Journal of Chromatography A (v.1104, #1-2)

Detection and quantification of low-molecular-weight aldehydes in pharmaceutical excipients by headspace gas chromatography by Zhong Li; Laura K. Jacobus; W. Peter Wuelfing; Mary Golden; Gregory P. Martin; Robert A. Reed (1-10).
The adverse effect of reactive chemical residues on the quality of drug products has necessitated the determination of low-molecular-weight aldehydes in pharmaceutical excipients. An analytical methodology for the detection of trace amounts of C1–C8 aliphatic aldehydes and benzaldehyde in excipients is described. The proposed procedure is based on the derivatization of aldehydes in aqueous solution with O-2,3,4,5,6-(pentafluorobenzyl) hydroxylamine hydrochloride (PFBHA), followed by static headspace gas chromatographic (SHS-GC) analysis of PFBHA aldehyde oximes with negative chemical ionization (NCI) MS detection. The method developed was demonstrated to be simple, selective, sensitive, and was successfully applied to the screening of aldehydes at sub-μg/g levels in over 30 typical excipients. The most abundant aldehydes found in the samples were formaldehyde and acetaldehyde, for which a rapid and reliable routine quantification method by readily available SHS-GC instrumentation coupled with flame-ionization detection was also developed and validated.
Keywords: Aldehydes; Excipients; Headspace; GC-MS; Negative chemical ionization; Pentafluorobenzylhydroxylamine hydrochloride;

Use of polyoxyethylene surfactants for the extraction of organochlorine pesticides from agricultural soils by Daura Vega Moreno; Zoraida Sosa Ferrera; José J. Santana Rodríguez (11-17).
Two non-ionic surfactant mixtures (POLE and Polyoxyethylene 10 Cetyl ether, POLE and Polyoxyethylene 10 Stearyl ether) have been used for microwave-assisted extraction of six organochlorine pesticides from agricultural soils prior to being determined by HPLC-UV. An experimental design was applied for the determination of variables which affect to recovery and to optimize the extraction parameters, surfactant concentration and volume, microwave time and power. Under the optimized conditions, the method was applied to different soil samples in order to analyze the influence of soil characteristics on the pesticides extraction. The results obtained indicate that most of these compounds can be recovered in good yields with RSD lower than 10% and detection limit ranged between 80 and 800 ng g−1 for the pesticides studied. The proposed method was applied to a reference soil sample and to aged soils.
Keywords: Organochlorine pesticides; Micellar medium; Extraction methods; Microwave; Soils; High performance liquid chromatography;

A continuous flow autoanalyzer for at-line monitoring of total grease and surfactant contents in alkaline degreasing baths is proposed. For this purpose, a simple, robust, automated configuration has been on-line coupled to an universal response detector, such as the evaporative light scattering one. The proposed autoanalyzer constitutes an advantageous alternative to manual procedures and achieves the determination of both indices in ca. 15 min. The parameters thus obtained can be used to evaluate the degree of exhaustion of the bath as therefore allows the timely decision-making about reusability, maintenance or renewal. The fractionation model has been validated using attenuated total reflection-Fourier transform-infrared (ATR-FT-IR) spectroscopy. The potential of the method was realized by applying it to the analysis of 15 real samples obtained from precleaning and cleaning alkaline baths from a production line of a metal industry.
Keywords: Industrial alkaline degreasing baths; Total grease and surfactant indices; Autoanalyzer; Evaporative light scattering detector;

Based on solid-phase microextraction (SPME) and on-fiber silylation, a method for simultaneous determinations of exogenous endocrine disrupting chemicals (EDCs) and endogenous steroid hormones in environmental aqueous and biological samples by gas chromatography–mass spectrometry (GC–MS) was developed. The selected target compounds were: octylphenol (OP), technical grade nonylphenol (t-NP), diethylstilbestrol (DES), dehydroisoandrosterone (DEHA), estrone (E1), 17β-estradiol (E2), testosterone (T) and pregnenolone (PREG). The optimization of operating conditions influencing the performances of SPME and derivatization were studied in detail. The average correlation coefficient of the calibration curves of the target compounds was 0.9968 and the linear ranges of most compounds spanned over three orders of magnitude. The LOD/(LOQ) values of the target compounds in river water and blood serum were in the range of 0.002–0.378/(0.008–1.261) μg L−1 and 0.004–0.474/(0.013–1.579) μg L−1, respectively, which were a bit higher than those in the pure water due to matrix effects. The developed method was applied to the determinations of target compounds in real samples. Exogenous OP, t-NP and DES were at 0.15, 4.67 and 0.02 μg L−1 in river water and 3.21, 12.17 and 0.15 μg L−1 in fish blood serum. Natural steroid hormones E1, E2 and T were at 0.18, 0.10 and 5.55 μg L−1 in river water, and in female fish serum, E1, E2 and PREG were at 1.61, 1.08 and 4.58 μg L−1, respectively. The proposed SPME method was compared with traditional SPE procedure and the results found using both methods were in the same order of magnitude and both are quite agreeable.
Keywords: Endocrine disrupting chemicals (EDCs); Steroid hormones; Solid-phase microextraction (SPME); Derivatization; Gas chromatography–mass spectrometry (GC–MS);

A simple liquid–liquid–liquid microextraction with automated movement of the acceptor and the donor phase (LLLME/AMADP) technique is described for the quantitative determination of five phenoxyacetic acids in water using a disposable and ready to use hollow fiber. The target compounds were extracted from the acidified sample solution (donor phase) into the organic solvent residing in the pores of the hollow fiber and then back extracted into the alkaline solution (acceptor phase) inside the lumen of the hollow fiber. The fiber was held by a conventional 10-μl syringe. The acceptor phase was sandwiched between the plunger and a small volume of the organic solvent (microcap). The acceptor solution was repeatedly moved in and out of the hollow fiber assisted by a programmable syringe pump. This repeated movement provides a fresh acceptor phase to come in-contact with the organic phase and thus enhancing extraction kinetics leading to high enrichment of the analytes. The microcap separates the aqueous acceptor phase and the donor phase in addition of being partially responsible for mass transfer of the analytes from donor solution (moving in and out of the hollow fiber from the open end of the fiber) to the acceptor solution. Separation and quantitative analyses were then performed using liquid chromatography (LC) with ultraviolet (UV) detection at 280 nm. Various parameters affecting the extraction efficiency viz. type of organic solvent used for immobilization in the pores of the hollow fiber, extraction time, stirring speed, effect of sodium chloride, and concentration of donor and acceptor phases were studied. Repeatability (RSD, 3.2–7.4%), correlation coefficient (0.996–0.999), detection limit (0.2–2.8 ng ml−1) and enichment factors (129–240) were also investigated. Relative recovery (87–101%) and absolute recoveries (4.6–13%) have also been calculated. The developed method was applied for the analysis of river water.
Keywords: Liquid–liquid–liquid microextraction; Automated movement of the acceptor and the donor phase; Phenoxyacetic acids; River water analysis;

The reliability of SPME combined with a chemical reaction for the analysis of short-chain aliphatic amines by liquid chromatography has been investigated. Different options to couple SPME and derivatization have been tested and compared: (i) derivatization of the analytes in solution followed by the extraction of the derivatives, (ii) extraction of the analytes and subsequent derivatization by immersing the SPME fibre onto a solution of the reagent, and (iii) extraction/derivatization of the analytes using fibres previously coated with the reagent. Methylamine (MA), dimethylamine (DMA) and trimethylamine (TMA) have been selected as a model of primary, secondary and tertiary amines, respectively. The analytes have been derivatized with the fluorogenic reagent 9-fluorenylmethyl chloroformate (FMOC), and the fibre coating was Carbowax-templated resin (CW-TR). The employment of fibres coated with FMOC to extract and derivatize the analytes was the best option, as compared with the other approaches tested the sensitivity was considerably improved. On the basis of these studies, a new procedure for the determination of MA, DMA and TMA in water is presented. To demonstrate the utility of the proposed conditions data on linearity, accuracy, repeatability and sensitivity are given. Results of the determination of the amines in tap, river and waste water are also presented.
Keywords: SPME; Derivatization; Amines; FMOC; Liquid chromatography;

Optimisation of stir bar sorptive extraction applied to the determination of volatile compounds in vinegars by Enrique Durán Guerrero; Ramón Natera Marín; Remedios Castro Mejías; Carmelo García Barroso (47-53).
Stir bar sorptive extraction (SBSE) was evaluated for analysing volatile compounds in vinegar. The extraction and desorption analytical conditions have been optimised using a two-level factorial design expanded further to a central composite design. This chemometric tool is very appropriate in screening experiments where the aim is to investigate several possibly influential and/or interacting factors. For the extraction step, the optimum analytical conditions were: sample volume 25 ml without dilution, sampling time 120 min, NaCl content 5.85 g, and stirring speed 1250 rpm. For the desorption step, the optimised analytical conditions were: desorption temperature 300 °C, cryofocusing temperature −140 °C, flow of helium 75 ml min−1, and desorption time 10 min. The SBSE procedure developed shows detection limits, and linear ranges adequate for analysing this type of compounds. The repeatability values obtained were lower than 10%.SBSE is a very simple, solvent-free, fast technique with better sensitivities, in general, than SPME. However, a disadvantage of this technique is that, up to now, the stir bar offers a limited enrichment capability for polar compounds because is only available with PDMS coating.
Keywords: Stir bar sorptive extraction; Vinegar; Volatile compounds; Two-level factorial design; Central composite design;

A variety of degradation products are produced upon dilute acid pretreatment of lignocellulosic biomass. Within this larger construct, organic acids, phenols and aromatic aldehydes represent important compound classes to investigate due to increasing evidence of their inhibitory effect on fermentative microorganisms. An analytical extraction procedure is presented, enabling isolation of potential analytes away from alternative products in biomass hydrolysates. Additionally, a reversed-phase high-performance liquid chromatography (HPLC) method has been developed and validated, affording simultaneous separation and quantitative determination of 32 potential analytes in water with UV detection at 210 nm. The method was subsequently employed to quantify a variety of aliphatic acid, aromatic acid, aldehyde and phenolic degradation products in a corn-stover hydrolysate at concentration levels ranging from 0.02 to 41 mM.
Keywords: Organic acids; Aliphatic acids; Aromatic acids; Aldehydes; Phenols; Biomass hydrolysates; Pretreatment degradation products; HPLC analysis;

Cellulose 3,5-dimethylphenylcarbamates bearing a low content of a vinyl group at the 6-position on the glucose units were synthesized by a previously developed regioselective method and chemically immobilized onto a vinylized silica gel as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC). The immobilization of the derivatives was performed through a radical polymerization reaction with AIBN as the initiator in the presence of toluene. The effects of vinyl monomers, such as isoprene, 2,3-dimethyl-1,3-butadiene (DMBD), ethylene glycol dimethacrylate (EDMA) and 1,5-hexadiene, on the immobilization and enantioselectivities of the derivatives were investigated. The effect of the temperature used for the radical polymerization reaction on the immobilization was also examined. In addition, the direct comparison of the chiral recognition abilities of the laboratory-made and commercially available columns was discussed.
Keywords: Cellulose derivative; Immobilization; Chiral stationary phase; Enantiomer; Radical polymerization; Chiral separation;

High-speed counter-current chromatography (HSCCC) was applied to the separation and purification of mangiferin, neomangiferin, cis-hinkiresinol and (-)-4′-O-methylnyasol from the Chinese medicinal herb Rhizoma Anemarrhenae. Five hundred milligrams of crude extracts were separated by using n-butanol–acetic acid (1%) (1:1, v/v) as the two-phase solvent system and yielded 35.3 mg of neomangiferin and 245.4 mg of mangiferin. During this separation, cis-hinkiresinol and (-)-4′-O-methylnyasol were still maintained in the stationary phase. The stationary phase was collected, evaporated to dryness and separated with light petroleum–ethyl acetate–methanol–water (1:1:1.2:0.8, v/v) and 1:1:1.4:0.6 (v/v) in gradient elution, which yielded 17.2 mg of cis-hinkiresinol and 12.4 mg of (-)-4′-O-methylnyasol. The purities of mangiferin, neomangiferin, cis-hinkiresinol and (-)-4′-O-methylnyasol were 96.3, 98.0, 97.3 and 98.2%, respectively, as determined by HPLC. The chemical structures of these components were identified by 1H NMR and 13C NMR.
Keywords: Rhizoma Anemarrhenae; HSCCC; Neomangiferin (7-O-β-d-glucopyranosyl-mangiferin); Mangiferin; cis-Hinkiresinol; (-)-4′-O-Methylnyasol;

A retention model based on stoichiometric approach has been developed in order to describe analyte retention of anions on latex-based pellicular ion exchanger. The chromatographic process entails two stepwise and complex equilibria, first is ion-pair forming of analyte or eluent ion with ion-exchange sites under the effect of electrostatic forces due to the sulfonic layer behind the aminated functional groups of stationary phase. Second component is the ion-exchange between the analyte and eluent ions. As a new parameter of the fractional electrostatic coefficient of the ion exchange capacity was introduced to develop retention profiles of anions. Analysis of the dependence of the capacity factors on the eluent concentrations at different values of fractional coefficient shed light on the possible complex mechanism. Extensive experimental retention data were obtained for 14 anions (formate, acetate, propionate, pyruvate, lactate, chloride, nitrate, oxalate, malonate, succinate, tartarate, fumarate, maleate, sulphate) using hydroxide eluents of varying concentration. The ion-pair formation and ion-exchange selectivity constants for analyte and eluent species are determined using derived retention equation from experimental data by nonlinear iterative calculation. The model was utilized to predict retention data under elution conditions of practical importance. The predicted and obtained retention factors are in good agreement, which confirms the predictive power of the model.
Keywords: Anion chromatography; Ion-pair forming; Ion exchange; Electrostatic potential; Equilibrium constants; Retention modeling;

Partition coefficients for 69 varied compounds were determined for the n-hexane–acetonitrile partition system and combined with 74 partition coefficients for (largely) terpenes, esters and alkylaromatic compounds determined by Isidorov and coworkers and 27 extraction p-values determined by Bowman and Beroza to derive a general model for the distribution of neutral compounds in the biphasic system. The partition coefficients, log  K p, were correlated through the solvation parameter model giving log  K p  = 0.097 (± 0.049) + 0.189 (±0.041)  E  − 1.332 (±0.056)  S  − 1.649 (±0.055)  A  − 0.966 (±0.074)  B  + 0.773 (±0.040)  V with a multiple correlation coefficient of 0.985, standard error of the estimate 0.114, and Fischer statistic 1087. The solute descriptor E is the excess molar refraction, S is the dipolarity/polarizability, A and B are the overall hydrogen-bond acidity and basicity, respectively, and V is McGowan's characteristic volume. The model is expected to be able to estimate further values of the partition coefficient to about 0.1 log units and is applicable to a wide range of compounds except for n-alkylcarboxylic acids, which have higher partition coefficients than predicted, most likely due to the formation of oligomers (e.g. dimers) in the n-hexane layer.
Keywords: Solvation parameter model; Partition coefficients; n-Hexane; Acetonitrile;

A perfusion reversed-phase high performance liquid chromatography (RP-HPLC) method has been designed to allow rapid (3.4 min) separations of maize proteins with high resolution. Several factors, such as extraction conditions, temperature, detection wavelength and type and concentration of ion-pairing agent were optimised. A fine optimisation of the gradient elution was also performed by applying experimental design. Commercial maize products for human consumption (flours, precocked flours, fried snacks and extruded snacks) were characterised for the first time by perfusion RP-HPLC and their chromatographic profiles allowed a differentiation among products relating the different technological process used for their preparation. Furthermore, applying discriminant analysis makes it possible to group the samples according with the technological process suffered by maize products, obtaining a good prediction in 92% of the samples.
Keywords: Food analysis; Maize; Multivariate analysis; Perfusion chromatography; Proteins;

A new approach to the construction and similarity analysis of chromatographic fingerprint for herbal medicine is presented in this paper. Samples of Chuanxiong, a herbal medicine for headache, from three producing areas of China were used to evaluate the utility of this study. The samples were analyzed with high-performance liquid chromatography (HPLC) and the peak areas of the chromatograms were used to construct the fingerprints of the herbal medicines. A vector of differences was defined between the two fingerprints. The scalar mean of the difference vector was taken as a statistic and both the t-test and Bayesian hypothesis testing were implemented to provide a one-to-one comparison of the fingerprints. Compared with principal component analysis (PCA), correlation coefficient and vector cosine, the new method offers a better differentiation of the similarity or difference between the fingerprints from same sample of Chuanxiong. When the new method was used in the similarity analysis of the fingerprints of Chuanxiong from different production areas, a clear-cut signature was obtained that reveals the significant difference between them.
Keywords: Fingerprint of herbal medicine; Chromatography; t-Test; Bayesian theorem;

An original method based on LC-MS for determination of the flame retardant tetrabromobisphenol A (TBBPA) in air is presented, as an alternative to the traditionally used GC-MS. The soft ionization in LC-MS makes it possible to monitor the intact molecule and to use 13C-labelled TBBPA as an internal surrogate standard, two features that improve both accuracy and precision of the analyses. Comparison of different acquisition modes in electrospray ionization showed that the lowest detections limit, 3.1 pg TBBPA injected, was obtained in SIM monitoring the molecular ions 542.7/544.7. A fragmentation pathway of TBBPA in LC-ESI-MS is suggested. The only sample clean-up steps required are solvent reduction and filtration of the sample extract. Recoveries were 93% at a 30 ng level and 75% at 3 ng. The new method was tested by analyses of air samples collected at a recycling plant for electronic equipment. The amount of TBBPA found was 13.8 ng/m3 with an RSD of 5.9%. Furthermore, it was found that TBBPA in a standard solution could be partially debrominated, if not carefully protected from light during storage.
Keywords: Air sampling; Tetrabromobisphenol A; TBBPA; Flame retardant; LC-MS;

A high-performance liquid chromatography–diode array detection (HPLC–DAD) method and a liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) method were developed to screen for the presence of synthetic phosphodiesterase-5 (PDE-5) inhibitors and their analogues, namely sildenafil, vardenafil, tadalafil, homosildenafil, acetildenafil and hydroxyhomosildenafil. The methods were applied to pre-market samples submitted to the Health Sciences Authority of Singapore (HSA) for testing. One sample was in the form of capsules while six other samples were pre-mixed bulk powder samples for dietary supplements to be repackaged or formulated into the final dosage forms (usually capsules). Identification of PDE-5 inhibitors and their analogues was achieved by comparing individual peak retention times, UV spectra and mass spectra with those of reference standards. The seven samples were found to contain at least one of the following compounds: sildenafil, vardenafil, hydroxyhomosildenafil, homosildenafil and acetildenafil. The five compounds were simultaneously determined by LC–ESI–MS/MS in multiple reactions monitoring (MRM) scan mode. The method has been validated for accuracy, precision, linearity and sensitivity.
Keywords: LC–ESI–MS/MS; PDE-5 inhibitor; Dietary supplement; Adulterants; Sildenafil; Vardenafil; Tadalafil; Homosildenafil; Hydroxyhomosildenafil; Acetildenafil;

Data-directed scan sequence for the general assignment of C-glycosylflavone O-glycosides in plant extracts by liquid chromatography-ion trap mass spectrometry by Geoffrey C. Kite; Elaine A. Porter; Fiona C. Denison; Renée J. Grayer; Nigel C. Veitch; Ian Butler; Monique S.J. Simmonds (123-131).
An ion trap LC–MS/MS method is described for the analysis of C-glycosylflavone O-glycosides in crude methanolic extracts of plants. The method employs survey scans with and without the application of up-front collision induced dissociation (CID) to generate diagnostic ions for data-directed MS/MS. The spectra acquired allow assignment of the C-linked sugar to either the C-6 or C-8 position of the aglycone and provide data on the molecular mass of the compound, the number and type of O-linked sugars and the molecular mass of the flavone aglycone. These data for the majority of C-glycosylflavone O-glycosides in an extract are obtained automatically in one LC–MS/MS analysis without manual pre-programming. Key to the assignment of the C-6 or C-8 site of C-glycosylation is the generation, by up-front CID, of the 0,1X+ product ion formed by internal cleavage of the C-linked sugar. MS/MS of this ion is found to have diagnostic value in addition to the 0,2X+ product ion described by other authors. Ion trap MS/MS spectra of [M + H]+ of the 6,8-di-C-glycosylflavones schaftoside and isoschaftoside show an additional and previously unreported diagnostic product ion that is useful in determining the type of sugar at the C-6 position. The product ion spectra of protonated kaempferol 3-O-glucosylrhamnosides show similarities to the spectra of C-glycosylflavone O-glycosides; this is a potential source of confusion if the analysis of such glycosides is limited solely to MS/MS of [M + H]+.
Keywords: C-Glycosylflavones; C-Glycosylflavone O-glycosides; Flavone C-glycosides; Vitexin; Isovitexin; Orientin; Isoorientin; Schaftoside; Isoschaftoside; Multiflorin A; Multiflorin B;

This paper presents a comparison between liquid chromatography with ultraviolet detection (LC–UV), liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods developed for the multiresidue determination of 8 quinolones, around their maximum residue levels (MRLs) in pig muscle. The procedure involves common extraction of the quinolones from the tissues by traditional extraction, a step for clean-up and preconcentration of the analytes by solid-phase extraction (SPE) and a subsequent liquid chromatographic analysis. The methods present satisfactory results of linearity, precision and limits of quantification much lower than the MRLs established by the European Union for quinolones in pig tissues.
Keywords: Liquid chromatography; Mass spectrometry; Pig muscle; Quinolones;

A highly sensitive HPLC with direct fluorescent detection (λ ex  = 235 nm, λ em  = 355 nm) was developed for Pb2+ and Cd2+ complexes with an aromatic polyaminocarboxylate, 1-(4-aminobenzyl)ethylenediamine-N,N,N′,N′-tetraacetate as a pre-column derivatizing agent. A reversed phase partition column pretreated by a cationic surfactant was employed. Although this ligand forms thermodynamically stable complexes with various metal ions, only peaks of Pb2+ and Cd2+ were detected with the ligand-centered emission in the HPLC due to the emissive activity and kinetic stability in the dissociation reaction. The detection limits obtained were 1.5 × 10−8 and 3.3 × 10−9  mol l−1 for Pb2+ and Cd2+, respectively.
Keywords: Lead ion; Cadmium ion; Aromatic polyaminocarboxylate; Dissociation kinetics; Pre-column derivatization; Fluorescent detection;

Detailed characterization of cationic hydroxyethylcellulose derivatives using aqueous size-exclusion chromatography with on-line triple detection by X. Michael Liu; Wei Gao; E. Peter Maziarz; Joseph C. Salamone; John Duex; Erning Xia (145-153).
A more complete understanding of polymeric, cationic cellulose derivatives, including polyquaterium-10 (Polymer JR), has become increasingly important in the eye care industry as thorough characterization of raw materials helps promote product quality and process control. Often such detailed information requires utilization of a combination of analytical techniques. In this work three Polymer JR samples with different viscosities were characterized using aqueous size exclusion chromatography (SEC) with a light scattering detector, a differential viscometer, and a differential refractometer (triple detection). Detailed molecular information such as absolute molecular weights, molecular weight distributions, intrinsic viscosities, and molecular conformations were obtained. One major challenge of analyzing cationic polymers is abnormal size exclusion separation, which could be caused by the ionic interaction between sample molecules and the column packing material. A selection of mobile phases varying in pH, buffer, organic solvent content, and molar concentration of salts was employed to evaluate the correlation of obtained molecular weight values and mobile phase composition. Universal calibration concept was used to examine the abnormal size exclusion separation phenomenon of Polymer JR samples when using different mobile phases. It was observed that the abnormal size exclusion was dependent on both the separation conditions and molecular weights of the samples. Despite the changes in separation parameters and uncharacteristic polymeric structure compared to conventional SEC samples, the use of aqueous SEC with triple detection provided reproducible and valuable molecular information of Polymer JR samples with low to medium molecular weights. By using a combination of high buffer content and adding organic solvent, the abnormal exclusion separation of high molecular weigh Polymer JR could be considerably reduced.
Keywords: Polymer JR; Cationic polymers; Absolute molecular weight; Molecular weight distribution; Intrinsic viscosity; Aqueous size exclusion chromatography; Triple detection;

The high-performance liquid chromatography–infrared spectroscopy (HPLC–IR) technique utilizing on-line flow through cell (FTC) detection has an inherent practical problem: strong absorption bands of the eluent may mask valuable analytical regions of the IR spectrum. The experimentalists’ answer to this challenge is physical elimination of the chromatographic eluent before spectroscopic detection, which however results in off-line measurement of spectra. In the present work, the capabilities of some chemometric algorithms using iteratively applied multi-way methods such as parallel factor analysis (PARAFAC) and PARAFAC2, developed with the aim of overcoming the problems of eluent elimination are examined and evaluated. Test calculations done on simulated liquid chromatographic infrared (LC–IR) data cubes have shown that although PARAFAC2 performs much better than the simple PARAFAC method, it does not give correct decompositions, just like multivariate curve resolution with alternative least squares (MCR–ALS) and related bilinear data based methods. In search for a better solution, a method named objective subtraction of solvent spectrum with iterative use of PARAFAC and PARAFAC2 (OSSS–IU–PARAFAC and OSSS–IU–PARAFAC2) has been developed. Calculations performed with the corresponding Matlab program developed by the authors and run with the appropriate functions in PLS_Toolbox yielded very promising results in evaluations of both simulated and real HPLC–IR data sets, after necessary data pretreatments.
Keywords: HPLC–IR; Flow through cell method; Solvent elimination; PARAFAC and PARAFAC2; OSSS–IU–PARAFAC; Chemometrics;

Routinely applied at both preparative and analytical scales, chiral ligand-exchange chromatography (CLEC) separates enantiomers capable of chelating a divalent transition-metal-ion through a pair of coordinating electronegative atoms. CLEC separation efficiencies are strongly dependent on column operating conditions, including temperature and mobile-phase solvent composition. Although previous empirical studies provide some useful guidelines for optimizing column operating conditions, the fundamental mechanisms underlying the unusually high sensitivity of CLEC performance to operating temperature and solvent composition remain poorly understood, limiting efforts to develop a comprehensive model for the technology. To address this problem, we report transport and chemical equilibria data for the separation of α-amino acids on a Nucleosil chiral-1 column presenting l-hydroxyproline as the immobilized ligand. Solute transport is found to be limited by pore diffusion at all column operating temperatures and solvent compositions, validating the existence of local equilibria throughout the column. Changes in separation performance are found to correlate with changes in chemical equilibria, emphasizing the need to carefully account for all speciation within the column when modeling CLEC and providing important fundamental data to achieve this goal. Each enantiomer participates in a large number of solution-phase complexes. As a result, the thermodynamic driving force for separation is unusually complex, allowing subtle changes in column operating conditions to mediate significant changes in speciation profiles and separation efficiency. A reaction-equilibria model accounting for all speciation within the CLEC column is proposed and used to estimate enantiomer partition coefficients and retention times.
Keywords: Chiral ligand-exchange chromatography (CLEC); Separations; Chemical reaction equilibria; Equilibrium formation constants;

Volatile organic amine was used as the mobile phase addictive during the separation of four bisphosphonates (alendronate, pamidronate, zoledronic acid and etidronate). An isocratic liquid chromatography method with evaporative light-scattering detection (ELSD) was developed for these bisphosphonates which are not retained on non-polar column and lack chromophore for detection. The analytes have sufficiently separated from each other on a Phenomenex C18 column. The effects of mobile phase composition and instrumental parameters of ELSD were studied. This newly developed method enables direct measurement for analysis of bisphosphonates without the need of derivatization. This developed method provides high separation and specificity to bisphosphonate analysis. In quantitative analysis, the method showed satisfactory precision (less than 2.8%) and accuracy (higher than 94.4%), good linearity (r  = 0.9991–0.9997) and sufficient sensitivity (15–18 μg/ml). It can be easily and conveniently adopted for the routine quality control analysis.
Keywords: Bisphosphonate; Column liquid chromatography; Evaporative light-scattering detector;

A method for the simultaneous quantification of reduced and oxidized glutathione in human plasma employing a two-dimensional chromatographic system with parallel porous graphitized carbon (PGC) columns coupled with fluorescence (FLD) and coulometric electrochemical detection (ED) has been developed. Post-sampling oxidation of reduced glutathione (GSH) was prevented by derivatizing the –SH group with monobromobimane (MBB) and the glutathione-bimane adduct (GSMB) was detected by FLD. Oxidized glutathione (GSSG) was detected by ED optimized to give lowest possible limits of detection (LOD). The method is fully validated and is currently used for determination of GSH, GSSG and its redox potential in different clinical studies.
Keywords: Glutathione; Electrochemical detection; Hypercarb; Plasma; GSSG; Chromatography;

Micellar liquid chromatography retention model based on mass-action concept of micelle formation by Lidia P. Loginova; Larisa V. Samokhina; Alexander P. Boichenko; Artem U. Kulikov (190-197).
Mass-action model of surfactant micelle formation has been used to develop a conceptual retention model in micellar liquid chromatography (MLC). The retention model bases on the consideration of the changes of the sorbate microenvironment at its transferring from the mobile phase (hybrid micellar eluent) to the stationary phase (a modified surface of alkyl-bounded sorbent). Principal retention equation contains the characteristics of hybrid micelles (critical micelle concentration, degree of counterion binding, partition coefficient of modifier between aqueous solution and micellar pseudo-phase) as well as three fitting parameters. The fitting parameters are an absolute term and coefficients that are equal to the number of molecules of surfactant and modifier, which are attached/detached by sorbate transferring from a hybrid micellar eluent to a modified surface of the stationary phase. On the MLC separation of five antibiotics of rubomicin derivatives and four esters of 4-hydroxybenzoic acid the model of the change of sorbate microenvironment has been tested. The adequateness of model to experimental data has been shown. A simple three-parameter function connecting log  k with log  c S and log  c R that provides a high goodness-of-fit follows from principal retention equation (c S and c R are the molar concentrations of surfactant and organic modifier in the micellar eluent, respectively).
Keywords: Micellar liquid chromatography; Retention model; Mass-action concept of micelle formation; Microenvironment of sorbate; Sodium dodecyl sulfate; 1-Pentanol; iso-Pentanol; Cytostatic rubomicin antibiotics; 4-Hydroxybenzoic acid esters;

Ultrahigh pressure liquid chromatography using elevated temperature by Yanqiao Xiang; Yansheng Liu; Milton L. Lee (198-202).
Fast liquid chromatographic (LC) methods are important for a variety of applications. Reducing the particle diameter (d p) is the most effective way to achieve fast separations while preserving high efficiency. Since the pressure drop along a packed column is inversely proportional to the square of the particle size, when columns packed with small particles (<2 μm) are used, ultrahigh pressures (>689 bar) must be applied to overcome the resistance to mobile phase flow. Elevating the column temperature can significantly reduce the mobile phase viscosity, allowing operation at higher flow rate for the same pressure. It also leads to a decrease in retention factor. The advantage of using elevated temperatures in LC is the ability to significantly shorten separation time with minimal loss in column efficiency. Therefore, combining elevated temperature with ultrahigh pressure facilitates fast and efficient separations. In this study, C6-modified 1.0 μm nonporous silica particles were used to demonstrate fast separations using a temperature of 80 °C and a pressure of 2413 bar. Selected separations were completed in 30 s with efficiencies as high as 220,000 plates m−1.
Keywords: Ultrahigh pressure; Elevated temperature; Fast separation; Mass transfer; Column efficiency;

Determination of steroids in human plasma using temperature-dependent inclusion chromatography for metabolomic investigations by Paweł K. Zarzycki; Kathrin M. Kulhanek; Roger Smith; Vicki L. Clifton (203-208).
Clinical and metabolomic investigations of complex human fluids require cost-effective methodologies that can rapidly assess the steroid hormone milieu of individual samples. The efficiency of quantification of many steroids is limited using immunoassays as these methods can only measure a single component of biological samples and are dependent upon the specificity of the antiserum used in the protocol. In this study, we optimised the solid-phase extraction protocol for the extraction of a range of steroids of varied polarity from estetrol to progesterone from human plasma. The final SPE procedure for efficient extraction of steroids was a washing mixture of 5 ml of 30% methanol and an elution solvent of 2 ml of 100% methanol using 0.5 g C-18 cartridges. This protocol resulted in a high recovery rate, ranging from 85.2 to 99.9% for both the internal standard (7,8-dimethoxyflavone) and steroids of interest. We also improved the separation methodology of our previous work using temperature dependent inclusion chromatography with a mobile phase composition of 35% acetonitrile and 12 mM of β-cyclodextrin at 29 °C. Under these conditions most of the fluid components including estetrol were detected in the first 10 min with progesterone appearing at 43 min. This method is simplistic, inexpensive and reproducible with the capabilities of accurate quantification of steroids. Therefore it could have numerous clinical and metabolomic applications.
Keywords: Solid-phase extraction; Inclusion chromatography; Diode-array detection; Temperature; Cyclodextrin; Estrogens; Progestagens; Steroid quantification; Metabolomics; Cord blood; Human plasma;

Stainless steel electrospray probe: A dead end for phosphorylated organic compounds? by R. Tuytten; F. Lemière; E. Witters; W. Van Dongen; H. Slegers; R.P. Newton; H. Van Onckelen; E.L. Esmans (209-221).
A study of the interaction of phosphorylated organic compounds with the stainless components of a liquid chromatography–electrospray ionisation–mass spectrometry system (LC–ESI–MS) was carried out to disclose a (forgotten?) likely pitfall in the LC–ESI–MS analysis of phosphorylated compounds. The retention behaviour of some representative compounds of different important classes of phosphorylated biomolecules such as nucleotides, oligonucleotides, phosphopeptides, phospholipids and phosphorylated sugars was investigated during their passage through the injector and the stainless steel electrospray capillary. It became clear that the stainless steel components within the LC–ESI–MS setup were able to retain and trap phosphorylated compounds when these compounds were introduced under acidic conditions (0.1% acetic acid). Their release from these stainless steel parts was accomplished by applying an extreme basic mobile phase (25–50% ammonium hydroxide, ca. pH 12). From the data collected one could conclude that the availability of a primary phosphate group appeared imperative but was not always sufficient to realise adsorption on a stainless surface. Furthermore, the number of phosphate moieties seemed to enhance the adsorption properties of the molecules and hence roughly correlated with the analyte fraction lost. Corrosion of the inner surface caused by the mobile phase and the electrospray process was found to be an important factor in the course of these adsorption phenomena.
Keywords: Electrospray; Phosphorylated compounds; Metal affinity; Adsorption; Phosphopeptides; Nucleotides; Oligonucleotides; Phospholipids; Phosphosugars; Corrosion;

Nanoflow liquid chromatography–electrospray ionization tandem–mass spectrometry (nanoLC–ESI–MS–MS) was applied for the characterization of intact phosphatidylcholine (PC) lipid molecules using a homemade reversed phase capillary column with a pulled tip for direct ESI at positive ion mode. Prior to the analytical column, a short capillary trapping column was utilized for on-line pre-concentration via microcross connection. Separation of intact phosphatidylcholines in the nanoflow LC column was carried out using a binary gradient elution method at 300 nL/min. The structures of the eluted PC components were determined by analysis of the typical fragment ions of PC molecules obtained from collision-induced dissociation (CID) after each precursor scan in mass spectrometry. In the current study, nanoflow LC–ESI–MS–MS analysis of PC molecules demonstrated the ability to obtain clear structural information, such as alkyl chain lengths and the degree of unsaturation with a protonated molecule ([M + H]+) and its characteristic fragment ions ([M + H―RCH2COOH]+, [M + H―RCH=C=O]+, and [M + H-184]+). Results from the nanoflow LC–ESI–MS experiment showed the limit of detection at 3.5 fmol for the 14:0/14:0-PC standard. This technique then was applied to intact PC extracts from soybean, bovine brain, and liver without derivatization and resulted in the identification of 28, 25, and 39 phosphatidylcholines, respectively. The LC–MS–MS method has been shown to be useful for the analysis of low concentration PC molecules in biological samples.
Keywords: Nanoflow liquid chromatography; Phosphatidylcholine; Separation of intact phosphatidylcholines; Electrospray ionization; Mass spectrometry; Tandem mass spectrometry; Soybean PC; Bovine brain PC;

A new method of analysis of 11 phenols, including five bibenzyls, three phenanthrenes, and three fluorenones, using high-performance liquid chromatography (HPLC)–diode array detection (DAD) was described. The separation of 11 phenols was effected by RP-HPLC (Beckman Coulter™ ODS column, 5 μm, 250 mm × 4.6 mm) using linear gradient elution systems of acetonitrile–1‰ trifluoroacetic acid (TFA). Satisfactory separation of these compounds was obtained in less than 45 min. The method was validated for linearity, repeatability, limits of detection (LOD) and limits of quantification (LOQ). Good results were obtained with respect to repeatability (relative standard deviation (RSD) < 3.5%) and recovery (85.77–104.92%). The developed method was applied to the simultaneous determination of 11 phenols from totally 31 Dendrobium species (mainly of medicinal plants) as well as other four samples from the similar genera as Pholidota, Flickingeria and Bulbophyllum. The range of the total amounts of bibenzyl, phenanthrene and fluorenone were found to from trace: 4.00, not detected (nd): 0.42 and nd: 0.24 μg mg−1, respectively.
Keywords: Dendrobium; Caulis Dendrobii; Phenols; Quality evaluation; HPLC–DAD;

Looking inside the pores of a chromatographic column by Bernd Trathnigg; Martin Veronik; Alexei Gorbunov (238-244).
The dependence of void volume and pore volume on mobile phase composition was studied on a reversed phase column based on poly(divinyl benzene) in mobile phases consisting of 100% tetrahydrofuran (THF) and 40–90% THF–water. Void volumes V 0 are obtained from a series of polymers with different molar mass by extrapolation of elution volume to M  = 0, the interstitial volume V i from the elution volume of polymers with a sufficiently high molar mass. The pore volume V p is obtained as the difference (V 0  −  V i). While V i does not depend on mobile phase composition, a dramatic decrease of the pore volume is observed, when the mobile phase is changed from THF to THF–water. This effect of the mobile phase composition on the pore volume is observed using PEG and PPG as test macromolecules, regardless the retention mechanism: at 40–50% THF both PEG (in exclusion mode) and PPG (in adsorption mode) experience the same reduced pore volume, which is considerably lower than that in 100% THF.
Keywords: Pore volume; Pore size; Chromatography; Polymer; PEG; PPG; THF; Size exclusion; Adsorption; Reversed phase;

New small-scale cross-axis coil planet centrifuge by Kazufusa Shinomiya; Kazuhiro Yanagidaira; Yoichiro Ito (245-255).
The cross-axis coil planet centrifuge (X-axis CPC) is useful for partitioning macromolecules with aqueous–aqueous polymer phase systems. The floor model we have built with a pair of separation columns had some shortcomings such as requirement of large space, short life of the flow tubes, and difficulty in installing columns. In order to improve the partition efficiency and the utility of counter-current chromatography (CCC), a new small-scale X-axis CPC was designed and fabricated in our laboratory. The down-sizing of the apparatus was done by reducing the scale to about 1/2 of our original model of X-1.5 L type with several improvements. Performance of the apparatus was evaluated on protein separation using an aqueous–aqueous polymer phase system composed of polyethylene glycol 1000 and dibasic potassium phosphate with four multilayer coiled columns. A series of experiments revealed that the combination of right- and left-handed coils produced the best partition efficiencies for both lower and upper mobile phases by selecting the revolution direction. The overall results indicate that the head–tail elution mode substantially affects to the peak resolution and stationary phase retention. This new X-axis CPC would be useful for the separation of various kinds of biologically active compounds.
Keywords: Counter-current chromatography; Instrumentation; Cross-axis coil planet centrifuge; Proteins; Polymer phase system;

Performance limits of monolithic and packed capillary columns in high-performance liquid chromatography and capillary electrochromatography by Sebastiaan Eeltink; Gert Desmet; Gabriel Vivó-Truyols; Gerard P. Rozing; Peter J. Schoenmakers; Wim Th. Kok (256-262).
A method is proposed for the comprehensive characterization and comparison of columns in the high-performance liquid chromatographic (HPLC) and capillary electrochromatographic (CEC) modes. Using this approach, column parameters such as the number of plates, the eddy-diffusion and mass-transfer contributions to peak broadening, the permeability, and the analysis time are incorporated in a single graph and a comparison in terms of efficiency and speed is obtained. The chromatographic performance of silica-based and polymer-based monolithic capillary columns is discussed and a comparison is made with the performance of packed columns. Also, the potential of ultra-high-pressure liquid chromatography is discussed in this context. In the HPLC mode, the best results were obtained with silica monoliths; in the CEC mode, the low-density methacrylate-ester-based monoliths showed the best performance.
Keywords: Column characterization; Performance limits; Microparticulate columns; Silica monoliths; Methacrylate monoliths; Efficiency;

Separation of cationic aracyl derivatives of betaines and related compounds by Malina K. Storer; Christopher J. McEntyre; Michael Lever (263-271).
Cationic aracyl esters of betaines can be formed by alkylation with aracyl halides or trifluoromethanesulfonates. HPLC on a non-endcapped strong cation exchange (SCX) column gave high retention of these derivatives. Cation exchange HPLC may be carried out on a normal-phase (silica or alumina) column using a polar organic solvent (acetonitrile, propan-2-ol) containing an aqueous buffer with an organic cation and a hydrophilic anion. Selectivity is affected by the choice of organic solvent and buffer, e.g. alcohols decrease the retention times of hydroxybetaines such as carnitine. Retention is reduced by increasing the water content and the buffer concentration. Capillary electrophoresis migration times are affected by the choice of buffer anion, with low pH citrate buffers favoured.
Keywords: Betaines; Separation; HPLC; CE; Mobile phase; 2-Naphthacyl triflate;

Size fractionation and characterization of natural colloids by flow-field flow fractionation coupled to multi-angle laser light scattering by M. Baalousha; F.V.D. Kammer; M. Motelica-Heino; H.S. Hilal; P. Le Coustumer (272-281).
Flow-field flow fractionation (FlFFF) coupled to multi-angle laser light scattering (MALLS) was evaluated for size and shape determination of standard spherical and arbitrarily shaped natural colloids. Different fitting methods for light scattering data retrieved from MALLS were evaluated to determine the particle size of spherical standards and natural colloids. In addition, FlFFF was optimized for best fractionation in connection to MALLS, minimal colloids-membrane interaction, and minimal sample losses. FlFFF, calibrated with standard particles, was used to determine hydrodynamic diameter, or radius (D h or R h), of the fractionated colloids, whereas the MALLS was used to determine root mean square radius of gyration (R g) for fractionated colloids. Combining both results, by calculating the R g/R h ratio, allows an estimation of colloid deviation from the shape of homogeneous sphere. Accordingly, this study demonstrates that, FlFFF–MALLS is a valuable technique for characterizing heterogeneous and arbitrarily shaped natural colloidal particles in terms of size and shape. To check the usefulness of FlFFF–MALLS in natural colloid studies, the technique was used to investigate the sedimentation behavior of extracted soil colloidal particles. Results illustrate that, in a silty till sample, carbonates function as cement between the colloidal particles, and consequently, change their sedimentation behavior. On the other hand, carbonate dissolution generates a more homogeneous colloidal sample.
Keywords: FlFFF; MALLS; Natural colloids; RMS radius of gyration; Hydrodynamic radius; Particle shape;

This paper examines geometric scaling models for field flow fractionation systems to understand how channel dimensions affect resolution and retention. Specifically, the changing contribution of the instrumental plate height during miniaturization of field flow fractionation (FFF) systems is reported. The work is directed towards determining the optimal geometrical parameters for miniaturization of field flow fractionation systems. The experimental relationship between channel height in FFF systems and instrumental plate heights is reported. FFF scaling models are modified to: (i) better clarify the dependence of plate height and resolution on channel height in FFF and (ii) include a more complete geometrical scaling analysis and model comparison in the low retention regime. Electrical field flow fractionation has been shown to benefit from miniaturization, so this paper focuses on that subtype, but surprisingly, the results also indicate the possibility of improvement in performance with miniaturization of other field flow fractionation systems including general FFF subtypes in which the applied field does not vary with channel height. This paper also discusses the potential role of more powerful microscale field flow fractionation systems as a new class of sample preparation units for micro-total-analysis systems (μ-TAS).
Keywords: Field flow fractionation; Scaling effects; Electrical field flow fractionation; Microfluidic separations; Micro-total-analysis-systems; Instrumental plate height;

Validation of a one-step extraction/methylation method for determination of fatty acids and cholesterol in marine tissues by Sonnich Meier; Svein A. Mjøs; Horaldur Joensen; Otto Grahl-Nielsen (291-298).
A simple and fast direct extraction/methylation with methanolic hydrogen chloride was validated for determination of fatty acids (FA) in marine tissues. Three parameters: reaction time, temperature and presence of non-polar solvent, were studied by an experimental 23 full factorial design. The method was validated for five different types of samples; cod liver (high lipid content >60%, mainly triacylglycerol), cod muscle (low lipid content, ≈1%, mainly phospholipids), cod plasma (lipid content, ≈2%, mainly lipoprotein complex, high water amount), cod testis (lipid content ≈3%, high levels of cholesterol), and herring muscle (lipid content ≈7%). The one-step procedure for extraction/methylation of wet tissues was compared with the traditional procedure of extraction of the lipids by the Folch method (chloroform/methanol, 2:1, v/v), followed by methylation. The two methods gave similar FA profiles. The one-step extraction/methylation procedure gave a higher recovery of the total FA than the traditional procedure. Problems with carry-over peaks of cholesterol from previous samples were avoided by application of extra long GC temperature programs. The cholesterol decomposed to some degree under the preceding methanolysis step, giving several peaks in the chromatograms. The decomposition peaks were identified by mass spectrometry as cholestdienes originating from dehydration of cholesterol, a metylether of cholesterol and a cholesteryl chloride. These cholesterol artefacts can be used for quantitative determination of cholesterol in the samples. Standard samples of cholesterol were determined with high accuracy, (R 2  > 0.99), and cholesterol in cod plasma was compared with good agreement (R 2  = 0.97) to an enzymatic method.
Keywords: Direct methylation; Folch extraction; Fatty acids; Cholesterol; Fatty acid methyl esters; Marine tissues; Cod (Gadus Morhua); Herring (Clupea harengus L.); Factorial design; Gas chromatography;

An iteration procedure is used to calculate revised solute descriptors for 103 varied compounds suitable for characterizing the retention properties of stationary phases for gas chromatography using the solvation parameter model. The iteration procedure utilizes a database of retention factors obtained on up to 39 open-tubular columns and up to five temperatures in the range 60–140 °C for the 103 solutes. The average of the standard deviation [Σ(log  k exp  – log  k calc)2/(n c  – 1)]0.5 where log  k exp is the experimental retention factor, log  k calc the model predicted retention factor, and n c the total number of retention factors) on all columns is 0.018 for the revised solute descriptors compared with 0.045 for the original values. When used to characterize the retention properties of six open-tubular columns selected to represent different selectivity groups the revised solute descriptors afford improved values for the multiple correlation coefficient and standard deviations of the system constants, and about a three-fold improvement in the standard error of the estimate compared with the original solute descriptors. The revised solute descriptors were used to model retention on the carborane–siloxane copolymer stationary phase Stx-500. This phase has low cohesion, is weakly electron lone pair repulsive, weakly dipolar/polarizable, and weakly hydrogen-bond basic. It has no hydrogen-bond acidity. Its separation properties are similar to those of the poly(diphenyldimethylsiloxane) stationary phases containing 5% diphenylsiloxane monomer, but it is not selectivity equivalent to these phases, being more dipolar/polarizable and a weaker hydrogen-bond base.
Keywords: Solvation parameter model; Gas chromatography; Stationary phase; Solute descriptors;

Analysis of the essential oil composition of eight Anthemis species from Greece by Vasiliki Saroglou; Nikos Dorizas; Zacharias Kypriotakis; Helen D. Skaltsa (313-322).
The volatile composition of eight Anthemis species has been studied. The essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus, and their analyses were performed by GC and GC–MS. Identification of the substances was made by comparison of mass spectra and retention indices with literature records. A total of 284 different compounds were identified and significant qualitative and quantitative differences and similarities were observed among the samples. The main constituents of the investigated populations of each taxon have been revealed as follows: A. altissima: (−)-linalool, trans-caryophyllene, cis-chrysanthenyl acetate; A. auriculata: spathulenol, trans-caryophyllene, β-eudesmol; A. chia: cis-chrysanthenyl acetate, trans-caryophyllene, germacrene-d; A. cotula: germacrene-d, spathulenol A. tinctoria: spathulenol, (−)-caryophyllene oxide, T-cadinol; A. melanolepis: p-cymene, chrysanthenone, trans-verbenol, (−)-caryophyllene oxide; A. tomentosa: (−)-linalool, 1,8-cineole; A. werneri subsp. werneri: nopol, terpineol-4, trans-caryophyllene. Sesquiterpene hydrocarbons were shown to be the main group of constituents of all taxa.
Keywords: Anthemis; Asteraceae; Volatile constituents; (−)-Linalool; cis-Chrysanthenyl acetate; trans-Caryophyllene; (−)-Caryophyllene oxide; Germacrene-d; trans-Verbenol; Spathulenol; GC; GC–MS; Chiral column; Chemotaxonomy;

This paper describes a new extraction method for the determination of aliphatic hydrocarbons (AHs) in soil and sediment samples, using continuous microwave-assisted extraction (MAE) combined with liquid–liquid extraction, for clean-up purposes. Analytical determinations were carried out by gas chromatography coupled with impact ionization mass spectrometry. The influence of the experimental conditions was tested using an agricultural soil spiked with standards (stored at 4 °C for 1 month) as reference soil. Maximum extraction efficiencies (80–90%) were achieved using 0.1–1.0 g of sample, 60 μl of water and 3 ml of n-hexane (extractant) and 5 min of extraction time; less than 70% of the most volatile hydrocarbons (C9–C12) were recovered since many evaporated during the drying step of the sample. MAE was compared with a conventional extraction method such as Soxhlet and a good agreement in the results was obtained (average recovery percentage value of 105% by comparing MAE against Soxhlet). Quality parameters such as linear range (0.5–800 μg/g), limits of detection (LODs) (0.1–0.2 μg/g) and precision (RSD, 4–6%) were determined using spiked soil samples. This method was successfully applied to the analysis of aliphatic hydrocarbons (C9–C27 including pristane and phytane) in contaminated real samples.
Keywords: Soil and sediment; Environmental analysis; Continuous microwave-assisted extraction; Aliphatic hydrocarbons;

The use of a supercritical carbon dioxide reaction medium for the determination of the (R)- and (S)-enantiomers of mandelic acid (MA) is proposed. The process involves a previous derivatization step under supercritical conditions by which the carboxyl group is esterified with methanol, then followed by acylation of the hydroxyl group in methyl MA with pentafluoropropionic anhydride in the absence of a catalyst. These derivatization steps cause no enantiomeric inversion. The derivatized enantiomers are extracted and quantified by gas chromatography. A BETA DEX 225 capillary column allows the separation of (R)-MA and (S)-MA as pentafluoropropionyl methyl esters with good resolution and precision. The overall method was used to determine both enantiomers in urine samples.
Keywords: Enantiomer separation; Supercritical fluid extraction; Gas chromatography; Mandelic acid; Urine samples;

Determination of melatonin (MT) (N-acetyl-5-methoxytryptamine) and related indole compounds using standard capillary electrophoresis (CE) system with UV detection was investigated. Satisfactory separations of six analytes i.e. l-tryptophan (l-TRP), 5-methoxyindoleacetic acid (5-MIAA), 6-hydroxymelatonin (6-HMT), MT, serotonin (SER) and 5-methoxytryptamine (5-MTRA) were performed employing micellar electrokinetic chromatography (MEKC). The optimal background electrolytes (BGE) used for separations were 20 mM tetraborate buffer (pH 9.2) and 20 mM phosphate buffer (pH 3.3) when employing techniques with normal and reverse migration of micelles, respectively. Fifty millimolar sodium dodecyl sulfate (SDS) was employed as the pseudostationary phase and voltage of ±20 kV was used throughout the investigation. On-line preconcentration techniques, stacking and sweeping, were applied in order to overcome high detection limits that are a serious drawback of CE with UV detection. A comparison of used techniques, concerning enhancement factors and limits of detection (LOD), is presented. Obtained results show that the use of stacking with reverse migrating micelles (SRMM) as one of preconcentration techniques allows obtaining the lowest estimated LODs for MT at the level of 30 ng/mL with injection time of 99 s at 0.5 psi. Estimated LODs for other analytes in these conditions were, 21, 26 and 100 ng/mL for l-TRP, 5-MIAA and 6-HMT, respectively. Signals of 5-MTRA and SER obtainable only with 10 s injection allowed reaching estimated LODs of 62.5 and 130 ng/mL, respectively. Analysis of spiked, diluted human serum was carried out as a preliminary application illustration of developed procedure.
Keywords: Melatonin; Capillary electrophoresis; MEKC; Stacking; Sweeping;

A novel method for single-cell analysis was developed by combining electroporation for intracellular immuno-reaction and capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection. Human interferon-gamma (IFN-γ) in natural killer (NK) cells was chosen as the test antigen. Two forms of IFN-γ in single cells could be well separated and detected with a limit of detection of zeptomole. In this assay, the anti-IFN-γ monoclonal antibody labeled with fluorescein isothiocyanate (Ab*) was introduced into NK cells by electrophoration for intracellular immuno-reaction. After completion of the intracellular immuno-reaction, the NK cells were chemically pre-perforated with digitonin to lyse easily. Then, one NK cell containing the complexes of IFN-γ isoantigens with Ab* was electrokinetically injected into the capillary. The cell adsorbed on the tip of capillary was lysed by ultrasonication. Finally, the complexes of the different forms of IFN-γ in the cell were separated and detected by CE–LIF detection.
Keywords: Single-cell analysis; Capillary electrophoresis; Laser-induced fluorescence detection; Immuno-reaction; Interferon-gamma;

Measurement of binding constant by chip electrophoresis is a very promising technique for the high throughput screening of non-covalent interactions. Among the different electrophoretic methods available that yield the binding parameters, continuous frontal analysis is the most appropriate for a transposition from capillary electrophoresis (CE) to microchip electrophoresis. Implementation of this methodology in microchip was exemplified by the measurement of inclusion constants of 2-naphtalenesulfonate and neutral phenols (phenol, 4-chlorophenol and 4-nitrophenol) into β-cyclodextrin by competitive assays. The issue of competitor choice is discussed in relation to its appropriateness for proper monitoring of the interaction.
Keywords: Frontal analysis; Binding constant; Microchip electrophoresis; Competitive assay;

A new type of diglycidyloxy-calix[4]arene coated fiber made by sol–gel method was initially prepared for capillary electrophoresis (CE) sample pretreatment. By using headspace solid-phase microextraction (SPME) combined with a novel back-extraction facility coupled off-line to capillary zone electrophoresis (CZE), the simultaneous determination of propranolol enantiomers in human urine was achieved. The clean up effect and preconcentration effect were realized for the first time without derivatization during the SPME process in terms of these strong polarity and thermal stable compounds. Ultrasonic back-extraction and field amplified sample injection (FASI) technologies were employed. Extraction and back-extraction parameters were optimized. Preconcentration of the sample by calix[4]arene fiber based SPME and FASI increased the sensitivity, yielding a limit of detection (LOD) of 0.01 μg/ml by CZE–diode array detection (DAD). Method repeatability (RSD < 6.5%) and fiber reusability (>150 extraction procedures) were observed over a linear range (0.05–10 μg/ml) in urine samples. Based on the superior thermal stability, high alkali- and solvent-resistant ability, marvelous repeatability and long lifetime of the novel fiber, this SPME–FASI–CZE procedure could meet the demand of minimum required performance limit (MRPL) set by the World Anti-doping Agency (WADA) for the detection of propranolol in urine samples.
Keywords: Calix[4]arene; Sol–gel; Solid-phase microextraction; Capillary electrophoresis; Propranolol enantiomers; Doping analysis;

Tanshinone IIA, the major component extracted from Radix salvia miltiorrhiza, has been observed to possess various kinds of pharmacological activities including antioxidant, prevention of angina pectoris and myocardial infarction and anticancer. Tanshinone IIA was incubated with rat liver microsomes and the resulting metabolites were identified by liquid chromatography/tandem mass spectrometry. The results showed the formation of three main hydroxyl metabolites. The three hydroxyl metabolites of tanshinone IIA were proved to be tanshinone IIB, hydroxytanshinone IIA and przewaquinone A by comparing the tandem mass spectra and the chromatographic retention time with that of the respective authentic compounds. Tanshinone IIB, hydroxytanshinone IIA and przewaquinone A are all the chemical components of total tanshinones. It was reasonable to presume that the three hydroxy metabolites of tanshinone IIA were pharmacologically active the same as tanshinone IIA and the total tanshinones.
Keywords: Tanshinone IIA; Metabolites identification; LC–MS–MS;

Author Index (371-373).

Subject Index (375-384).