Journal of Chromatography A (v.1218, #38)

Chiral separation of agricultural fungicides by Virginia Pérez-Fernández; Maria Ángeles García; Maria Luisa Marina (6561-6582).
Fungicides are very important and diverse environmental and agricultural concern species. Their determination in commercial formulations or environmental matrices, requires highly efficient, selective and sensitive methods. A significant number of these chemicals are chiral with the activity residing usually in one of the enantiomers. The different toxicological and degradation behavior observed in many cases for fungicide enantiomers, results in the need to investigate them separately. For this purpose, separation techniques such as GC, HPLC, supercritical fluid chromatography (SFC) and CE have widely been employed although, at present, HPLC still dominates chromatographic chiral analysis of fungicides. This review covers the literature concerning the enantiomeric separation of fungicides usually employed in agriculture grouping the chiral separation methodologies developed for their analysis in environmental, biological, and food samples.
Keywords: Fungicides; Chiral separation; Capillary electrophoresis; Gas chromatography; High performance liquid chromatography; Supercritical fluid chromatography;

A high performance liquid chromatography–high resolution mass spectrometry (HPLC–HRMS) method for simultaneous screening and quantification of 28 drugs was developed and validated for 2.5 mg hair samples. Target drugs and their metabolites included amphetamines, cocaine, opioids, benzodiazepines, antidepressants, and hallucinogens. After decontamination, hair samples were extracted with 200 μL of a mixture of water: acetonitrile:1 M trifluoroacetic acid (80:10:10, v/v) using a 5 min simultaneous pulverization/extraction step. The extracts were analysed by HPLC–HRMS in an Orbitrap at a nominal resolution of 60,000, with concomitant in source collisional experiments (in source CID). Gradient elution on an Atlantis T3 column resolved 28 target compounds and 5 internal standards. Total chromatographic run time was 26 min. Calibration was achieved by linear regression analysis utilizing six calibration points; R 2 ranged from 0.9964 to 0.9999, the limits of quantification were 0.1 ng/mg for 8 compounds, 0.2 ng/mg for 16 compounds and 0.5 ng/mg for 4 compounds; mean relative errors from −21% to +23% were obtained; relative standard deviation, used to estimate repeatability and intermediate reproducibility at three concentrations, was always less than 20%. Process efficiency and recoveries for all analytes were better than 65 and 73%, respectively, at any concentration. The method was applied to hair samples from forensic investigations that contained a broad assortment of drugs of abuse and pharmaceuticals. The use of concomitant HRMS full scan and CID afforded the possibility of retrospective analysis for discovering untargeted drugs.
Keywords: HPLC–HRMS; Orbitrap; Hair; Drugs of abuse; Pharmaceuticals; Pulverization; Extraction;

Quantification of human growth hormone by amino acid composition analysis using isotope dilution liquid-chromatography tandem mass spectrometry by Ji-Seon Jeong; Hyuk-Min Lim; Sook-Kyung Kim; Hyung-Keun Ku; Kyung-Hwa Oh; Sang-Ryoul Park (6596-6602).
We describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution–HPLC–mass spectrometry (ID–HPLC–MS) method. Sample purity was confirmed using capillary zone electrophoresis, HPLC and MS. The analyte protein, human growth hormone (hGH), was effectively hydrolyzed by incubation with 8 M hydrochloric acid at 130 °C for 48 h, where at least 1 μM of hGH was treated to avoid possible degradation of released amino acids during hydrolysis. Using a reversed-phase column, the analytes (isoleucine, phenylalanine, proline and valine) were separated within 5 min using an isocratic eluent comprising 10% acetonitrile containing 0.1% trifluoroacetic acid. The detection limit (signal to noise ratio of 3) of amino acids was 5.5–6.2 fmol per injection. The quantification precision (RSD) of amino acids for intra- and inter-day assays was less than 0.98% and 0.39%, respectively. Comparison with other biochemical and instrumental methods revealed substantially higher accuracy and reproducibility of the ID–HPLC–MS/MS method as expected. The optimized hydrolysis and analytical conditions in our study were suitable for accurate quantification of hGH.
Keywords: Amino acid (AA) analysis; HPLC–MS/MS; Isotope dilution mass spectrometry (IDMS); Acid hydrolysis; Protein quantification; Human growth hormone;

A novel and low solvent consumption method for the sensitive determination of fungicide residues in wine samples is proposed. Analytes were extracted by dispersive liquid–liquid microextraction (DLLME) and further determined by gas chromatography–mass spectrometry (GC–MS). Under optimized conditions, a binary mixture of acetone and 1-undecanol (0.5 and 0.05 mL, respectively) was used to extract target compounds from diluted (1:1) wine samples. After centrifugation, the floating phase of 1-undecanol was solidified and separated from the liquid hydro-alcoholic matrix. Thereafter, it was allowed to melt at room temperature and injected in the GC–MS system. The method showed relative standard deviations (RSDs, %) below 13%, limits of quantification (LOQs) between 0.2 and 3.2 ng mL−1 and linear responses for concentrations up to 300 ng mL−1. The efficiency of the liquid-phase microextraction process was scarcely affected by the characteristics of wine samples, consequently pseudo-external standard calibration (using matrix matched standards of red and white wine) sufficed to achieve acceptable accuracy values: relative recoveries from 81 to 120%. The applicability of the method was demonstrated with commercial wine samples.
Keywords: Dispersive liquid–liquid microextraction; Non-chlorinated extractant; Fungicides; Wine analysis; Gas chromatography–mass spectrometry;

Selective enrichment of the degradation products of organophosphorus nerve agents by zirconia based solid-phase extraction by Pankaj K. Kanaujia; Deepak Pardasani; Vijay Tak; Ajay K. Purohit; D.K. Dubey (6612-6620).
Selective extraction and enrichment of nerve agent degradation products has been achieved using zirconia based commercial solid-phase extraction cartridges. Target analytes were O-alkyl alkylphosphonic acids and alkylphosphonic acids, the environmental markers of nerve agents such as sarin, soman and VX. Critical extraction parameters such as modifier concentration, nature and volume of washing and eluting solvents were investigated. Amongst other anionic compounds, selectivity in extraction was observed for organophosphorus compounds. Recoveries of analytes were determined by GC–MS which ranged from 80% to 115%. Comparison of zirconia based solid-phase extraction method with anion-exchange solid-phase extraction revealed its selectivity towards phosphonic acids. The limits of detection (LOD) and limit of quantification (LOQ) with selected analytes were achieved down to 4.3 and 8.5 ng mL−1, respectively, in selected ion monitoring mode.
Keywords: Solid-phase extraction; Phosphonic acids; Carboxylic acids; Zirconia; Nerve agents; HybridSPE;

This research paper presents a comparative study of the performance of conventional, ultrasound-assisted (UA), and temperature-controlled (TC) ionic liquid (IL) dispersive liquid-phase microextraction (IL-DLLME). Various parameters that affect extraction efficiency, such as type and volume of extraction and disperser solvent, centrifugation time, salt addition, effect of temperature on TC-IL-DLLME, and effect of sonication time on UA-IL-DLLME, were evaluated. UA-IL-DLLME was found to provide the best extraction efficiency. Under optimized conditions, great enrichment factors (506–515) and good recoveries (101.2–103.0%) were obtained by analyte extraction in real samples. The limit of detections (LODs) ranged from 0.21 to 0.38 μg L−1. Good linearity was obtained in the range of 0.5–200 μg L−1 for ethofenprox and tetramethrin, and 1–200 μg L−1 for meperfluthrin and alpha-cypermethrin. Based on optimized conditions, the UA-IL-DLLME method was applied and combined with high-performance liquid chromatography with diode array detection (HPLC-DAD) to determine the presence of ethofenprox, tetramethrin, meperfluthrin, and alpha-cypermethrin in honey samples.
Keywords: Ultrasound-assisted ionic liquid dispersive; Liquid–liquid microextraction; Pyrethroids; Liquid chromatography; Ionic liquid;

2H NMR has been used as a tool for probing the state of water in hydrophilic stationary phases for liquid chromatography at temperatures between −80 and +4 °C. The fraction of water that remained unfrozen in four different neat silicas with nominal pore sizes between 60 and 300 Å, and in silicas with polymeric sulfobetaine zwitterionic functionalities prepared in different ways, could be determined by measurements of the line widths and temperature-corrected integrals of the 2H signals. The phase transitions detected during thawing made it possible to estimate the amount of non-freezable water in each phase. A distinct difference was seen between the neat and modified silicas tested. For the neat silicas, the relationship between the freezing point depression and their pore size followed the expected Gibbs–Thomson relationship. The polymeric stationary phases were found to contain considerably higher amounts of non-freezable water compared to the neat silica, which is attributed to the structural effect that the sulfobetaine polymers have on the water layer close to the stationary phase surface. The sulfobetaine stationary phases were used alongside the 100 Å silica to separate a number of polar compounds in hydrophilic interaction (HILIC) mode, and the retention characteristics could be explained in terms of the surface water structure, as well as by the porous properties of the stationary phases. This provides solid evidence supporting a partitioning mechanism, or at least of the existence of an immobilized layer of water into which partitioning could be occurring.
Keywords: HPLC; Hydrophilic interaction chromatography; Retention mechanisms;

This study deals with the application of the Wilke–Chang equation to the estimation of molecular diffusivity (D m) in liquid phase systems including polar solutes and/or solvents. First, D m of benzene in six different organic solvents was experimentally measured by the peak parking method. The value of association coefficient (α) was calculated from the D m values by assuming that D m can be represented by the Wilke–Chang equation. Then, the α value was correlated with the solubility parameter (δ) and E T of the solvents. Two different curved correlations were observed between α and the two physico-chemical parameters. This means that α of given solutes and solvents can be obtained from the values of δ and/or E T. Finally, D m was estimated by a modified Wilke–Chang equation, which is derived by considering the aggregation of not only solvent molecules but also solute molecules. Although α is necessary for the estimation, it was calculated from δ for various solutes and solvents. The D m values estimated were compared with those reported in literature. The mean square deviation between the D m values was calculated less than 19% for 71 D m data. It was demonstrated that the modified Wilke–Chang equation can be used for estimating D m in liquid phase systems containing polar solutes and solvents.
Keywords: Molecular diffusivity; Wilke–Chang equation; Association coefficient; Solubility parameter; Mass transfer kinetics;

Simmitecan (L-P) is an anticancer ester prodrug, which involves activation to chimmitecan (L-2-Z). In the current study, a liquid chromatography/tandem mass spectrometry-based method was developed for simultaneous determination of L-P and L-2-Z in various plasma samples. Because L-P is rapidly converted to L-2-Z by blood carboxylesterase during and after sampling, which hampers accurate determination of L-P and L-2-Z in the biological samples, different carboxylesterase inhibitors were tested. As a result, bis(4-nitrophenyl)phosphate gave the best results with respect to inhibitory capability, hemolysis, and matrix effects and was used to deactivate blood carboxylesterases when sampling. The plasma samples were precipitated with acetonitrile and the resulting supernatants were separated using a pulse gradient method on a C18 column. Irinotecan and camptothecin were used as internal standards for quantification of L-P and L-2-Z, respectively. Protonated L-P, L-2-Z and their internal standards were generated by electrospray ionization and their precursor-product ion pairs (m/z 599 → 124, 405 → 361, 587 → 195, and 349 → 305, respectively) were used for measurement. The newly developed bioanalytical assay processed favorable accuracy and precision with lower limits of quantification of 2.1 nM for L-P and 3.4 nM for L-2-Z, and was successfully applied to pharmacokinetic studies in tumor-bearing nude mice, rats, and dogs. There are substantial species differences in the ester activity. The experimental strategies illustrated in our report may be adopted for measurement of other prodrugs (including irinotecan) or drugs subject to ester hydrolysis, as well as their metabolites, in biological matrices.
Keywords: Simmitecan; Chimmitecan; Irinotecan; Camptothecin; Prodrug; Carboxylesterase; Bis(4-nitrophenyl)phosphate; Pulse gradient chromatography; Matrix effects;

The effect of the addition of 25%, 50% and 75% (weight percent, wt%) of larger particles (resp. 3 and 5 μm) to a commercial batch of 1.9 μm particles has been investigated as an academic exercise to study the effects of particle size distribution on the kinetic performance of packed bed columns in a magnified way. Comparing the performance of the different mixtures in a kinetic plot, it could be irrefutably shown that the addition of larger particles to a commercial batch of small particles cannot be expected to lead to an improved kinetic performance. Whereas the addition of 25 wt% of larger particles still only has a minor negative effect, a significantly deteriorated performance is obtained when 50 or 75 wt% of larger particles are added. In this case, separation impedance number increases up to 200% were observed. Studying the packing structure through computational packing simulations, together with the experimental determination of the external porosity, helped in understanding the obtained results. This showed that small particles tend to settle in the flow-through pores surrounding the larger particles, leading to very high packing densities (external porosities as low as 32% were observed) and also negatively influencing the column permeability as well as the band broadening (because of the broadened flow-through pore size range).
Keywords: Particle size distribution (PSD); Kinetic plot; Particle packing; Total Pore Blocking method; External porosity;

This paper presents a rapid analytical method for the simultaneous determination of flonicamid and its metabolites N-(4-trifluoromethylnicotinoyl) glycine (TFNG), 4-trifluoromethylnicotinic acid (TFNA), and 4-trifluoromethylnicotinamide (TFNA-AM) in vegetables using QuEChERS by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Samples were extracted with acetonitrile. The extract was purified through QuEChERS method with primary secondary amine (PSA) and graphite carbon black (GCB). Then the extract was diluted with 0.1% formic acid in water, and analyzed by LC–MS/MS on a Waters Acquity BEH C18 column with methanol/0.1% formic acid in water as mobile phase with gradient elution. The linearity of the analytical response across the studied range of concentrations (0.20–500 μg/L) was excellent, obtaining correlation coefficients higher than 0.998. No significant matrix effects were observed. Recovery studies were carried out on spiked spinach and cucumber blank samples, at four concentration levels (0.01, 0.05, 0.50 and 2.0 mg/kg) performing six replicates at each level. Mean recoveries of 81.3–94.8% with CVs of 2.4–7.0% were obtained. The method demonstrated to be suitable for the simultaneous determination of flonicamid and its metabolites in vegetables.
Keywords: Flonicamid; Insecticide; Vegetables; LC–MS/MS; QuEChERS;

Simultaneous enantioselective determination of fenbuconazole and its main metabolites in soil and water by chiral liquid chromatography/tandem mass spectrometry by Yuanbo Li; Fengshou Dong; Xingang Liu; Jun Xu; Jing Li; Zhiqiang Kong; Xiu Chen; Wencheng Song; Yunhao Wang; Yongquan Zheng (6667-6674).
A novel and sensitive method was developed for the simultaneous determination of fenbuconazole and its main metabolites enantioselectively using chiral liquid chromatography coupled with tandem mass spectrometry. The separation and determination were performed using reversed-phase chromatography on a cellulose chiral stationary phase, a Chiralcel OD-RH (150 mm × 4.6 mm) column, under isocratic conditions at 0.5 mL/min flow rate. The effects of three cellulose-based columns and three amylose-based columns on the separation were also investigated. The elution orders of the eluting enantiomers were identified by an optical rotation detector. The QuEChERS (acronym for Quick, Easy, Cheap, Effective, Rugged and Safe) method and solid-phase extraction (SPE) were used for the extraction and clean-up of the soil and water samples, respectively. Parameters including the matrix effect, linearity, precision, accuracy and stability were evaluated. Under optimal conditions, the mean recoveries for all enantiomers from the soil samples were 82.5–104.1% with 2.7–9.5% intra-day relative standard deviations (RSD) and 5.7–11.2% inter-day RSD at 5, 25 and 50 μg/kg levels; the mean enantiomer recoveries from the water samples were 81.8–104.6% with 2.6–11.4% intra-day RSD and 5.3–10.4% inter-day RSD at 0.25, 0.5 and 2.5 μg/L levels. Coefficients of determination R 2  ≥ 0.9991 were achieved for each enantiomer in the soil and water matrix calibration curves within the range of 1.0–125 μg/L. The limits of detection (LOD) for all enantiomers in the soil and water were less than 0.8 μg/kg, whereas the limit of quantification (LOQ) did not exceed 2.5 μg/kg. The results of the method validation confirm that this proposed method is convenient and reliable for the enantioselective determination of the enantiomers of fenbuconazole and its main metabolites in soil and water.
Keywords: Fenbuconazole; Metabolite; Enantioselectivity; LC–MS/MS; Chirality; Enantiomers;

In this study, we examined the effect of first dimension column selectivity in reversed phase (RP) online comprehensive two dimensional liquid chromatography (LC × LC). The second dimension was always a carbon clad metal oxide reversed phase material. The hydrophobic subtraction model (HSM) and the related phase selective triangles were used to guide the selection of six different RP first dimension columns. Various kinds of samples were investigated and thus two different elution conditions were needed to cause full elution from the first dimension columns. We compared LC × LC chromatograms, contours plots, and f coverage plots by measuring peak capacities, peak numbers, relative spatial coverage, correlation values, etc. The major finding of this study is that the carbon phase due to its rather different selectivity from other reversed phases is reasonably orthogonal to a variety of common types of bonded reversed phases. Thus quite surprisingly the six different first dimension stationary phases all showed generally similar separation patterns when paired to the second dimension carbon phase. This result greatly simplifies the task of choosing the correct pair of phases for RP × RP.
Keywords: Two dimensional liquid chromatography; Selectivity; Reversed phase; LC × LC method development; Hydrophobic subtraction method; Carbon stationary phases;

An interesting adsorption behavior of racemic methyl mandelate on a tris-(3,5-dimethylphenyl)carbamoyl cellulose chiral stationary phase was theoretically and experimentally investigated. The overloaded band of the more retained enantiomer had a peculiar shape indicating a type V adsorption isotherm whereas the overloaded band of the less retained enantiomer had a normal shape indicating a type I adsorption behavior. For a closer characterization of this separation, adsorption isotherms were determined and analyzed using an approach were Scatchard plots and adsorption energy distribution (AED) calculations are combined for a deeper analysis. It was found that the less retained enantiomer was best described by a Tóth adsorption isotherm while the second one was best described with a bi-Moreau adsorption isotherm. The latter model comprises non-ideal adsorbate–adsorbate interactions, providing an explanation to the non-ideal adsorption of the more retained enantiomer. Furthermore, the possibility of using the Moreau model as a local model for adsorption in AED calculations was evaluated using synthetically generated raw adsorption slope data. It was found that the AED accurately could predict the number of adsorption sites for the generated data. The adsorption behavior of both enantiomers was also studied at several different temperatures and found to be exothermic; i.e. the adsorbate–adsorbate interaction strength decreases with increasing temperature. Stochastic analysis of the adsorption process revealed that the average amount of adsorption/desorption events increases and the sojourn time decreases with increasing temperature.
Keywords: Chiral separation; Perturbation peak method; Adsorption energy distribution; Stochastic modeling; Tóth adsorption model; Moreau adsorption model;

Energetics of lysozyme adsorption on mesostructured cellular foam silica: Effect of salt concentration by Jungseung Kim; Rebecca J. Desch; Stephen W. Thiel; Vadim V. Guliants; Neville G. Pinto (6697-6704).
The heat of lysozyme adsorption on mesostructured cellular foam (MCF) silica was measured using flow microcalorimetry (FMC) to investigate the influence of a neutral salt, sodium sulfate. At concentrations up to 0.5 M sodium sulfate, a complex initial exotherm was followed by an endotherm. Protein surface coverage, the magnitudes of the exothermic heat signals and the magnitudes of the net heat of adsorption increased with sodium sulfate concentration. These observations suggest that electrostatic interactions are the principal driving force at low ionic strengths; van der Waals interactions become dominant at higher salt concentrations. Each exotherm could be deconvoluted into two exotherms, indicating multiple modes of lysozyme attachment to the silica surface. The endothermic peak, associated with protein desorption, disappeared at the highest sodium sulfate concentration (1.0 M), indicating irreversible adsorption of the protein on the MCF silica surface. The data are consistent with an adsorption mechanism in which the initial attachment of lysozyme to the surface is followed by a reorientation and formation of a secondary or stronger attachment to the surface.
Keywords: Flow microcalorimetry (FMC); Mesostructured cellular foam (MCF) silica; Lysozyme; Adsorption energetics; Sodium sulfate;

Widely used as flame retardants, organophosphate esters (OPEs) are now broadly present in the indoor and outdoor environments. Currently available liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods share some drawbacks with gas chromatography (GC) methods, including time consuming, limited target OPEs, incomplete separation capability for some OPEs and low throughput. In this study, a fast and high throughput LC–MS/MS method was developed. For the first time, all the twelve OPEs that have been studied in literature, ranging from the very polar and volatile trimethyl phosphate to the very hydrophobic and non-volatile tris(2-ethylhexyl) phosphate, were separated within 11 min. Different from previous studies, we found that the blank contamination was mainly from organic mobile phase rather than the enrichment process, and it can be efficiently eliminated by using acetonitrile rather than methanol as the organic phase of the mobile phase. The signal to noise ratio (S/N) was significantly improved by using 0.1% formic acid as an organic modifier. The method exhibited high throughput and sensitivity and can baseline separate 11 of the 12 OPEs studied within 11 min with LOQs ranging from 2 to 6 ng/L. The relative standard deviations were in the range of 2–10%. For both reagent water and river water, the spiked recoveries of OPEs ranged from 70 to 110%, except for the very polar and volatile trimethyl phosphate that has recovery below 10%. The developed procedure was successfully applied to study the OPE contamination of the Songhua River, and it was found that all the target OPEs were detected with total concentrations of around 1 μg/L in the river waters.
Keywords: Organophosphate esters; Flame retardants; LC–MS/MS; High throughput; Songhua River;

A multiresidue method for screening of emerging contaminants in aquatic environments was developed. The method was based on sample pretreatment with solid phase extraction (SPE) and analysis with an ultra performance liquid chromatograph–time-of-flight mass spectrometer (UPLC–TOF-MS). The method was optimized and tested with standard solutions of model compounds containing 84 pesticides and pharmaceuticals. Four different SPE sorbents were evaluated to gain maximum recovery for the analytes. For the final procedure a combination of two different sorbents was chosen. In spite of high matrix suppression, the method quantification limits (MQLs) were acceptable. Therefore, the method can be used for screening known target compounds. The applicability of the method for posttarget and nontarget screening will be reported later. To preliminarily assess the quantitative performance of the method, some compounds in wastewater effluent were quantified using the standard addition method. Three pesticides and eight pharmaceuticals were found in concentrations up to ∼2200 ng/L.
Keywords: Liquid chromatography–time-of-flight-mass spectrometry; Multiresidue method; Solid phase extraction; Emerging contaminants;

Standard operating procedures (SOPs) are of paramount importance in the analytical field to ensure the reproducibility of the results obtained among laboratories. SOPs gain special interest when the aim is the analysis of potentially unstable compounds. An SOP for analysis of lipid hydroperoxides (HpETEs) is here reported after optimization of the critical steps to be considered in their analysis in human serum from sampling to final analysis. The method is based on automated hyphenation between solid-phase extraction (SPE) and liquid chromatography–mass spectrometry (LC–MS). The developed research involves: (i) optimization of the SPE and LC–MS steps with a proper synchronization; (ii) validation of the method—viz. accuracy study (estimated as 86.4% as minimum value), evaluation of sensitivity and precision, which ranged from 2.5 to 7.0 ng/mL (0.25–0.70 ng on column) as quantification limit and precision below 13.2%), and robustness study (reusability of the cartridge for 5 times without affecting the accuracy and precision of the method); (iii) stability study, involving freeze–thaw stability, short-term and long-term stability and stock solution stability tests. The results thus obtained allow minimizing both random and systematic variation of the metabolic profiles of the target compounds by correct application of the established protocol.
Keywords: Targeting metabolomics; Standard operation protocols (SOPs); Lipid hydroperoxides; Stability analysis; SRM; Automation;

In this study, a method for enantioseparation of terbutaline and salbutamol was established using Chirobiotic V column as a stationary phase. Polar ionic mode applying mobile phase containing ammonium nitrate in 100% ethanol, pH 5.1 was found to give the best separation. The salt concentration in the mobile phase and pH value were found to be the most important chromatographic factors affecting separation. Separation of enantiomers of these two basic analytes was complete in less than 10 min without applying ammonium trifluoroacetate (ATFA) or triethylamine (TEA) salts.
Keywords: Enantioseparation; Terbutaline; Salbutamol; Chirobiotic V; Polar ionic mode;

Using current data analysis techniques, even the most advanced LC–MS instrumentation can identify only a small fraction of compounds found in typical biological extracts. Augmenting MS information with HPLC retention information allows many more to be identified. In fact, our calculations indicate that a quadrupole MS is able to identify more compounds than an FTICR-MS when the quadrupole spectrum is augmented with retention information. Unfortunately, retention information is extremely difficult to harness for compound identification. Here, we demonstrate the first use of isocratic data measured on one LC–MS to “project” gradient retention on to different LC–MS systems. Using 35 chemically diverse solutes chosen to encompass the full range of reversed-phase alkylsilica interactions, and using experimental conditions typical of metabolomics experiments, gradient retention was projected from one instrument to another with only 1.2–2.6% error—enough accuracy to considerably improve compound identification. Besides accounting for nonlinear relationships of retention versus solvent composition as well as dead time versus solvent composition, accounting for the precise shape of the gradient profile (not just the dwell volume) improved projection accuracy on one instrument by up to 4 fold whereas flow rate non-idealities likely caused considerable error on the other instrument. Thus, these two factors must be taken into account to accurately project retention on diverse instrumentation.
Keywords: Retention prediction; Chemical identification; Isocratic to gradient conversion; Retention projection; Gradient profile; Cross-instrument;

Isocratic retention data should make a suitable foundation for an accurate, cross-instrument LC retention prediction system. Our previous work suggested that in order to accurately calculate (or “project”) gradient retention times on a wide range of HPLC systems using a single set of isocratic retention data, the precise shape of both the gradient and flow rate profiles produced by each instrument must be properly taken into account. However, accurate measurement of these system properties is difficult and time-consuming. In this work, we describe an approach that uses the measured gradient retention times of a set of standard solutes spiked into the sample along with their known isocratic retention vs. eluent composition relationships to determine the effective gradient and flow rate profiles by back-calculation. Retention “projections” of 20 other solutes using these back-calculated profiles, under various chromatographic conditions typical of metabolomics experiments, were remarkably accurate (as good as 0.23% of the gradient time, R 2 up to 0.99996), being very near the level of retention reproducibility. Our calculations suggest that this level of accuracy will allow a quadrupole MS to identify 38-fold more compounds out of a simulated mixture of 7307; it would allow an FTICR-MS to improve its identification rate nearly two-fold with the same mixture. Moreover, very little effort is required of the user. This approach provides a simple way to correct for all instrument-related factors affecting retention, allowing dramatically streamlined and improved retention projection across gradients, flow rates, and HPLC instruments.
Keywords: Retention prediction; Chemical identification; Gradient profile; Retention projection; Cross-instrument;

Pressure-assisted electrokinetic supercharging for the enhancement of non-steroidal anti-inflammatory drugs by Michelle M. Meighan; Mohamed Dawod; Rosanne M. Guijt; Mark A. Hayes; Michael C. Breadmore (6750-6755).
Electrokinetic supercharging (EKS) combines field-amplified sample injection with transient isotachophoresis (tITP) to create a powerful on-line preconcentration technique for capillary electrophoresis. In this work, EKS is enhanced with a positive pressure (pressure-assisted EKS, or PA-EKS) during injection to improve stacking of non-steroidal anti-inflammatory drugs (NSAIDs). Several parameters, including buffer composition and concentration, terminating electrolyte, organic modifier, and injection voltage and injection time of both terminating electrolyte and sample were optimized. Detection limits for seven NSAIDs were determined and an enhancement in sensitivity of almost 50,000-fold was obtained. The PA-EKS method has the potential to be a simple MS compatible preconcentration method to improve the sensitivity of CE.
Keywords: Capillary electrophoresis; Pressure-assisted; Electrokinetic supercharging; Stacking; Non-steroidal anti-inflammatory drugs; Water samples;

Highly sensitive detection of S-nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence by Siyang Wang; Magdalena L. Circu; Hu Zhou; Daniel Figeys; Tak Y. Aw; June Feng (6756-6762).
S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S–NO) to SH in cysteine using the “fluorescence switch” assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8 ± 0.2 and 10.4 ± 0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5 ± 0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7 ± 0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions.
Keywords: S-nitrosylation; Fluorescence switch assay; CGE-LIF; Alzheimer's disease transgenic mice model;

Natural nanoparticles, including both natural organic matter (NOM) and inorganic mineral-like phases, have been broadly characterized using Flow Field-Flow Fractionation (FlowFFF). Calibration with polystyrene sulfonate (PSS) standards was generally carried out in order to determine the molecular weight distribution of the NOM, however if the analyzed sample has a different charge density compared to the PSS standards, the resulting molecular weight distribution may become meaningless. The presented study therefore investigates and compares the influences of ionic strength and sample load on the retention time and recovery of both PSS standards and natural nanoparticles from a variety of sources. The minimum ionic strength in the carrier solution and the maximum injected sample load required for satisfactory separation depend on the molecular weight of the PSS standards and on the nature of the NOM. The degree to which results depend on conditions and parameters within the FlowFFF varies significantly between the different natural nanoparticle samples. We found that it may be necessary to calibrate the channel under different conditions from the actual sample runs. Under well controlled and documented conditions this could represent an important move away from the paradigm of “same conditions for standards and sample”. From all conditions tested, the most reliable molecular weight calibrations were obtained at elevated ionic strengths in the carrier solution (>0.04 M) and low injected mass of PSS. However, even under these optimized conditions variations of up to 20% occur in the calculated molecular weights, and the recovery of NOM falls by up to 50% at high ionic strengths. Many applications aim for both correct molecular weight distribution and the measurement of low concentrations of elements bound to natural nanoparticles. We conclude, however, that finding conditions that are equally optimal for both of these analytical tasks is not always feasible.
Keywords: Flow Field-Flow Fractionation; Ionic strength; Natural nanoparticles; Natural organic matter; Molecular weight calibration; Particle recovery;

Thermal field-flow fractionation coupled with online multiangle light scattering, differential refractive index and quasielastic light scattering (ThFFF-MALS/dRI/QELS) was used to simultaneously determine the molecular weight (MW) and composition of polystyrene–poly(n-butyl acrylate) (PS–PBA) and polystyrene–poly(methyl acrylate) (PS–PMA) copolymers. The online measurement of the normal diffusion coefficient (D) by QELS allowed calculation of the copolymer thermal diffusion coefficient (D T ) of sample components as they eluted from the ThFFF channel. D T was found to be independent of MW for copolymers with similar compositions and dependent on composition for copolymers with similar MW in a non-selective solvent. By using a solvent that is non-selective to both blocks of the copolymer, it was possible to establish a universal calibration plot of D T versus mole fraction of one of the monomer chemistries comprising the copolymer. PS–PBA and PS–PMA linear diblock polymers were determined to vary in composition from 100/0 to 20/80 wt% PS/acrylate and ranged in MWs between 30 and 360 kDa. The analysis of a PS–PBA miktoarm star copolymer revealed a polydisperse material with a weight percent PBA of 50–75% and MW ranging from 100 to 900 kDa. The presented ThFFF-MALS/dRI/QELS method allowed rapid characterization of polymers with MW and chemical distributions in a single analysis.
Keywords: Copolymer; Composition; Thermal field-flow fractionation; Thermal diffusion; Polyacrylate; Light scattering;

Extraction of pesticides, dioxin-like PCBs and PAHs in water based commodities using liquid–liquid microextraction and analysis by gas chromatography–mass spectrometry by Soma Dasgupta; Kaushik Banerjee; Sagar Utture; Parijat Kusari; Sameer Wagh; Kondiba Dhumal; Sanjay Kolekar; Pandurang G. Adsule (6780-6791).
Water based samples such as flavored drinks, juices and drinking water may contain contaminants at ultra trace level belonging to different chemical classes. A novel, simple, low-cost and fast method was developed and validated for trace residue extraction of pesticides, dioxin-like PCBs and PAHs from water and water based samples followed by analysis through gas chromatography (GC) coupled with time-of-flight mass spectrometry (ToFMS). The extraction solvent type, volume; sample volume and other extraction conditions were optimized. This was achieved by extracting 10 mL sample with 250 μL chloroform by vortexing (1 min, standing time of 2 min) followed by centrifugation (6000 rpm, 5 min). The bottom organic layer (200 μL) was pipetted out, evaporated to near dryness and reconstituted in 20 μL of ethyl acetate + cyclohexane (1:9) mixture resulting in an enrichment factor of 400. The recoveries of all compounds were within 76–120% (±10%) with the method detection limit (MDL) ranging from 1 to 250 ng/L depending on the analyte response. The MDLs were 400 times lower than the instrument quantification limits that ranged from 0.4 to 100 ng/mL. The method was further validated in water based drinks (e.g. apple, lemon, pineapple, orange, grape and pomegranate juice). For the juices with suspended pulp, the extraction was carried out with 400 μL chloroform. The extract was analyzed by GC-ToFMS at both 1D and GC × GC modes to chromatographically separate closely eluting interfering compounds the effect of which could not be minimized otherwise. The resulting peak table was filtered to identify a range of compounds belonging to specific classes viz. polycyclic aromatic hydrocarbons, chlorinated, brominated, and nitro compounds. User developed scripts were employed on the basis of identification of the molecular ion and isotope clusters or other spectral characteristics. The method performed satisfactorily in analyzing both incurred as well as market samples.
Keywords: Microextraction; Gas chromatography time-of-flight mass spectrometry; Method validation; Measurement uncertainty; Dioxin-like polychlorinated biphenyls; Polyaromatic hydrocarbons; Pesticide residues;

Comparative analysis of peak-detection techniques for comprehensive two-dimensional chromatography by Indu Latha; Stephen E. Reichenbach; Qingping Tao (6792-6798).
Comprehensive two-dimensional gas chromatography (GC×GC) is a powerful technology for separating complex samples. The typical goal of GC×GC peak detection is to aggregate data points of analyte peaks based on their retention times and intensities. Two techniques commonly used for two-dimensional peak detection are the two-step algorithm and the watershed algorithm. A recent study compared the performance of the two-step and watershed algorithms for GC×GC data with retention-time shifts in the second-column separations. In that analysis, the peak retention-time shifts were corrected while applying the two-step algorithm but the watershed algorithm was applied without shift correction. The results indicated that the watershed algorithm has a higher probability of erroneously splitting a single two-dimensional peak than the two-step approach. This paper reconsiders the analysis by comparing peak-detection performance for resolved peaks after correcting retention-time shifts for both the two-step and watershed algorithms. Simulations with wide-ranging conditions indicate that when shift correction is employed with both algorithms, the watershed algorithm detects resolved peaks with greater accuracy than the two-step method.
Keywords: Two-dimensional chromatography; Comprehensive two-dimensional gas chromatography (GC×GC); Chemometrics; Peak detection; Watershed algorithm; Two-step peak detection;

This study describes the development of a multiresidue method based on gas chromatography–electron ionization-tandem mass spectrometry (GC–EI-MS/MS) for the detection of sixteen polycyclic aromatic hydrocarbons (PAHs), five phthalate esters (PEs), seven polychlorinated biphenyls (PCBs), six polybrominated diphenyl ethers (PBDEs), six alkylphenols (APs), three organochlorined pesticides and their isomers or degradation products (OCPs) and bisphenol A in seawater, river water, wastewater treatment plant (WWTP) effluents, sediments and mussels. Solid phase extraction (SPE) was used for the extraction of target analytes in aqueous samples, and ultrasound assisted extraction for solid samples. GC–EI-MS/MS acquisition conditions in selected reaction monitoring (SRM) using two transitions per compound were optimized. In this way, quantification and unequivocal identification of organic micropollutants were performed in compliance with the Decision 2002/657/EC. Good linearity responses with coefficients of determination higher than 0.99 were obtained. Methodological detection limits (MDLs) in seawater ranged from 0.1 to 6 ng L−1; in river water from 0.1 to 4.8 ng L−1; in WWTP effluents from 1 to 75 ng L−1; in sediments from 1 to 150 ng g−1 and in mussels from 1 to 125 ng g−1. MDLs and recovery yields were compared with other published methods and similarities or even improvements were achieved. The optimized method was applied to analyze five samples from each matrix collected in coastal areas, showing its potential use for marine pollution monitoring.
Keywords: GC–EI-MS/MS; Organic micropollutants; Multiresidue determination; Marine ecosystem; Monitoring; SPE and ultrasound assisted extraction;

Study of the surface ionization detector for gas chromatography by Weiwei Li; Dapeng Wu; Shiheng Chen; Hong Peng; Yafeng Guan (6812-6816).
The structure of the surface ionization detector (SID) and the operation parameters of GC–SID were investigated to reduce peak tailing and to enhance sensitivity. The performances of the GC–SID, including its repeatability, linearity, sensitivity, selectivity, and tolerance towards water vapor, were evaluated systematically. Compared with nitrogen–phosphorus detector (NPD), the SID was able to detect fg level triethylamine, and selectively respond to alkylamines, some anilines, and some nitrogen heterocyclic compounds. Among alkylamines, the SID sensitivity to diisobutylamine was rather small. Even so, it was also still 10 times higher than that on NPD. The SID selectivity, defined as the sensitivity ratio between triethylamine and various tested non-nitrogen compounds, was higher than 106. It was found that the SID is highly tolerant towards water vapor, allowing direct injection of water sample. Finally, the GC–SID was applied to directly measure trace amines in headspace gases of rotted meat and trace simazine in tap water. The SID sensitivity to simazine was proven to be 5 times higher than that on flame ionization detector (FID). This study suggests that the SID is a promising GC detector.
Keywords: Surface ionization detector; GC; Quartz liner; Thermo ionization; Amines; Selectivity;

Furaneol is an important aroma compound. It is very difficult to extract furaneol from food matrices and separate it on a gas chromatography column due to its high polarity and instability. A new quantitative method was developed to quantify furaneol in aqueous samples by the use of derivatization/solid phase microextraction (SPME) coupled with gas chromatography/mass spectrometry (GC/MS). The derivatization was carried out by reacting pentafluorobenzyl bromide with furaneol in basic solutions at elevated temperatures. The derivative was stable and less polar so that SPME-GC/MS could be applied for extraction, separation and detection. The automated analytical method had a limit of detection (LOD) of 0.5 ng mL−1, a limit of quantification (LOQ) of 2 ng mL−1, a repeatability of 9.5%, and a linear range from 2 to 500 ng mL−1. The method was applied to analyze fruit samples. And it was found that the concentrations of furaneol in tomato ranged from 95 to 173 μg kg−1, in strawberries ranged from 1663 to 4852 μg kg−1. The results were verified with a LC procedure. To facilitate analytical method development, some physico-chemical parameters for furaneol were determined in this work. Its solubility in water was determined as 0.315 g mL−1 (25 °C). Its Log  D in water and Log  P in 0.1 M phosphate buffer were −0.133 and 0.95 (20 °C), respectively. Its pK a was 8.56 (20 °C).
Keywords: Furaneol; Solid phase microextraction (SPME); Solubility; Log  D; Log  P; pK a;

The retention behavior of components analyzed by chromatography varies with instrumental settings. Being able to predict how changes in these settings alter the elution pattern is useful, both with regards to component identification, as well as with regards to optimization of the chromatographic system. In this work, it is shown how experimental designs can be used for this purpose. Different experimental designs for response surface modeling of the separation of fatty acid methyl esters (FAME) as function of chromatographic conditions in GC have been evaluated. Full factorial, central composite, Doehlert and Box–Behnken designs were applied. A mixture of 38 FAMEs was separated on a polar cyanopropyl substituted polysilphenylene-siloxane phase capillary column. The temperature gradient, the start temperature of the gradient, and the carrier gas velocity were varied in the experiments. The modeled responses, as functions of chromatographic conditions, were retention time, retention indices, peak widths, separation efficiency and resolution between selected peak pairs. The designs that allowed inclusion of quadratic terms among the predictors performed significantly better than factorial design. Box–Behnken design provided the best results for prediction of retention, but the differences between the central composite, Doehlert and Box–Behnken designs were small. Retention indices could be modeled with much better accuracy than retention times. However, because the errors of predicted t R of closely eluting peaks were highly correlated, models of resolution (R s) that were based on retention time had errors in the same range as corresponding models based on ECL.
Keywords: Gas chromatography; Fatty acid methyl esters; Experimental design; Response surface modeling; Retention indices;

Methadone (MET) metabolism has been largely demonstrated with a high inter-individual variability and, therefore, quantification of MET is very important for therapeutic drug monitoring. A cation-selective exhaustive injection and sweeping MEKC (CSEI-Sweeping) was first developed to analyze MET and its two metabolites, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline (EMDP), in human serum. After pretreatment, the samples were electrokinetically injected into capillary (10 kV, 500 s) and swept by the separation phosphate buffer (100 mM, pH 4.0) containing 20% tetrahydrofuran and 100 mM SDS at −15 kV. The LODs were 200 pg/mL for MET and EMDP, and 400 pg/mL for EDDP. Ten volunteers were administered MET (5.0–120.0 mg/day) orally for 84 days and serum samples were taken after the daily dose of MET (days 1, 2, 7, 14, 28, 56 and 84) individually. This method was used for monitoring MET and its metabolites in heroin addicts and for pharmacokinetic investigations.
Keywords: CSEI-Sweeping; Methadone; Metabolites; Human serum; Capillary electrophoresis;

Identification of impurities in artemisinin, their behavior in high performance liquid chromatography and implications for the quality of derived anti-malarial drugs by Rodger W. Stringham; Michael Pennell; Walter Cabri; Giulio Carzana; Fabrizio Giorgi; Silvana Lalli; Giuseppe Marazzi; Marco Torri (6838-6842).
Previous work on the HPLC analysis of artemisinin tentatively identified the two impurities present above trace levels. This identification was based on LC–MS results and NMR of impurities isolated from artemisinin. In this work the impurities have been synthesized allowing verification of their identity by LC–MS. It is found that the previously suggested elution order is incorrect. A determination of relative response factors strongly impacts suggested limits on impurity levels and explains the erroneous peak assignment. The fates of the identified impurities are explored in the transformation of artemisinin to its derivative active pharmaceutical ingredients. A survey of a wide variety of artemisinin samples isolated from different geographical regions, different growing seasons, different plant backgrounds and using different extraction and purification approaches showed that artemisinin has sufficient purity for its intended use as a raw material for anti-malarial drug products.
Keywords: Artemisinin; Artemisitene; Epiartemisinin; Artemether; Artesunate; Dihydroartemisinin;

Bi-level optimizing control of a simulated moving bed process with nonlinear adsorption isotherms by Kiwoong Kim; Jin-Il Kim; Hyukmin Park; Yoon-Mo Koo; Kwang Soon Lee (6843-6847).
A bi-level optimizing control scheme originally proposed for a simulated moving bed (SMB) with linear isotherms has been extended to an SMB with nonlinear isotherms. Cyclic steady state optimization is performed in the upper level to determine the optimum switching period and time-varying feed/desorbent flow rates, and repetitive model predictive control is run in the lower level for purity regulation, taking the decision variables from the upper level as feed-forward information. Experimental as well as numerical study for an SMB process separating a high-concentration mixture of aqueous l-ribose and l-arabinose solutions showed that the proposed scheme performs satisfactorily against various disturbances. In contrast, an alternative scheme based on an SMB model with linear isotherms showed a limitation in the control performance; this scheme was apt to fail in purity regulation.
Keywords: SMB; Nonlinear adsorption isotherm; PowerFeed; Repetitive control; RMPC; Optimizing control;

It is shown theoretically that a claim in the literature about the overall separation efficiency of asymmetrical flow FFF channels being improved by geometries that permit a uniform channel flow velocity throughout the channel length is untrue.
Keywords: Flow field-flow fractionation; Asymmetrical flow FFF; Separation efficiency; Trapezoidal channel geometry; Flow velocity gradient; H-value;