Anna Rocco

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.

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.

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.

Analysis of phenolic compounds in extra virgin olive oil by using reversed-phase capillary electrochromatography.

In this work, the simultaneous separation of ten phenolic compounds (protocatechuic, p‐coumaric, o‐coumaric, vanillic, ferulic, caffeic, syringic acids, hydroxytyrosol, tyrosol and oleuropein) in extra virgin olive oils (EVOOs) by isocratic RP CEC is proposed. A CEC method was optimized in order to completely resolve all the analyzed compounds by studying several experimental parameters. The influence of the stationary phase type (C18 and C8 modified silica gel), buffer concentration and pH as well as the organic modifier content of the mobile phase on retention factors, selectivity and efficiency were evaluated in details. A capillary column packed with Cogent® bidentate C18 particles for 23 cm and a mobile phase composed by 100 mM ammonium formate buffer pH 3/H2O/ACN (5:65:30 v/v/v) allowed the baseline resolution of the compounds under study in less than 35 min setting the applied voltage and temperature at 22 kV and 20°C, respectively. A study, evaluating the intra‐ and interday precision as well as LOD and LOQ and method linearity was developed in accordance with the analytical procedures for method validation. LODs were in the range of 0.015–2.5 μg/mL, while calibration curves showed a good linearity (r2 >0.997). The CEC method was applied to the separation and determination of these compounds in EVOO samples after a suitable liquid–liquid extraction procedure. The mean recovery values of the studied compounds ranged between 87 and 99%.

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 μm×30 cm) packed with a 3 μm 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 ∼200 nL/min. Under optimized CEC‐ESI‐MS conditions, separation of the investigated drugs was performed within 13 min. CEC‐MS and CEC‐MS2 spectra were obtained by providing the unambiguous confirmation of these drugs in urine samples. Method precision was determined with RSDs values ≤3.3% for retention times and ≤16.3% for peak areas in both intra‐day and day‐to‐day experiments. LODs were established between 0.78 and 3.12 ng/mL for all compounds. Linearity was satisfactory in the concentration range of interest for all compounds (r2≥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%.

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.

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

β‐Blocker drugs or β‐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 β‐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 β‐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.

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.

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.

Enantiomeric separation of acidic compounds by nano‐liquid chromatography with methylated‐β‐cyclodextrin as a mobile phase additive

Some racemic nonsteroidal anti-inflammatory drugs, namely naproxen, indoprofen, ketoprofen, flurbiprofen, carprofen, cicloprofen, flunoxaprofen and suprofen were separated into their enantiomers by nano-LC. Chiral recognition was achieved adding to the mobile phase heptakis (2,3,6-tri-O-methyl)-beta-cyclodextrin (TM-beta-CD). Capillary columns of 100 microm id, packed with different RP particles were used for experiments. Effect of experimental parameters such as mobile phase composition, stationary phase type and length of packed capillary column on retention factor and chiral resolution of analytes were studied. The stationary phase type played a very important role in the enantiorecognition process. Best results in terms of highest enantioresolution factor and largest number of separated enantiomers were obtained reducing the particles size to 3 microm with RP(18) stationary phase. Most favourable mobile phase for enantiodiscrimination was obtained using relatively low concentrations of ACN (30%, v/v), 30 mM of TM-beta-CD and pH value of 3.0. The retention time of all studied enantiomers decreased by increasing the CD derivative concentration. The retention factors of selected studied compounds, specifically flurbiprofen, naproxen and suprofen, were measured employing TM-beta-CD concentrations in the range 0-40 mM. Assuming a 1:1 enantiomer/CD ratio, the apparent association constants of the studied enantiomers were calculated.