Journal of Chromatography A (v.1190, #1-2)

The fundamental difficulties currently impeding the elucidation of retention mechanisms in RPLC are reviewed. The various definitions and conventions concerning void volume and excess adsorption in liquid–solid systems are compared and contrasted. The utility and integrity of various chromatographic methods for the measurement of excess adsorption isotherms of HPLC eluents are discussed. Finally, literature methods for obtaining absolute adsorption data from excess quantities are reviewed and evaluated.
Keywords: Liquid–Solid adsorption; Reversed-phase liquid chromatography;

Weak cation-exchange restricted-access material for on-line purification of basic drugs in plasma by Yoshiaki Sato; Eiichi Yamamoto; Susumu Takakuwa; Takashi Kato; Naoki Asakawa (8-13).
A methylcellulose-immobilized weak cation-exchange (MC-WCX) silica-based restricted-access material (RAM) was developed. The MC-WCX consists of an MC outer surface and 2-carboxyethyl phase internal surface, allowing for direct analysis of basic drugs in plasma. The retention properties of the MC-WCX were evaluated for sulpiride, quinidine, ranitidine, and desipramine. The MC-WCX retained model drugs by cation-exchange, and retained drugs were eluted with the mobile phase containing small amount of acids or salts compared with the MC strong cation-exchanger (MC-SCX). These results indicated the ease of use of the MC-WCX solid-phase extraction (SPE) column when coupled to a reversed-phase analytical column in column-switching high-performance liquid chromatography (HPLC), and various detection principals. Further direct analysis of model drugs in plasma using the MC-WCX SPE column in a column-switching HPLC system successfully performed with sufficient recovery. It is concluded that the MC-WCX is useful for the analysis of basic drugs in plasma.
Keywords: Weak cation-exchange restricted-access material; Plasma; Basic drugs; Column-switching; Solid-phase extraction;

Microwave hydrodiffusion and gravity, a new technique for extraction of essential oils by Maryline Abert Vian; Xavier Fernandez; Franco Visinoni; Farid Chemat (14-17).
A new process design and operation for the extraction of essential oils was developed. Microwave hydrodiffusion and gravity (MHG) is a combination of microwaves for hydrodiffusion of essential oils from the inside to the exterior of biological material and earth gravity to collect and separate. MHG is performed at atmospheric pressure without adding any solvent or water. MHG has been compared with a conventional technique, hydrodistillation (HD), for the extraction of essential oil from two aromatic herbs: spearmint (Mentha spicata L.) and pennyroyal (Mentha pulegium L.) belonging to the Labiatae family. The essential oils extracted by MHG for 15 min were quantitatively (yield) and qualitatively (aromatic profile) similar to those obtained by conventional hydrodistillation for 90 min. MHG also prevents pollution through potential 90% of energy saved which can lead to greenhouse gas emission benefits.
Keywords: Microwave; Hydrodiffusion; Earth gravity; Essential oil; Extraction; Mentha spicata; Mentha pulegium;

Protein recognition via ion-coordinated molecularly imprinted supermacroporous cryogels by Nilay Bereli; Müge Andaç; Gözde Baydemir; Ridvan Say; Igor Yu Galaev; Adil Denizli (18-26).
Molecular imprinting is a method for making selective binding sites in synthetic polymers using a molecular template. The aim of this study is to prepare lysozyme-imprinted supermacroporous cryogels which can be used for the purification of lysozyme (Lyz) from egg white. N-Methacryloyl-(l)-histidinemethylester (MAH) was chosen as the metal-coordinating monomer. In the first step, Cu2+ was complexed with MAH and the lysozyme-imprinted poly(HEMA–MAH) [Lyz-MIP] cryogel were produced by free radical polymerization initiated by N,N,N′,N′-tetramethylene diamine (TEMED) in an ice bath. After that, the template (i.e., lysozyme) was removed using 0.05 M phosphate buffer containing 1 M NaCl (pH 8.0). The maximum lysozyme adsorption capacity was 22.9 mg/g polymer. The relative selectivity coefficients of Lyz-MIP cryogel for lysozyme/bovine serum albumin and lysozyme/cytochrome c were 4.6 and 3.2 times greater than non-imprinted poly(HEMA–MAH) (NIP) cryogel, respectively. Purification of lysozyme from egg white was also monitored by determining the lysozyme activity using Micrococcus lysodeikticus as substrate. The purity of the desorbed lysozyme was about 94% with recovery about 86%. The Lyz-MIP cryogel could be used many times without decreasing the adsorption capacity significantly.
Keywords: Cryogels; Molecular imprinting; Molecular recognition; Lysozyme purification; Protein adsorption; Affinity binding;

Ultrasound-assisted emulsification–microextraction of emergent contaminants and pesticides in environmental waters by Jorge Regueiro; Maria Llompart; Carmen Garcia-Jares; Juan C. Garcia-Monteagudo; Rafael Cela (27-38).
The analytical use of ultrasound-generated emulsions has recently found a growing interest to improve efficiency in liquid–liquid extraction since they increase the speed of the mass transfer between the two immiscible phases implied. Thus, dispersed droplets can act as efficient liquid–liquid microextractors in the continuous phase, and later they can be readily separated by centrifugation. A novel method based on ultrasound-assisted emulsification–microextraction (USAEME) and gas chromatography coupled to mass spectrometry (GC/MS) has been developed for the analysis of synthetic musk fragrances, phthalate esters and lindane in water samples. Extraction conditions were optimized using a multivariate approach. Compounds were extracted during 10 min in an acoustically emulsified media formed by 100 μL chloroform and 10 mL sample (enrichment factor = 100). The method performance was studied in terms of accuracy (recovery = 78–114%), linearity (R 2  ≥ 0.9990) and repeatability (RSD ≤ 14%). Limits of detection (LODs) were at the pg mL−1 level for most of compounds, and at the sub-ng mL−1 level for the most ubiquitous phthalate esters. USAEME is proposed as an efficient, fast, simple and non-expensive alternative to other extraction techniques such as SPE, SPME and LPME for the analysis of environmental waters including bottled, tap, river, municipal swimming pool, sewage and seaport water samples. Since no matrix effect has been found for any of the water types analyzed, quantification could be carried out by using conventional external calibration, thus allowing a higher throughput of the analysis in comparison with other microextraction techniques based on equilibrium such as solid-phase microextraction.
Keywords: Ultrasound-assisted emulsification–microextraction; Microextraction; Synthetic musk fragrances; Phthalate esters; Lindane; Factorial experimental design; Water analysis;

“On the Collander equation”: Protein partitioning in polymer/polymer aqueous two-phase systems by Pedro P. Madeira; José A. Teixeira; Eugénia A. Macedo; Larissa M. Mikheeva; Boris Y. Zaslavsky (39-43).
Distribution coefficients of randomly selected proteins were measured in aqueous two-phase systems (ATPSs) formed by different combinations of Dextran-75 (Dex), Ficoll-70, polyethylene glycol-8000 (PEG), hydroxypropyl starch-100 (PES), and Ucon50HB5100 (Ucon, a random copolymer of ethylene glycol and propylene glycol) at particular polymer concentrations, all containing 0.15 M NaCl in 0.01 M phosphate buffer, pH 7.4. Most of the proteins in the PEG-Ucon system precipitated at the interface. In the other ATPSs, namely, PES-PEG, PES-Ucon, Ficoll-PEG, Ficoll-Ucon, and in Dex-PEG and Dex-Ucon described earlier the distribution coefficients for the proteins were correlated according to the solvent regression equation: ln  K i  =  a io  ln  K o  +  b io , where K i and K o are the distribution coefficients for any protein in the ith and oth two-phase systems. Coefficients a io and b io are constants, the values of which depend upon the particular compositions of the two-phase systems under comparison.
Keywords: Aqueous two-phase systems; Protein partitioning; Collander equation;

The environmental fate of phenols represents a diachronic scientific consideration mainly due to their high toxicity and diverse physicochemical properties rendering them difficult to be analyzed as unity. Ion-pair-assisted extraction and microextraction techniques in association with a dedicated derivatization reaction are possible to lead to enhanced selectivity and sensitivity in gas chromatography. Phase-transfer catalytic liquid–liquid extraction–derivatization and ion-pair-assisted single-drop microextraction with in-drop derivatization are successfully employed for the analysis of 15 phenolic compounds. The analytes that react at room temperature with p-toluenesulfonyl chloride into the bulk of the organic phase are subsequently determined by GC–MS in selective-ion monitoring mode. Aiming at maximizing the derivatization yields obtained from the 15 analytes in a reasonable time period, the optimum experimental parameters were established along with the figures of merit of the methods. The limits of detection ranged from 0.48 to 1.5 ng/ml and from 0.20 to 0.28 ng/ml respectively, while the limits of quantitation ranged from 1.4 to 4.5 ng/ml and from 0.59 to 0.84 ng/ml for the two methods with the techniques under study. The overall procedure presented satisfactory analytical features with the liquid–liquid extraction protocol being easier to carry out while the single-drop one, presented higher sensitivity and significant reduction of the organic solvent employed. By comparison with other methods for the analysis of phenols, the proposed methods exhibit adequately low detection limits, good precision, short derivatization time and low solvent, sample and reagent consumption.
Keywords: Phenols; p-Toluenesulfonyl chloride; Phase-transfer catalysis; Ion-pair; Single-drop microextraction; Preconcentration; Derivatization;

A microwave-assisted extraction (MAE) method has been developed and optimized for the extraction of six tricyclic antidepressants (TCAD; nordoxepin, nortriptyline, imipramine, amitriptyline, doxepin, dezypramine) from human serum. Optimal parameters of MAE (solvent and extraction temperature) for water solution of these drugs were defined. The microwave-assisted procedure developed was validated by extraction of serum samples at two concentration levels and then successfully applied to the analysis of reference material. Limit of quantification, precision and recovery were found for the studied compounds in the ranges 0.04–0.15 μg/mL, 1.57–4.34% (RSD) and 94–105%, respectively.
Keywords: Microwave-assisted extraction; Tricyclic antidepressants; Biological samples; HPLC; Validation;

Rapid linear scale-up of a protein separation by centrifugal partition chromatography by I.A. Sutherland; G. Audo; E. Bourton; F. Couillard; D. Fisher; I. Garrard; P. Hewitson; O. Intes (57-62).
The scaling up of the separation of two proteins with an aqueous two-phase system (ATPS) from 176 mg with a 500 ml laboratory scale centrifugal partition chromatography (CPC) column to 2.2 g with a 6.25 litre pilot-scale column is presented. A model sample system of a mixture of lysozyme and myoglobin was chosen for this study using an ATPS system comprising 12.5% (w/w) PEG-1000:12.5% (w/w) K2HPO4. It was found that the maximum sample concentration possible without precipitation was 2.2 mg/ml for each constituent. The optimisation of rotor speed, mobile phase flow rate and sample loading was performed on a laboratory-scale device. It was found that a centrifuge speed of 2000 rpm (224 ‘g’), 10 ml/min mobile phase flow rate with a 43 ml (10% of active column volume) sample volume gave optimum operating conditions. This was linearly scaled up to pilot scale by increasing mobile phase flow rate, fraction size and sample loading in the ratio of the system capacities (i.e. 12.5:1). Flow rate was therefore increased from 10 ml/min to 125 ml/min, fraction size from 10 ml to 125 ml and sample loading from 43 ml to 500 ml. Rotor speed however was reduced from 2000 rpm on the laboratory device to 1293 rpm on the pilot-scale device to maintain the same 224 ‘g’ field in each chamber, as the pilot-scale CPC unit has a larger rotor radius than the laboratory one. Resolution increased from Rs = 1.28 on the 500 ml rotor to Rs = 1.88 on the 6.25 litre rotor, giving potential throughputs in batch mode of over 40 g/day.
Keywords: Aqueous two-phase systems, ATPS; Centrifugal partition chromatography, CPC; Counter-current chromatography, CCC; Lysozyme; Myoglobin; Protein separation;

Separation of betalains from berries of Phytolacca americana by ion-pair high-speed counter-current chromatography by Gerold Jerz; Tanja Skotzki; Kathrin Fiege; Peter Winterhalter; Sławomir Wybraniec (63-73).
The first preparative fractionation of betalain pigments by means of ion-pair high-speed counter-current chromatography (IP-HSCCC) from berry extracts of Phytolacca americana (Phytolaccaceae) is presented. A novel HSCCC solvent system consisting of 1-butanol–acetonitrile–water (5:1:6, v/v/v) was applied using ion-pair forming trifluoroacetic acid at low concentration (0.7%, v/v). Affinity of polar betacyanins and betaxanthins to the organic stationary phase of the biphasic HSCCC solvent mixture was considerably improved. Partitioning coefficient values and influence of increasing trifluoroacetic acid additions to the biphasic solvent mixture were measured for all identified betacyanins and betaxanthins. Gentle separation by IP-HSCCC of the injected pigment extract (900 mg) yielded sufficient amounts of the principal pigments 15S-betanin/15R-isobetanin. The pure epimers separated by C18-HPLC were immediately studied by one- and two-dimensional NMR. In the recovered fractions, minor concentrated betacyanins and betaxanthins were significantly enriched by IP-HSCCC and were detected for the first time in the extracts of P. americana. IP-HSCCC and C18-HPLC were shown to be complementary techniques in the isolation procedure of recovering minor concentrated, highly polar and chemically instable betacyanins and betaxanthin from complex plant matrices. Altogether, identification of 17 betalains was achieved by HPLC-diode array detection-electrospray ionization MS/MS in the HSCCC fractions with their respective isomers, also resulting in the tentative elucidation of betacyanins with novel salicylic acid substitution pattern in the berry extracts of P. americana.
Keywords: Betanin; Betalains; Betacyanins; Betaxanthins; Ion-pair counter-current chromatography; IP-HSCCC; HPLC-DAD-ESI-MS/MS; NMR; Phytolacca americana; Phytolaccaceae;

Hexabromocyclododecanes (HBCDs), used as additive brominated flame retardants, are of high concern due to their widespread use and increasing levels in various environmental systems. High-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC–MS/MS) was developed for the determination of HBCD diastereoisomers. A detailed study was carried out to optimize the composition of the mobile phase involving methanol/acetonitrile/water, and the values of MS/MS parameters. It was found that the mobile phase could simultaneously affect the chromatographic separation and sensitivity. The instrumental limits of detection (LODs) on column in this study were 0.5, 0.3 and 0.3 pg for α-HBCD, β-HBCD and γ-HBCD, respectively. The effects of extracted matrix components on HBCD determination were investigated by spiking air and soil sample extracts with three 13C-labelled individual stereoisomers. The results indicated that the responses of the HBCD analysis in air and soils were not significantly affected by matrix effects. The method reported here was further applied to the air and soil samples. Three HBCD diastereoisomers were detected in all the air and soil samples, with levels ranging from 1.2 to 1.8 pg/m3 and 1.7 to 5.6 ng/g dry weight, respectively.
Keywords: Hexabromocyclododecane; Brominated flame retardants; LC–MS/MS; Diastereoisomers; Matrix effects;

By optimizing extraction, separation and analytical conditions, a reliable and accurate high-performance liquid chromatographic (HPLC) method coupled with photodiode array detector (DAD) at room temperature is developed for simultaneous determination of three diarylheptanoids (juglanin A, juglanin B, rhoiptelol) and an α-tetralone derivative (regiolone) in methanol extracts from the green walnut husks (Juglans regia L.) The sample pretreatment process involved the reflux extraction using methanol as the extract with a ratio of liquor to sample of 15 mL/g. The separation was achieved on a SinoChrom ODS-AP C18 column with gradient elution using acetonitrile and 2% (v/v) acetic acid in water. The intra-day and inter-day precision (RSD%) for the analytes ranged from 1.08 to 1.51 and 0.60 to 1.13, respectively. The average recoveries obtained were from 88.4% to 96.2% for the analytes with RSDs below 3.13%. The correlation coefficients of the calibration curve exceeded 0.999. The detection limits were 0.51, 0.25, 0.32 and 0.35 ng at a signal-to-noise ratio of 3, respectively. Quantitative analyses of the samples from different grown sites and in obtained different months showed that the contents of the analytes varied significantly. The method was then successfully applied for the detection and isolation of a new diarylheptanoid derivative in the green walnut husks (J. regia L.). The structure of the new compound was elucidated by various spectroscopic methods including 2D NMR techniques (COSY, HMQC, HMBC), HR-ESI-MS and X-ray single-crystal diffraction analysis.
Keywords: Juglans regia L.; Diarylheptanoids; α-Tetralone; HPLC;

Five new chiral derivatizing reagents 5-hydrazino-2,4-dinitrophenyl-l-alaninamide (HDNP-l-Ala-NH2), 5-hydrazino-2,4-dinitrophenyl-l-phenylalaninamide (HDNP-l-Phe-NH2), 5-hydrazino-2,4-dinitrophenyl-l-valinamide (HDNP-l-Val-NH2), 5-hydrazino-2,4-dinitrophenyl-l-leucinamide (HDNP-l-Leu-NH2) and 5-hydrazino-2,4-dinitrophenyl-l-phenylglycinamide (HDNP-d-Phg-NH2) were synthesized by straightforward two-step synthesis starting from 1,5-difluoro-2,4-dinitrobenzene. Nucleophilic substitution of one fluorine atom in DFDNB with different amino acid amides yielded Marfey's reagent (5-fluoro-2,4-dinitrophenyl-l-alaninamide) and its structural variants (5-fluoro-2,4-dinitrophenyl-l-phenylalaninamide, 5-fluoro-2,4-dinitrophenyl-l-valinamide, 5-fluoro-2,4-dinitrophenyl-l-leucinamide and 5-fluoro-2,4-dinitrophenyl-d-phenylglycinamide). Chiral hydrazine reagents were prepared by nucleophilic substitution of remaining fluorine atom in Marfey's reagent and its variants with hydrazine under basic conditions. These reagents react quantitatively with chiral carbonyl compounds under mild conditions (30 °C, 30 min) to form hydrazone diastereomers. The labeling reaction occurs only in the presence of acid which has a catalytic action and diastereomers have strong absorbance around 348 nm. The separation of diastereomers was tried on a reversed-phase C18 HPLC column using different binary solvent combinations. Excellent separation was achieved in case of cyclic ketones having substitution at α-position. Optimization for derivatization yield, limit of detection, limit of quantification, linearity, accuracy and precision was carried out with respect to HDNP-l-Val-NH2. Studies related to effects of structural modification in reagents and analytes on chromatographic behavior of diastereomers were also analyzed.
Keywords: Chiral hydrazines; Chiral tagging reagents; Chiral carbonyl compounds; Diastereomeric separation; RP-HPLC;

High-performance liquid chromatography separation of enantiomers of flavanone and 2′-hydroxychalcone under reversed-phase conditions by Roberto Cirilli; Rosella Ferretti; Emiliana De Santis; Bruno Gallinella; Leo Zanitti; Francesco La Torre (95-101).
A reversed-phase high-performance liquid chromatography (HPLC) method was developed for evaluating the chiral discrimination ability of Chiralpak IA chiral stationary phase (CSP) towards flavanone. The effect of the nature and pH buffer as well as nature of alcohol modifier on enantioselectivity was investigated. Comparative study of enantioseparation in reversed-phase and polar organic conditions indicated a significative improvement in resolution when aqueous-based eluents were used. The developed reversed-phase chromatographic method was able to separate the enantiomers of flavanone from its isomeric form, the 2′-hydroxychalcone. The stereochemical stability of flavanone was studied by classical off-column HPLC kinetic procedures in aqueous and non-aqueous media. It was clearly demonstrated that the 2′-hydroxychalcone was involved as intermediate in the on-column and off-column enantiomerization process of flavanone.
Keywords: Flavanone; 2′-Hydroxychalcone; Chiral stationary phase; Chiralpak IA; Chiroptical properties; Reversed-phase enantioseparation; Configurational stability; Racemization;

Univariate method for background correction in liquid chromatography–Fourier transform infrared spectrometry by Guillermo Quintás; Bernhard Lendl; Salvador Garrigues; Miguel de la Guardia (102-109).
An univariate method is proposed for background correction in on-line gradient liquid chromatography–Fourier transform infrared (LC–FTIR) spectrometry using acetonitrile:water as mobile phase components. The method is based on the calculation of the ratio of absorbances (AR) at two characteristic wavenumbers for each spectrum. This parameter is subsequently used to locate the most appropriated eluent spectrum within a reference spectra matrix (RSM) to be subtracted from each spectrum included in the sample chromatogram. To correct minor changes in eluent spectra intensity during the elution of analytes, a correction factor (K f), defined as the ratio of the absorbance of the sample and the selected eluent spectrum at a defined wavenumber was determined. The performance of the procedure was evaluated by correcting an on-line gradient LC–FTIR injection of a mixture of two pesticides (Atrazine and Diuron). Using the AR of the absorbance at 2248.6 and 2256.3 cm−1 and a K f at 2248.6 cm−1, the correlation factors between FTIR spectra extracted at the peak apex from the LC–FTIR chromatogram and those obtained from pure pesticide standards were 0.975 and 0.94 for Atrazine and Diuron, respectively.
Keywords: On-line LC–FTIR; Chemometrics; Background correction; Reference spectra matrix;

The key interactions of a chiral solute, norephedrine or 2-amino-1-phenyl-1-propanol (PPA), with three commercially important polysaccharide-based chiral stationary phases, amylose Tris(3,5-dimethylphenylcarbamate) (ADMPC), amylose Tris((S)-α-methylbenzylcarbamate) (ASMBC) and cellulose Tris(3,5-dimethylphenylcarbamate) (CDMPC) are studied in detail using different experimental techniques and molecular simulations. The HPLC retention factors of the enantiomers of PPA in n-hexane/2-propanol (90/10, v/v) at 298 K vary significantly with these sorbents. The enantioselectivities of −PPA versus +PPA are 2.4, 1.0, and 0.8 (reversal in the elution order), respectively. The observed changes in the wavenumbers and the intensities of the amide bands of these polymers in the attenuated total reflection-infrared spectroscopy (ATR-IR) spectra upon absorption of each enantiomer are different. The IR wavenumbers, and the H-bonding interaction energies of the polymer side chains with each enantiomer (polymer–solute) in four different binding configurations are estimated and ranked using the density functional theory (the DFT/B3LYP/6-311+g(d,p) level of theory). X-ray diffraction (XRD) results show that the polymer crystallinity increases significantly upon absorption of each enantiomer. The helical pitches and the inter-rod packing for these polymers are inferred from the XRD results and incorporated into the molecular dynamics (MD) simulations. The elution orders predicted for the enantiomers of PPA using MD simulations of the polymer–PPA binary systems are consistent with the chromatography results. The enantioselectivity observed in ADMPC is hypothesized to be due to having three simultaneous interactions (two H-bond and one π–π) of the polymer with −PPA versus two interactions (one H-bond and one π–π) with +PPA.
Keywords: Amylose; Cellulose; Polysaccharide; Carbamate; Norephedrine; 2-Amino-1-phenyl-1-propanol; Stereo-specific; Chiral chromatography; Chiralpak;

Biodiesel (a mixture of fatty acid esters) is normally analyzed using gas chromatography/flame ionization detection, as specified by the ASTM D6584 and EN14105 standards. This paper proposes a binary gradient method for analyzing biodiesel mixtures using non-aqueous reverse phase HPLC with a UV detector capable of overcoming the drawbacks of the gas chromatographic technique normally used. The new analytical method was developed by means of a statistical sensitivity analysis applied to the main parameters influencing the recording, using the full factorial design method combined with the Yates algorithm and the steepest ascent optimization procedure. The present study shows the influence of the main biodiesel mixture separation analysis parameters. The resulting tool proved valid for analyzing not only biodiesel but also any traces of unreacted oil.
Keywords: Biodiesel; HPLC; Sunflower oil; Factorial design; Optimization; Transesterification;

This paper details the advancements made in the modeling of open column and packed bed pressure-flow. The theoretical description is a one-dimensional elasticity model. By accounting for the loss of intra-particle porosity through empiricism, and by systematically selecting the functional form of the elastic modulus from stress–strain data, this model can accurately predict several kinds of large-scale behavior from small-scale data: packed pressure-flow, open column pressure-flow, and critical velocity. The robustness of the model has been demonstrated for MabSelect, SP 650M, Butyl Sepharose 4 FF and several other agarose-based and polymethacrylate-based resins. The predicted critical velocities are on average within ±5% of observations. A simple modification to the Blake–Kozeny permeability expression allows accurate prediction of packed bed pressure-flow explicitly from compression factor, packed bed height, and settled bed inter-particle porosity. The model provides limits on mobile phase velocity and on operating pressure-flow as a function of bed height, particle size, and resin rigidity, and allows exploration of commercial manufacturing scenarios to identify scalable process time and cycle number.
Keywords: Commercial-scale chromatography; Pressure drop; Scale-up; Modeling; Column packing; Process time; Number of cycles; Protein A; Protein purification;

A novel qualitative analytical method for peak tracking in impurity profiling control by the correlation of spectra was established. Two-dimensional (2D) standard spectrochromatographic data produced by high-performance liquid chromatography with diode array detection (HPLC-DAD) were compared with sample data to develop two-dimensional chromatographic spectral correlative maps. Taking full advantage of separation efficiency of HPLC and spectral specificity of the analytes, the method was successfully used to recognize impurities in quinolone antibacterials, when in combination with relative retention times (RRTs). For the comparison of spectra was expanded to three-dimensional space, simultaneous identification of the chromatographic peaks can be obtained rapidly without preparation and injection of a reference solution, even when the mobile phase changed or the peaks of multi-component samples overlapped.
Keywords: Two-dimensional chromatographic spectral correlative map; HPLC-DAD; Identification; Impurities;

Determination of low-molecular-mass aliphatic carboxylic acids and inorganic anions from kraft black liquors by ion chromatography by Jaana M. Käkölä; Raimo J. Alén; Jukka Pekka Isoaho; Rose B. Matilainen (150-156).
An ion chromatographic (IC) method with suppressed conductivity detection (CD) was developed and validated for the quantitative determination of several low-molecular-mass aliphatic mono- and dicarboxylic acids as their carboxylate anions together with some inorganic anions (chloride, sulfate, and thiosulfate) from kraft black liquors. To confirm the identification of some carboxylate anions which lack commercial model substances, a qualitative IC method with suppressed electrospray ionization mass spectrometry (ESI-MS) was also developed. The separations were performed on an IonPac AS 11-HC anion-exchange column operated at 25 °C within 25 min by a gradient elution with aqueous potassium hydroxide (suppressed CD in the AutoRegen mode) or sodium hydroxide (suppressed ESI-MS in the pressurized bottle mode). In the validation process a mixture of carboxylic acids and inorganic anions in aqueous media and in seven different types of wood and non-wood black liquor samples were quantitatively analyzed by IC-CD. As a result, calibration lines with correlation coefficients of 1.00 for all analytes were achieved at a concentration range from 0.05 to 105 mg L−1. In black liquor samples intra-day (n  = 6) precision values ranged from 0.9 to 5%. Day-to-day (n 1  = 3) and intermediate precision values were less than 5% for all other compounds except sulfate and thiosulfate. The variability in the thiosulfate and sulfate results is due in large part to the oxidation of sulfide and thiosulfate, respectively. Recoveries were close to 100% with standard deviations less than 8%. Depending of the analyte, the limits of detection and quantification were, respectively, between 1 and 8 μg L−1 and between 3 and 27 μg L−1 for standard compounds in aqueous media and between 6 and 106 μg L−1 and between 14 and 148 μg L−1 for black liquor samples. These validation results clearly indicated that with respect to selectivity, linearity, limits of detection and quantification, precision, and accuracy, the IC-CD method showed good applicability in the determinations described above.
Keywords: Aliphatic carboxylic acids; Inorganic anions; Ion chromatography; Conductivity detection; Mass spectrometry; Method validation; Black liquor;

Analysis of phenolic compounds in Epimedium plants using liquid chromatography coupled with electrospray ionization mass spectrometry by Hai-Yu Zhao; Jiang-Hao Sun; Miao-Xuan Fan; Li Fan; Lei Zhou; Zhi Li; Jian Han; Bao-Rong Wang; De-An Guo (157-181).
A new method based on HPLC coupled with electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) has been established for the analysis of phenolic compounds in Epimedium plants. The characteristic fragmentation pathways of 29 phenolic compounds were studied, and 126 compounds were identified or tentatively characterized based on their mass spectra from fourteen samples including the aerial samples and the underground parts of the seven species. Furthermore, the differences in phenolic compound profiles between different Epimedium plants and two parts of the seven species were reported for the first time. Due to their significant chemical differences, these Epimedium species should be used separately in clinical practice.
Keywords: Phenolic compounds; Epimedium; HPLC–ESI-MS/MS;

Separation orthogonality has been explored with respect to comprehensive two-dimensional liquid chromatography (2D-LC) for different reversed-phase stationary phases. The outcome of this study points out an SB-CN × BEH-C18 combination, used in the first and the second dimension, respectively, as the most orthogonal chromatographic system for the samples assayed. The present investigation reports the employment of an ultra high pressure liquid chromatography system (UPLC) as the second chromatographic dimension, increasing the sensitivity and the speed, completing the whole chromatographic separation in a reasonable time frame. Finally, an off-line 2D-LC method with diode array detection based on the UPLC has been optimized, allowing the separation and minimizing the run time. SB-CN and BEH-C18 were employed as first and second dimension, respectively, with gradient elution applied in each dimension. Alprazolam degraded tablets were studied as a proof of concept of the utility of this type of setups for impurity profiling of complex samples.
Keywords: Alprazolam; Impurity profile; Orthogonality; Two-dimensional liquid chromatography; Ultra performance liquid chromatography;

Several cultured strains of Gymnodinium catenatum isolated worldwide have been shown to produce important proportions of the recently discovered benzoate paralytic shellfish poisoning (PSP) toxins GC1 through GC3. These toxins pose a new challenge for the HPLC analysis of shellfish predating during blooms of this microalga because due to their hydrophobicity are retained along the C18 solid-phase extraction step employed to eliminate interferences. The production of GC toxins was confirmed in a clone of G. catenatum isolated from the Portuguese Northwest coast during the winter bloom of 2005, in addition to a clone from 1989 reported previously by other authors. The major peroxide oxidation products of GC1+2 and GC3 were, respectively, dcGTX2+3 and dcSTX. The search of benzoate analogues in bivalves contaminated during the winter 2005 bloom showed these analogues constituted a minor component of the N1-H containing toxins, as selectively detected by peroxide oxidation. While in G. catenatum GC1-3 were the major components after C1+2 and B1, in bivalves dcGTX2+3 and dcSTX were the major components after C1+2 and B1. Similar conclusions were later extended to more shellfish species naturally contaminated during the autumn bloom of 2007. In the gut content of sardines GC toxins were present, while in crabs predating upon shellfish, these were absent. A generalised conversion of GC toxins into decarbamoyl analogues was confirmed by in vitro incubations of bivalve's digestive glands with semi-purified GC toxins. This is the first report of widespread carbamoylase activity in shellfish, exclusively targeted at benzoate PSP analogues and that is heat-inactivated. Despite the high proportion of benzoate analogues produced by G. catenatum, analyses of bivalves contaminated with PSP toxins seem to be simplified due to the important conversion of benzoate into decarbamoyl analogues that occurs in bivalves. These last analogues are detected by common HPLC methods used for food protection.
Keywords: Gymnodinium catenatum; Benzoate toxins; Carbamoylase; PSP; Shellfish; GC toxins; Liquid chromatography; Fluorescence detection;

We developed a technique controlling contaminant Al3+ level using a combination of kinetics and thermodynamics at a pre-column derivatizing step in HPLC. The technique involves modifying a conventional HPLC of 8-quinolinol (Ox) complex with Al3+. The contaminant suppressing reagents, dihydroxyazobenzene (DHAB) and 1,2,3-trihydroxybenzene (pyrogallol) were added to the Ox and pH buffer solutions to convert contaminant Al3+ in these solutions to inactive complexes. The Al3+ in the sample selectively formed Ox complexes with fast kinetics. After that, the labeled fluorescent complexes in the resultant metastable state were separated in the HPLC. This technique successfully suppressed contamination by a factor of 17. This method allowed for an improvement in the detection limits and also provided a stable blank.
Keywords: Contamination suppression; Pre-column derivatization; Aluminum; Dihydroxyazobenzene; Pyrogallol; 8-Quinolinol; Fluorescent detection;

Polyomavirus VP1 protein in pentamer form was expressed in E. coli and purified using glutathione-S-transferase (GST) affinity chromatography. Purified GST-tagged protein was found to exist as soluble aggregates with a size distribution of 1-52 tagged pentamers (340–1800 × 103  kDa), as determined by asymmetrical flow field flow fractionation with multiple angle light scattering (AFFFF-MALS). Aggregation did not inhibit tag removal by enzymatic cleavage, implying that the quaternary structure of the VP1 pentamers had been maintained. Elution gel filtration (EGF) was utilized to prepare a solution enriched with protein small enough to access resin pores (LMWe) as well as solution enriched with protein excluded from resin pores (HMWe). Material size distributions within both solutions were determined using AFFFF-MALS (radius of gyration LMWe: 5–10 nm; HMWe: 10–35 nm) and dynamic light scattering (DLS) (hydrodynamic diameter LMWe: 10–90 nm; HMWe: 20–300 nm). DLS and AFFFF-MALS analysis of each fraction of affinity chromatography purified material identified the elution profiles of large and small aggregate structures. DLS readings of all fractions were significantly affected by the presence of high molecular weight aggregates, with Z-average hydrodynamic diameter values reflecting the mass ratio of large and small aggregate structures in a solution. The methods utilized in this study have the potential to be used during chromatographic purification of all proteins that exist as soluble aggregates to determine size distribution. The finding that GST-tagged viral proteins exist as soluble aggregates has implications for existing immunological studies that utilize them.
Keywords: Aggregate; Soluble aggregate; Glutathione-S-transferase; Virus; Field-flow-fractionation; Light scattering; Particle; Protein;

Separation of complex branched polymers by size-exclusion chromatography probed with multiple detection by Marianne Gaborieau; Julien Nicolas; Maud Save; Bernadette Charleux; Jean-Pierre Vairon; Robert G. Gilbert; Patrice Castignolles (215-223).
Size-exclusion chromatography (SEC) separates polymers by hydrodynamic volume (the universal calibration principle). Molecular weights can be determined using viscometry (relying on universal calibration) and light scattering (independent of universal calibration). In the case of complex branched polyacrylates with tetrahydrofuran as eluent, universal calibration is valid, although the separation in term of molecular weight is incomplete: a given elution slice contains a range of molecular weights, described in terms of a ‘local polydispersity’. The local polydispersity index decreases when the number of branches per chain increases and complete separation is reached for highly branched chains.
Keywords: Multiple-detection size-exclusion chromatography (SEC); Separation; Polyacrylate; Complex branched polymer; Molecular weight; Hydrodynamic volume;

Trace analysis of fumagillin in honey by liquid chromatography-diode array–electrospray ionization mass spectrometry by Ma.J. Nozal; J.L. Bernal; Ma.T. Martín; J. Bernal; A. Álvaro; R. Martín; M. Higes (224-231).
In this work a new liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD–ESI-MS) method has been developed for the determination of fumagillin residues in honey. This procedure involves a solid-phase extraction on polymeric cartridges for the isolation of fumagillin from diluted honey. Chromatographic separation of fumagillin was performed in isocratic mode, on a C18 column (150 mm × 4.60 mm i.d., 5 μm), the mobile phase consisted of a mixture of ammonium formate 20 mM in water and acetonitrile (61/39, v/v), at 35 °C and the flow rate was set at 1.0 mL/min. Average analyte recoveries, influenced by the botanical origin were from 88 to 96% in replica sets of fortified honey samples. The detection limits of the LC-DAD–ESI-MS method were between 24 and 1 μg/kg for clear honeys (rosemary) and between 45 and 4 μg/kg for dark honeys (heather). The developed method has been applied to the analysis of fumagillin residues in honey samples collected from veterinary treated beehives, infected by Nosema ceranae and fed with the technical product at different doses.
Keywords: Fumagillin; Honey; Determination; Residues; LC-DAD; LC-ESI-MS;

Direct synthesis of hierarchical monolithic silica for high performance liquid chromatography by Hua Zhong; Guiru Zhu; Peiyuan Wang; Jian Liu; Jie Yang; Qihua Yang (232-240).
The silica-based monolith exhibiting a hierarchical bimodal porous structure has been directly synthesized via lytropic mesophase. The hydrolysis and condensation of tetramethoxysilane (TMOS) in the presence of poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (P123) and acetic acid results in silica monolith with MSU-type mesoporous structure embedded in the skeleton of the interconnected macropore. The silica monolith with bimodal porous structure can separate benzene and phenol with high flow rate and low back-pressure. Moreover, the chromatographic property of C18-grafted silica monolith is investigated in the separation of aromatic molecules. Our primary result shows that the silica monolith with interconnected macropore and MSU-type mesopore is a promising packing material as stationary phase for high performance liquid chromatography.
Keywords: Silica monolith; Bimodal porous structure; Macroporous; High performance liquid chromatography;

In this work multiple linear regression (MLR) and artificial neural network (ANN) were used to predict the gradient retention times of diverse sets of organic compounds in four separate data sets. Descriptors which were used as inputs of these models are five linear free energy relationship (LFER) solute parameters including E, S, A, B and V. In the first step eight separate multiple linear regression and artificial neural network models were used to predict the gradient retention time for each gradient condition separately. Results obtained in this step reveal that there are significant relations between LFER parameters and gradient retention times of solutes in liquid chromatography. Then MLR and ANN were applied to develop more general models in which several different gradient elution conditions were used. The performances of these models are compared in terms of their standard errors and also correlation analysis. The results obtained reveal that although there are no significant differences between ANN and MLR in separate modeling of the gradient retention times, ANN has a significant superiority over MLR models in developing the general models for various gradient elution conditions. The results of sensitivity analysis on ANN models indicate that the order of importance for input terms in separate ANN models is V x  >  B  >  S  >  E  >  A and in the case of combined ANN model is V x  >  B  >  t g  >  S  >  E  >  A, which are in agreement with the order of percentage of significance terms that obtained from the MLR models.
Keywords: Artificial neural network; LFER parameters; Linear free energy relationship; Quantitative structure–retention relationship; Gradient retention; Multiple linear regression;

Determination of household chemicals using gas chromatography and liquid chromatography with tandem mass spectrometry by Rebecca A. Trenholm; Brett J. Vanderford; Jörg E. Drewes; Shane A. Snyder (253-262).
A method has been developed for the determination of 24 household high production volume (HPV) chemicals in municipal wastewater systems using solid-phase extraction (SPE) and analyses using both gas chromatography and liquid chromatography, each with tandem mass spectrometry (GC–MS/MS and LC–MS/MS). Target compounds include pesticides, antioxidants, fragrances, plasticizers, preservatives and personal care products. Method reporting limits ranged from 0.1 to 100 ng/L in water and recoveries for most compounds were between 54 and 112%. Household HPVs were consistently detected in raw sewage entering three full-scale wastewater treatment plants. Compounds such as vanillin, DEET, benzophenone, 3-indolebutyric acid, bisphenol A, triclosan and triclocarban were detected in all wastewater influent and effluent samples, but were significantly lower in the effluent. Many of the remaining compounds were detected in the influent, but below detection in effluent samples. Menthol and phenoxyethanol had the highest observed concentrations in influent samples ranging from 1.5 to 13 μg/L for menthol, and 8.8 to 22 μg/L for phenoxyethanol. MGK-11, methylresorcinol, trifluralin, hexabromododecane, acriflavin and atrazine were not detected in any samples. The method described here detects a broad range of HPV chemicals with great sensitivity and selectivity.
Keywords: Household chemicals; High production volume (HPV) chemicals; Solid phase extraction; Gas chromatography; Liquid chromatography; Tandem mass spectrometry;

A new polystyrene-based monolithic stationary phase, which was prepared by single step in situ copolymerization of styrene, divinylbenzene and vinylbenzenesulfonic acid (VBSA), was developed as a separation column for capillary electrochromatography, in which VBSA was employed as the charge-bearing monomer. Polymerization time of the polystyrene-based monolith had slightly influenced the separation time of the tested analytes, but it effectively altered their separation resolutions. Furthermore, baseline separation for a wider range of acetonitrile levels of mobile phase was achieved when a monolithic column prepared by a longer polymerization time was used. This novel polystyrene-based monolithic column provided an adequate electroosmotic flow either in basic or acidic mobile phase when VBSA level was maintained at 2.6% (w/w). Finally, this proposed polystyrene-based column allowed seven tested analytes to achieve a reproducible baseline separation within 2.2 min with theoretical plate numbers higher than 164 000 plates/m.
Keywords: CEC; Vinylbenzenesulfonic acid; Polystyrene-based monolithic stationary phase;

Use of specific surface areas in inverse gas chromatography studies at zero surface coverage by M.C. Almazán-Almazán; M. Pérez-Mendoza; I. Fernández-Morales; M. Domingo-García; F.J. López-Garzón (271-277).
Inverse gas chromatography (IGC) is frequently used to study adsorption processes at zero surface coverage on microporous activated carbons. This allows to determine the thermodynamic adsorption parameters as equilibrium constants, V S, standard enthalpies of adsorption, Δ H A ° , standard free energy of adsorption, Δ G A ° , and so on. Nevertheless, the surface areas of the adsorbents (microporous carbons in this case) are needed for this purpose. The experimental determination of the surface areas of microporous solids is not univocal and the results depend on the adsorbate employed in the measurements, usually N2 or CO2. This means that the thermodynamic parameters obtained by IGC are subjected to a degree of uncertainty depending on whether N2 or CO2 is used to determine the surface area values. The aim of this paper is to discuss which of the two surface area values is more appropriate to be used in IGC measurements at zero surface coverage. Experimental and theoretical considerations are supplied in a thorough discussion which supports that CO2 surface area value is more appropriate. Thus, it is proposed that this should be used instead of the more generally extended nitrogen specific surface area obtained by the BET equation.
Keywords: Inverse gas chromatography; Zero surface coverage; Nitrogen and carbon dioxide surface areas; Thermodynamic parameters;

The medical commission of the International Olympic Committee forbids the use of anabolic androgenic steroids to improve sporting performances. Nine anabolic steroids (androsterone (A), nandrolone, estradiol, testosterone propionate, nandrolone-17 propionate, dydrogesterone, testosterone, epitestosterone, boldenone) and α-cholestane as internal standard were studied by gas chromatography coupled with mass spectrometry (GC/MS). The derivatisation reagent employed for the derivatisation of anabolic steroids was a mixture of N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), ammonium iodide and 2-mercaptoethanol (1000:2:6, v/w/v). Trimethylsilyl (TMS) derivatives were obtained. Anabolic steroids can be derivatised into one or two forms, mainly for androsterone into A-monoTMS and A-diTMS. The aim of this study was to research the optimization conditions of the derivatisation process (maximum yield of silylation reaction) of each anabolic steroid into only one form. A two-level factorial Doelhert design was used to determine the influence of different parameters and their interactions on each compound, thanks to response surface methodology. The parameters to be optimized were the reaction time and the temperature. The interaction “temperature–reaction time” is significant and has a positive effect on the improvement of the effectiveness of the derivatisation. Considering the large amount of information, often not convergent, a global desirability function was applied for multi-responses optimization. Thus, the optimized temperature and the reaction time of silylation were 85 °C and 24 min, respectively. Several GC/MS analytical parameters were also studied: linearity (regression coefficient upper than 0.99 for each compound, sensibility (range of concentration 0.05–0.30 μg/ml). Confirmatory experiments were applied to check the predicted values and to validate the model. The confirmatory assay responses are relatively close to the responses predicted. We observed satisfactory resolutions by GC/MS and a run lower than 12 min.
Keywords: Derivatisation; Anabolic steroids; Optimization; Experimental design; Doelhert design; Desirability;

A new sampling method for the determination of polycyclic aromatic hydrocarbons (PAHs) in ambient air is described. The method is based on active sampling on sorption tubes consisting of polydimethylsiloxane (PDMS) foam, PDMS particles and a TENAX TA bed. After sampling, the solutes are quantitatively recovered by thermal desorption and analysed by capillary GC–MS. The new sampling method has been compared to the classical method using high-volume sampling on a glass fiber filter followed by polyurethane foam for 24 h sampling of ambient air. Volumes enriched were 144 l on the mixed bed and 1296 m3 with the classical method. The concentrations measured using the new method were significantly higher that the values obtained using the classical method, i.e. a factor 1.2–3 for the high molecular weight PAHs and up to 35 times for naphthalene and 23 times for acenaphthene. The total toxicity equivalence value (TEQ) for PAHs was ca. two times higher compared to the conventional method, illustrating that the concentrations of PAHs in ambient air have been underestimated until now. Some figures of merit (mean value for 17 PAHs) of the method are repeatability 7.4%, detection limit 13 pg/m3, accuracy 105.6% and linearity 0.996. The method also opens interesting perspectives for the determination of other semi-volatile persistent organic pollutants (POPs) in air as illustrated with the analysis of polychlorinated biphenyls (PCBs) at a workplace during removal of transformer oil.
Keywords: Capillary GC–MS; Thermal desorption; Air monitoring; Multi-bed sampling; Polycyclic aromatic hydrocarbons;

Quantitative analysis of 3-alkyl-2-methoxypyrazines in juice and wine using stable isotope labelled internal standard assay by Y.S. Kotseridis; M. Spink; I.D. Brindle; A.J. Blake; M. Sears; X. Chen; G. Soleas; D. Inglis; G.J. Pickering (294-301).
A solid phase microextraction (HS-SPME)–GC–MS methodology was established for the analysis of 3-alkyl-2-methoxypyrazines (MPs) in wine using a stable isotope dilution assay. The compounds analysed were 3-isobutyl-2-methoxypyrazine (IBMP), 3-sec-butyl-2-methoxypyrazine (SBMP), and 3-isopropyl-2-methoxypyrazine (IPMP) using their respective deuterated analogues ([2H3]-IBMP, [2H3]-SBMP, [2H3]-IPMP) as internal standards, synthesised during this work. A divinylbenzene/carboxene/polydimethylsiloxane (DVB/CAR/PDMS) fibre was selected for isolation of MPs and the effects of matrix parameters such as pH and ethanol concentration were examined in the development of the method. Best results were obtained at a pH of approximately 6 and with a wine dilution factor of 1:2.5, resulting in an ethanol concentration of approximately 5% (v/v). Relative standard deviations (RSDs) of replicate samples were 5.6–7% for all MPs at 5 ng L−1 and <5% for 15 and 30 ng L−1 samples. The limit of detection was <0.5 ng L−1 in juice and 1–2 ng L−1 in wine. The recovery efficiencies for spiked wine samples were between 99 and 102% for all three MPs. Using this method, we investigated the impact of the Multicoloured Asian Lady Beetle (MALB) on MPs in wine. In red wine fermented with live MALB, IPMP is the most prevalent MP detected, although SBMP concentrations are also increased and IBMP is unchanged from background levels. MALB that have been dead for 1 day before addition to juice can still contribute to elevated SBMP concentrations in wine, but not if they have been dead for 3 days or longer. Clarifying juice prior to fermentation leads to substantially lower IPMP concentration in the subsequent wine when compared with unclarified juice.
Keywords: 3-Alkyl-2-methoxypyrazine; Wine; Cabernet sauvignon; Headspace solid phase microextraction; GC–MS; Deuterated analogues; Multicoloured Asian Lady Beetles; Harmonia axyridis; Ladybug taint;

Affordable and sensitive determination of artemisinin in Artemisia annua L. by gas chromatography with electron-capture detection by Shuoqian Liu; Na Tian; Zhonghua Liu; Jianan Huang; Juan Li; Jorge F.S. Ferreira (302-306).
Artemisinin demand has increased sharply since the World Health Organization recommended its use as part of the artemisinin combination therapies in 2001. The area for the crop cultivation has expanded in Africa and Asia and simpler and affordable methods for artemisinin analysis are needed for crop quality control. This work presented a novel chromatographic method of artemisinin analysis using gas chromatography with electron-capture detection. The sample extraction and preparation involved a single-solvent one-step extraction, with samples being analyzed in the extraction solvent directly after extraction. This method was accurate and reproducible with over 97% recoveries. The limit of detection was less than 3 μg/mL and the limit of quantification was less than 9 μg/mL, allowing samples as low as 100 mg dry weight to be analyzed for artemisinin. The method can be applied to quality control of commercial plant extracts and to artemisinin-derived pharmaceuticals.
Keywords: Chromatographic analysis; GC-ECD; Quantification; Qinghaosu;

Study of the chemical derivatization of zearalenone and its metabolites for gas chromatography–mass spectrometry analysis of environmental samples by Said Kinani; Stéphane Bouchonnet; Sophie Bourcier; Jean-Marc Porcher; Sélim Aït-Aïssa (307-315).
This study compares different silylation procedures of zearalenone and its metabolites: α-zearalenol, β-zearalenol, zearalanone, α-zearalanol and β-zearalanol for gas chromatography–mass spectrometry (GC–MS) analysis. Four silylating agents among the most frequently used to derivatize polar organic compounds were tested: N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA), N,N-diethyltrimethylsilylamine (TMSDEA) and a commercial mixture of N,O-bis(trimethylsilyl)acetamide, trimethylchlorosilane and N-trimethylsilyimidazole. Previous studies showed that the addition of polar and/or basic solvents can significantly improve the yield of a reaction of derivatization. In this work, four solvents were tested: pyridine, dimethylformamide, acetonitrile and acetone. The influence of each solvent was studied as a function of the silylating agent/solvent ratio. The influences of the temperature and of the reaction time on the reaction yields were also evaluated. A GC–MS quantitation method associating methanol chemical ionization and selected ion storage with three ions was developed and successfully tested on a reconstituted sediment spiked in zearalenone and its metabolites.
Keywords: Zearalenone; α-Zearalenol; β-Zearalenol; Zearalanone; α-Zearalanol; β-Zearalanol; Silylation; Chemical derivation; Gas chromatography; Mass spectrometry;

The applicability of programmable temperature vaporizer (PTV) solvent vent injection to the gas chromatographic (GC) determination of pesticide residues in fruits and vegetables was evaluated with the aim of miniaturizing the current multiresidue method. For that purpose 24 pesticides representing different chemical classes were initially chosen for optimisation of the large volume injection (LVI) parameters. Various parameters related to the optimum injector performance were tested for several types of packed and empty liners using both fast (at-once) and speed-controlled PTV solvent vent injection of standard solutions in ethyl acetate. In the next step, several packed and empty liners were evaluated for their suitability for pesticide multiresidue analysis. Parameters identified as optimal were then applied for PTV solvent vent injection of sample extracts prepared using the miniaturized multiresidue method to assess the long-term stability of the system. The combined use of large volume injection of 10 μl ethyl acetate extract into an empty multi-baffled or a CarboFrit packed liner using PTV injectors and GC–MS analysis enabled the detection and quantification of 124 pesticides in fruit and vegetable samples at the 0.01 mg/kg level using miniaturized reversed-phase solid-phase extraction (RP-SPE) of diluted acetone extract and clean-up on a small anion-exchange SPE column.
Keywords: Programmed-temperature vaporizer (PTV); Large volume injection (LVI); Pesticide residue analysis; Multiresidue method; GC–MS;

Comparison of four extraction/methylation analytical methods to measure fatty acid composition by gas chromatography in meat by M. Juárez; O. Polvillo; M. Contò; A. Ficco; S. Ballico; S. Failla (327-332).
Four different extraction–derivatization methods commonly used for fatty acid analysis in meat (in situ or one-step method, saponification method, classic method and a combination of classic extraction and saponification derivatization) were tested. The in situ method had low recovery and variation. The saponification method showed the best balance between recovery, precision, repeatability and reproducibility. The classic method had high recovery and acceptable variation values, except for the polyunsaturated fatty acids, showing higher variation than the former methods. The combination of extraction and methylation steps had great recovery values, but the precision, repeatability and reproducibility were not acceptable. Therefore the saponification method would be more convenient for polyunsaturated fatty acid analysis, whereas the in situ method would be an alternative for fast analysis. However the classic method would be the method of choice for the determination of the different lipid classes.
Keywords: Beef; Intramuscular; Fat; Gas chromatography;

Domestic and office dust samples (n  = 37) were analyzed for hexabromocyclododecanes (HBCDs) using gas chromatography–electron-capture negative ionization–mass spectrometry (GC–ECNI/MS) and liquid chromatography–electrospray tandem mass spectrometry (LC–ESI/MS/MS). To determine the best method to quantify HBCDs using GC–ECNI/MS, BDE 128 was used as internal standard (I.S.) in all samples, while 13C-labeled α-HBCD was used as I.S. in some samples. Total HBCD concentrations (sum of α-, β-, and γ-HBCD diastereomers) were calculated using response factors (RFs) for α- and γ-HBCD as individual diastereomers and using an average RF for both diastereomers. Statistical comparison showed that concentrations obtained via GC–ECNI/MS were statistically indistinguishable (p  > 0.05) from those obtained using LC–ESI/MS/MS. The closest match between the two techniques was obtained using [13C]α-HBCD as I.S. and the average RF for α- and γ-HBCDs. Excellent linear correlations (Pearson coefficient values r  > 0.9) were obtained between the GC–ECNI/MS and LC–ESI/MS/MS results, with slopes ranging from 0.76 to 1.36. Pentabromocyclododecenes (four isomers) and tetrabromocyclododecadienes (two isomers) were detected in the studied samples and were identified as degradation products of HBCDs after separation from the parent compound on the basis of both retention time and mass spectrum. This finding suggests that the elimination of HBr is the major degradation pathway for HBCDs in dust.
Keywords: Liquid chromatography; Gas chromatography; Mass spectrometry; Hexabromocyclododecanes; HBCDs; Degradation products; Indoor dust;

Headspace solid-phase microextraction combined with gas chromatography and mass spectrometry was used for the quantification of 32 volatiles which represent the typical chemical reactions that can occur during beer ageing. Detection was accomplished by employing on-fibre derivatisation using o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA) and normal HS-SPME extraction. The procedures were optimised for SPME fibre selection, PFBHA loading temperature and time, extraction temperature and time, and effect of salt addition. Interference of matrix effects was overcome by calibrating according to the standard addition method and by using internal standards. Afterwards, the method was validated successfully and was applied to study the flavour stability of different beer types.
Keywords: Beer; Flavour stability; Solid-phase microextraction; On-fibre derivatisation; Gas chromatography; Mass spectrometry;

Optimization of separation and detection conditions for the multiresidue analysis of pesticides in grapes by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry by Kaushik Banerjee; Sangram H. Patil; Soma Dasgupta; Dasharath P. Oulkar; Shubhangi B. Patil; Rahul Savant; Pandurang G. Adsule (350-357).
A comprehensive GC×GC–TOFMS method was optimized for multiresidue analysis of pesticides using a combination of a non-polar (RTX-5MS, 10 m × 0.18 mm × 0.2 μm) and a polar capillary column (TR-50MS, 1 m × 0.1 mm × 0.1 μm), connected in series through a dual stage thermal modulator. The method resolved the co-elution problems as observed in full scan one-dimensional GC–MS analysis and allowed chromatographic separation of 51 pesticides within 24 min run time with library-searchable mass spectrometric confirmation. Four pesticides, viz. chlorpyrifos-methyl, vinclozoline, parathion-methyl and heptachlor could be baseline separated on GC×GC, which were otherwise closely eluting and interfering each other's detection in 1D GC–MS run. Similarly, it could be possible to separate myclobutanil, buprofezin, flusilazole and oxyfluorfen on GC×GC. Although in 1D GC–MS, these closely eluting compounds could be identified through deconvolution algorithm and ‘peak-find’ option of the Chromatof® software but the spectral purity significantly improved on GC×GC analysis. Thorough optimization was accomplished for the oven temperature programming, ion source temperature and GC×GC parameters like modulation period, duration of hot pulses, modulation-offset temperature, acquisition rate, etc. to achieve best possible separation of the test compounds. The limit of detection significantly improved by 2–12 times on GC×GC–TOFMS against GC–TOFMS because of sharper and narrower peak shapes. The method was tested for grape matrix after preparing the samples using previously described method and recoveries of the entire test pesticides were within 70–110% at 10 ng/g level of fortification. GC×GC–TOFMS was found to be an excellent technique for library-based screening of pesticides with high accuracy and sensitivity.
Keywords: Grape; Pesticide residues; Multiresidue analysis; GC×GC–TOFMS; Deconvolution; Recovery;

For resolution of multicomponent overlapping GC/MS signals, a window independent component analysis (WICA) is proposed. In WICA, the window information, i.e., the existing interval along the retention time, of each component is obtained. Then, with the data in the selective (non-overlapping) window, the mass spectrum of the first component is extracted by using independent component analysis (ICA), and with the data of selective (non-overlapping) ions, chromatographic profile of the component is calculated. With a subtraction of the extracted information from the data matrix, the information of the next component can be obtained. In this way, mass spectrum and chromatographic profile of each component can be extracted sequentially along the retention time. Due to the usage of the window information, the limitation of ICA in resolution of multicomponent overlapping analytical signals is overcome. Two experimental GC/MS data are analyzed with the proposed WICA. Results show that both mass spectra and chromatographic profiles of the components in the overlapping GC/MS signal are accurately extracted.
Keywords: Resolution of overlapping signal; GC/MS; Independent component analysis (ICA); Immune algorithm (IA);

A monolithic stationary phase was prepared by in situ copolymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and methacrylic acid (MAA), in a binary porogenic solvent consisting of toluene and 1-dodecanol. The resulting monolith was evaluated as a hydrophilic interaction-capillary electrochromatography (HI-CEC) stationary phase under the mode of pressurized capillary electrochromatography (pCEC). Effects of the buffer pH, salt concentration and the mobile phase composition on the electroosmotic flow (EOF) velocity and the retention factors of the compounds were investigated. The generation of cathodic EOF under a broad pH range was attributed to the presence of the carboxyl groups on the surface of the polar stationary phase. The carboxyl groups offered at the same time the possibility of weak electrostatic interaction with analytes. The separation mechanism of the monolithic column was discussed in detail. It was found that the separation mechanism of charged solutes could be attributed to a mixed mode of HI and weak electrostatic interaction, as well as the effect of electrophoresis, while the separation of neutral solutes was based on the hydrophilic interaction at high acetonitrile (ACN) content.
Keywords: Hydrophilic interaction; Electrostatic interaction; Methacrylate-based; Monolith; Pressurized capillary electrochromatography;

Human erythropoietin (hEPO) is a glycoprotein hormone produced primarily by the kidney, which stimulates red blood cell production. Recombinant human erythropoietin (rhEPO), generally produced in Chinese hamster ovary (CHO) cells, can be used as not only a therapeutic protein but also a doping agent in sports. Profiling of EPO glycoforms is a critical means for quality control in pharmaceutical industrial and anti-doping analysis of misuse in sports. However, the existing methods for the analysis of EPO are associated with either time consuming or poor resolution. In this work, a rapid and high-resolution glycoform profiling method was presented based on capillary isoelectric focusing (cIEF) with whole column imaging detection (WCID). Experimental conditions that influence the separation were investigated. Under optimized conditions, rhEPO from three different sources were resolved into distinct populations within 5 min with excellent reproducibility. As compared with existing methods, the presented method exhibited the advantages of speed and high resolution. If combined with an effective sample enrichment step and a much more sensitive WCID version, the method can be a potential alternative for the detection of rhEPO misuse in sports.
Keywords: Erythropoietin; Capillary isoelectric focusing; Whole column imaging detection; Glycoform profiling; Separation;

In this paper capillary electrochromatography of alkali and alkaline-earth metal cations in open tubular capillary columns is described. Capillary columns are prepared by coating fused silica capillaries of 75 μm I.D. with poly(butadiene-maleic acid) copolymer (PBMA) in multiple layers. Thermally initiated radical polymerization is used to crosslink the stationary phase. Capillary columns with different number of stationary phase layers can be prepared and allow for the adjustment of separation selectivity in the electrochromatographic mode. Fast and sensitive separations of common inorganic cations are achieved in less than 6 min in a 60 cm capillary column with on-column capacitively coupled contactless conductivity detector. Limits of detection (S/N = 3) for the determination of alkali and alkaline-earth metal cations range from 0.3 to 2.5 μM and repeatability is better than 0.5, 4.5 and 6.1% for migration times, peak heights and peak areas, respectively.
Keywords: Capillary electrochromatography; Open tubular columns; Contactless conductivity detection; Inorganic cations; Ion chromatography;

This paper describes an automatic rapid approach for in-capillary derivatization of ephedrine (E) and pseudoephedrine (PE) and subsequent sensitive determination of the derivatives by micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) detection using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) as fluorescent reagent. The unique feature of this method is the capillary being used as a small reaction chamber, in which the sample, derivatization buffer and reagent solutions were injected directly into the capillary by tandem mode, followed by an electrokinetic step (5 kV, 15 s) to enhance the mixing efficiency of analytes and reagent plugs. Standing a specified time of 1 min for reaction, the derivatives were then immediately separated and determined. Several parameters for in-capillary derivatization and subsequent MEKC separation were systematically investigated. Under these optimized conditions, a baseline separation of the two analytes was achieved within 10 min and the derivatization concentration limits of detection were found to be 4.8 ng mL−1 for E and 1.6 ng mL−1 for PE, respectively. The method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of the two alkaloids in ephedra herb and its preparations.
Keywords: In-capillary derivatization; 4-Fluoro-7-nitro-2,1,3-benzoxadiazole; Laser-induced fluorescence; Micellar electrokinetic chromatography; Ephedrine; Pseudoephedrine;

A sensitive liquid chromatography–electrospray tandem mass spectrometry method combined with solid-phase extraction and silica cartridge cleanup was established for 16 sulfonamides and trimethoprim in various water matrices. Signal suppression of all target analytes in sewage treatment plant influent, effluent and river water was improved by this method developed in this study. The method detection limits for 17 analytes were 20–200 pg/L for influent, 16–120 pg/L for effluent and 8.0–60 pg/L for river water with overall mean recoveries of 62–102% in all studied matrices. This method was used to analyze residual sulfonamides and trimethoprim in wastewater and river samples from Japan, and 8 analytes (0.08 (sulfadimethoxine)–161 ng/L (sulfapyridine) in wastewater and 10 (0.03 (sulfamethizol)–8.9 ng/L (sulfaquinoxaline) in river samples were detected.
Keywords: Environmental analysis; Silica cartridge cleanup; Sulfonamides; LC–ESI-MS/MS;

Grapefruits were found to contain d-glucaric acid, which has anticancer properties. In the present investigation, a method has been developed for the quantification of d-glucaric acid in grapefruit by high-performance liquid chromatography (HPLC) using a simple isocratic mobile phase with detection at 210 nm. Grapefruit samples were homogenized, centrifuged and filtered through a 0.45 μm membrane and injected into a HPLC system. The developed method was used for the quantification of d-glucaric acid in nine widely used grapefruit varieties. Furthermore, the identity of d-glucaric acid was confirmed by mass spectra. Seasonal variation of d-glucaric acid within the individual varieties were also measured in fruits harvested during November, February and May. The overall trend of d-glucaric acid level was increased from early to late season fruits. The developed method has a sensitivity of d-glucaric acid as low as 0.05 μg with an accuracy and precision >95%. This method was found to be simple, fast, accurate and reproducible. Moreover, the identity of d-glucaric acid was confirmed by mass spectra. Additionally, the labor intensity and cost of sample preparation were greatly reduced as compared to reported methods. This is the first report on quantification of d-glucaric acid in different varieties of grapefruits from three harvesting sessions.
Keywords: d-Glucaric acid; Method development; HPLC; Grapefruit;

Author Index (399-402).