exchange cartridge followed by CEC-MS. The CEC experiments were performed in fused silica capillaries (100 microm x 30 cm) packed with a 3 mum cyano derivatized silica stationary phase. A laboratory-made liquid junction interface was used for CEC-MS coupling. The outlet capillary column was connected with an emitter tip that was positioned in front of the MS orifice. A stable electrospray was produced at nanoliter per minute flow rates applying a hydrostatic pressure (few kPa) to the interface. The coupling of packed CEC columns with mass spectrometer as detector, using a liquid junction interface, provided several advantages such as better sensitivity, low dead volume and independent control of the conditions used for CEC separation and ESI analysis. For this purpose, preliminary experiments were carried out in CEC-UV to optimize the proper mobile phase for CEC analysis. Good separation efficiency was achieved for almost all compounds, using a mixture containing ACN and 25 mM ammonium formate buffer at pH 3 (30:70, v/v), as mobile phase and applying a voltage of 12 kV. ESI ion-trap MS detection was performed in the positive ionization mode. A spray liquid, composed by methanol-water (80:20, v/v) and 1% formic acid, was delivered at a nano-flow rate of approximately 200 nL/min. Under optimized CEC-ESI-MS conditions, separation of the investigated drugs was performed within 13 min. CEC-MS and CEC-MS(2) spectra were obtained by providing the unambiguous confirmation of these drugs in urine samples. Method precision was determined with RSDs values or=0.995). The developed CEC-MS method was then applied to the analysis of drugs of abuse in spiked urine samples, obtaining recovery data in the range 80-95%.

Optical isomer separation of flavanones and flavanone glycosides by nano-liquid chromatography using a phenyl-carbamate-propyl-beta-cyclodextrin chiral stationary phase.

In this paper a phenyl-carbamate-propyl-beta-cyclodextrin stationary phase was employed for the enantioseparation of several flavonoids, including flavanones and methoxyflavanones by using nano-liquid chromatography (nano-LC). The same stationary phase was also used for the diastereoisomeric separation of two flavanone glycosides. The compounds: flavanone, 2'-hydroxyflavanone, 4'-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, 4'-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, hesperetin, hesperidin, naringenin and naringin were studied using reversed, polar organic and normal elution modes. The effect of the nature and composition of the mobile phase (organic modifier type, buffer and water content in the reversed phase mode) on the enantioresolution (R(s)), retention factor (k) and enantioselectivity (alpha) were investigated. Baseline resolution of all studied flavonoids, with the exception of 2'-hydroxyflavanone and naringin, was achieved in reversed phase mode using a mixture of MeOH/H(2)O at different ratios as mobile phase. Good results, in terms of peak efficiency and short analysis time, were obtained adding 1% triethylammonium acetate pH 4.5 buffer to MeOH/H(2)O mixture. The separation of the studied compounds was also performed in polar organic mode. By using 100% of MeOH as mobile phase, the resolution was achieved for the studied analytes, except for 7-hydroxyflavanone, 2'-hydroxyflavanone, naringenin, hesperidin and naringin. Normal mode was tested employing a mixture of EtOH/hexane/TFA as mobile phase achieving the enantiomeric and diastereomeric separation of only hesperetin and hesperidin, respectively. The use of nano-LC technique for the resolution of flavanones optical isomers allowed to achieve good resolutions in shorter analysis time compared to the results reported in literature with conventional HPLC.

Analysis of hesperetin enantiomers in human urine after ingestion of blood orange juice by using nano-liquid chromatography.

Hesperetin (HT) is a flavanone abundantly found in citrus fruits. It has been reported that HT possesses significant antioxidant, anticancer, anti-inflammatory and analgesic activities. This explains the necessity of developing new methods more powerful and sensitive for analyzing HT in biological fluids. Taking into account the chiral nature of HT, the study of the stereospecific kinetics of in vitro and in vivo metabolism and tissue distribution could be a useful tool for further understanding stereoselective biotransformations in human body. A simple nano-liquid chromatographic method for the determination of the enantiomeric composition of hesperetin in human urine was developed. Chiral separation was achieved using a 100 microm I.D. capillary, packed with phenyl-carbamate-propyl-beta-cyclodextrin stationary phase, employing a mobile phase composed by a mixture of triethylammonium acetate buffer (1%, v/v, pH 4.5) and water/methanol (30:70, v/v) at room temperature. The detection was done by using on-column UV detector at 205 nm. Calibration curves were linear in the studied concentration range from 0.25 to 25 microg/mL (r(2)>0.999). Precision assay was <4.5% and was within 3% at the limit of quantification (0.5 microg/mL). The recovery of 7-ethoxycoumarin (IS), R- and S-hesperetin was greater than 82.48%, utilizing a liquid-liquid extraction procedure. The developed method was successfully applied to the determination of hesperetin enantiomers in urine samples obtained from a male volunteer, after the ingestion of 1L of a commercial blood orange juice.

Food analysis: a continuous challenge for miniaturized separation techniques.

One of the current trends of modern analytical chemistry is the miniaturization of the various tools daily used by a large number of researchers. Ultrafast separations, consumption of small amounts of both samples and reagents as well as a high sensitivity and automation are some of the most important goals desired to be achieved. For many years a large number of research laboratories and analytical instrument manufacturing companies have been investing their efforts in this field, which includes miniaturized extraction materials, sample pre-treatment procedures and separation techniques. Among the separation techniques, capillary electromigration methods (which also include CEC), microchip and nano-LC/capillary LC have received special attention. Besides their well-known advantages over other separation tools, the role of these miniaturized techniques in food analysis is still probably in an early stage. In fact, applications in this field carried out by CEC, microchip, nano-LC and capillary LC are only a few when compared with other more established procedures such as conventional GC or HPLC. The scope of this review is to gather and discuss the different applications of such miniaturized techniques in this field. Concerning CE, microchip-CE and CEC works, emphasis has been placed on articles published after January 2007.