Analytica Chimica Acta (v.633, #2)

Improvements of reliability for methylmercury determination in environmental samples by Dan-Yi Yang; Hoang-Yen Thi Truong; Yu-Wei Chen; Nelson Belzile (157-164).
The determination of methylmercury (MeHg) in environmental samples by ethylation derivation–gas chromatography–atomic fluorescence spectrometry (ED–GC–AFS) is associated with an intimate problem of water moisture accumulation introduced in the ethylation step, which enters the detection system and cause a spectroscopic interference. With a simple modification on the GC–AFS system, this problem was eliminated and the analytical quality of the measurements was significantly improved. The presence of dissolved sulfide in samples can also cause serious chemical interference in the ethylation step resulting in lower or total loss of the MeHg signal. It was found that a masking system of CuSO4–Na2C2O4 was able to eliminate this interference. With this system, the accurate determination of trace amount of MeHg in high dissolved sulfide containing samples was achieved. Satisfactory analytical results were obtained with the certified reference sediment IAEA405, sulfate reducing bacteria culture and sulfide containing water samples. The limit of detection and quantitation of this masking system is 0.01 and 0.04 ng L−1 respectively. Other factors affecting ethylation are also discussed.
Keywords: Methylmercury determination; Ethylation derivation–gas chromatography–atomic fluorescence spectrometry; Water vapour interference; Sulfide interference; Masking agents;

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for the quantitative imaging of Cu and other essential elements (such as K, Mg, Mn, P, S and B) in the leaves of a Cu-tolerant plant Elsholtzia splendens treated with the enriched 65Cu isotope tracer (isotope abundance of 89.2%). The leaves (newly formed, fully grown and oldest) were scanned directly with a focused Nd:YAG laser in the laser ablation chamber. The ablated material was transported with argon as carrier gas to a quadrupole-based ICP-MS (ICP-QMS), and the ion intensities of 65Cu+, 39K+, 24Mg+, 55Mn+, 31P+, 34S+ and 11B+ were measured by ICP-QMS to study the accumulation of Cu and other elements of interest. Standard reference material NIST SRM 1515 Apple Leaves doped with known concentrations of analytes (from 0.1 to 2000 mg L−1) was measured together with the samples by LA-ICP-MS and was used for the quantification of the analytical data. Notable accumulation of Cu in the newly formed leaves was clearly identified by imaging LA-ICP-MS. The increased isotope ratios of 65Cu/63Cu measured by LA-ICP-MS demonstrated the path of Cu uptake and accumulation via the petiole and main veins in the leaves. Cu stress-induced accumulation of K, Mg, Mn, P and S in the newly formed leaves was observed, while B was not significantly affected. In the present study, the concentrations of K, Mg, Mn, P and S were not obviously changed in the fully grown leaves after short-term treatment. Along with the treatment, a visible decrease of K and P was found in the oldest leaves, while other elements were not influenced by Cu stress.
Keywords: Imaging; Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS); Elsholtzia splendens; 65Cu; Nutrient elements; Plant leaves;

Multivariate calibration analysis of colorimetric mercury sensing using a molecular probe by Javier Pérez-Hernández; Josep Albero; Xavier Correig; Eduard Llobet; Emilio Palomares (173-180).
Selectivity is one of the main challenges of sensors, particularly those based on chemical interactions. Multivariate analytical models can determine the concentration of analytes even in the presence of other potential interferences. In this work, we have determined the presence of mercury ions in aqueous solutions in the ppm range (0–2 mg L−1) using a ruthenium bis-thiocyanate complex as a chemical probe. Moreover, we have analyzed the mercury-containing solutions with the co-existence of higher concentrations (19.5 mg L−1) of other potential competitors such as Cd2+, Pb2+, Cu2+ and Zn2+ ions. Our experimental model is based on partial least squares (PLS) method and other techniques as genetic algorithm and statistical feature selection (SFS) that have been used to refine, beforehand, the analytical data. In summary, we have demonstrated that the root mean square error of prediction without pre-treatment and with statistical feature selection can be reduced from 10.22% to 6.27%.
Keywords: Mercury quantification; Enhanced selectivity; Variable selection; Multivariate calibration;

Complexes of tetra-tert-butyl-tetraazaporphine with Al(III) and Zr(IV) cations as fluoride selective ionophores by Łukasz Górski; Monika Mroczkiewicz; Mariusz Pietrzak; Elżbieta Malinowska (181-187).
In this work, complexes of Zr(IV) and Al(III) cations with 2,7,12,17-tetra-tert-butyl-5,10,15,20-tetraazaporphine (TAP) were tested as ionophores in plasticized PVC membranes of ion-selective electrodes. It was found that both tested ionophores show enhanced affinity towards fluoride anion. High fluoride selectivity was observed in the presence of anionic or cationic additives in the membrane, which indicates that proposed compounds work according to charged or neutral carrier mechanism, depending on membrane composition and pretreatment. tert-Butyl substituents, present in the structure of tested compounds, were supposed to prevent formation of ionophore dimers within the membrane phase. This process was found to be responsible for some unfavorable potentiometric properties of electrodes based on complexes of Zr(IV) and Al(III) cations with porphyrins (compounds closely related to tetra-tert-butyl-5,10,15,20-tetraazaporphine). As it was shown using spectrophotometrical measurements, Al(III)–TAP was not susceptible to dimerization, while dimer formation was observed for Zr(IV)–TAP. In full agreement with these observations, electrodes with membranes containing Al(III)–TAP responded in near-Nernstian and fast manner towards fluoride anion, while the employment of Zr(IV)–TAP as ionophore resulted in super-Nernstian and sluggish response. Plasticized PVC membranes doped with Al(III)–TAP and 20 mol% of lipophilic anionic additives shown remarkable F selectivity, with selectivity coefficients, log K F − , Y − pot. , as follows: −4.4 (Y =Br), −4.3 (Cl), −4.2 (NO3 ), −3.6 (SCN), −2.9 (ClO4 ).
Keywords: Tetraazaporphine; Ion-selective electrode; Fluoride-selective ionophore; Potentiometry;

Modern extraction technique-pressurized liquid extraction (PLE) was optimised for extraction of lycorine and galanthamine (Amaryllidaceae alkaloids) from Narcissus jonquilla ‘Pipit’. Crude extracts were purified on Oasis MCX cartridges, and the alkaloids eluted with 80–100% recoveries using methanol–10% ammonia solution (3:1, v/v). Quantitative results were obtained by both HPTLC–densitometry on silica gel plates and RP-HPLC with diode array (DAD) on XTerra C18 stationary phase. Both methods were fully validated in terms of specificity, precision (including intra- and inter-day measurements), LOD and LOQ values, correlation of UV spectra and linearity of calibration curves. The methods were also well correlated each other with correlation coefficients (r) 0.98823 and 0.99081, respectively, for the mean values of galanthamine and lycorine.Among the investigated solvents methanol and 1% tartaric acid methanolic solution at default conditions (120 °C, p  = 60 bar, time: 10 min, one static cycle) permit the highest yields of the total sum of the alkaloids, whereas for toluene the lowest amounts were measured. Lycorine to galanthamine mean ratios were dependant on the type of solvent used, and in toluene galanthamine and related alkaloids were preferably extracted.In temperature experiments for galanthamine, the levels of this compound increased from the temperature of 20 till 150 °C in the investigated solvent systems, then decreased with slight increase from the temperature of 175 to 200 °C in 1% tartaric acid methanolic solution. When lycorine was analysed, similar trends were observed, however the maximum of the concentration was measured at a temperature about 125 °C. The ratios of the mean values of these two compounds differed in temperature-dependant experiments in both solvent systems.Further more, two TLC with bioautography approaches were used in screening for anticholinesterese properties of the extracts. No qualitative differences were found among the different solvent extracts, and AChE inhibition was correlated with galanthamine and related compounds.In conclusion, optimised PLE was much more effective than previously applied hot-solvent extraction, microwave-assisted extraction (MAE) or ultrasound-assisted extraction (USAE).
Keywords: Lycorine; Galanthamine; Pressurized liquid extraction; Thin-layer chromatography; High-performance liquid chromatography; Bioautography;

An organically modified silicate molecularly imprinted solid-phase microextraction device for the determination of polybrominated diphenyl ethers by Maggie Ka-Yi Li; Ngai-Yu Lei; Chengbin Gong; Yijun Yu; Ka-Ho Lam; Michael Hon-Wah Lam; Hongxia Yu; Paul Kwan-Sing Lam (197-203).
An organically modified silicate (ORMOSIL) SPME stationary phase molecularly imprinted with BDE-209 has been successfully fabricated by conventional sol–gel technique from phenyltrimethoxysilane and tetraethoxysilane. The thickness of the ORMOSIL-SPME stationary phase, on fused-silica optical fibres, was measured to be ca. 9.5 μm with a volume of ca. 0.12 μL. Rebinding assays and Scatchard analysis revealed that the imprinted ORMOSIL-SPME stationary phase possessed a binding affinity, K B , of 7.3 ± 1.7 × 1010  M−1 for BDE-209, with a receptor site density, B max, of 1.2 × 10−3  pmol per SPME device. Besides its molecular template, the ORMOSIL-SPME stationary phase also showed good affinity (log  K B  ≥ 9.5) for smaller BDE congeners commonly found in the natural environment. The density of receptor sites within the imprinted matrix for those smaller BDE congeners was even higher than that for BDE-209. This may be attributable to the binding site heterogeneity of the imprinting process that creates deformed binding sites that are suitable for the accommodation of the smaller BDE congeners. Compared to the commercially available polyacrylate and polydimethylsiloxane SPME stationary phases, the imprinted ORMOSIL-SPME devices showed much higher pre-concentration ability towards polybrominated diphenyl ethers (PBDEs), even in direct immersion sampling at room temperature. Coupled with GC–NCI-MS and GC-μECD, the imprinted ORMOSIL-SPME device was able to achieve detection sensitivity of 0.2–3.6 pg mL−1 and 1–8.8 pg mL−1, respectively, for commonly occurring BDE congeners, including medium to high molecular weight PBDEs. The imprinted ORMOSIL-SPME device has been successfully applied to monitor PBDE contents in municipal wastewaters.
Keywords: Molecular imprinting; Organically modified silicate; Solid-phase microextraction; Polybrominated diphenyl ether determination; Natural waters; Municipal wastewaters;

Due to the lack of one universally applicable and commonly used reference method, sample preparation in isoflavone (IF) analysis has been performed by many different methods which renders comparison and quality assessment of published IF contents in foodstuffs difficult.In the present work, the impact of different experimental parameters on the IF concentrations determined in soybeans, tofu, soy drink and textured vegetable protein by different extraction and hydrolysis methods was assessed and IF contents obtained by optimized orthogonal methods were compared. Chromatographic analysis was performed by HPLC-UV-ESI-MS. If possible sources of error – which are also pointed out in this work – are avoided, IF contents obtained by extraction, acid-, base- and enzymatic hydrolysis are similar. However, these sample preparation methods differ in the amount of time, standard compounds and instruments required, ruggedness, and in their applicability to analysis of complex composite samples containing soy as minor ingredient. Enzymatic hydrolysis with Helix pomatia juice after extraction by sonication with first 50, then 80% aqueous acetonitrile in the presence of zinc sulfate heptahydrate and after adjustment to ≤10% organic solvent turned out to be the method of choice if only aglucone equivalent contents are required. The advantages of this method are short chromatographic run times, smallest danger of coelution, lowest achievable limits of quantitation and therefore best suitability for work-up of complex composite samples and that only aglucone standards are needed for quantitation.
Keywords: Isoflavones; Sample preparation; Method development; Analysis; Soy foods; High performance liquid chromatography with ultraviolet and mass spectrometric detection;

Development of a derivatisation method for the analysis of aldehyde modified amino acid residues in proteins by Fourier transform mass spectrometry by Mostafa Pournamdari; Ahmed Saadi; Elizabeth Ellis; Ruth Andrew; Brian Walker; David G. Watson (216-222).
A method was developed for the analysis of amino acids within bovine serum albumin (BSA) which had been modified by reaction with different enals. BSA was reacted with the aldehydes and the reaction products were stabilised by reaction with NaBH4. The protein was then hydrolysed with 6N HCl and the hydrolysis products were analysed by liquid chromatography–mass spectrometry (LC–MS). The modified amino acids were derivatised with propylchloroformate. High resolution mass spectrometry carried out using an LTQ-Orbitrap instrument which was able to characterise a wide range of adducts. In addition double adducts were observed to be formed with 4-hydroxynonenal (HNE) and lysine or lysine + histidine. Qualitatively it was possible to consistently observe a pyridinium adduct formed between lysine and pentenal in human plasma from normal subjects.
Keywords: Aldehyde; Protein; Adduct; Fourier transform mass spectrometry; Human plasma;

The efficiencies of three derivatisation reagents that react with either the amine (9-fluorenylmethyl chloroformate (FMOC)) or the carboxylic acid group (butanol) of amino acid or with both types of functional groups (propyl chloroformate) were compared in the analysis of amino acids by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI–MS/MS). Separation of 20 amino acids derivatised with these three reagents was studied on reversed-phase chromatography. Linearity, repeatability and limits of detection of the LC-ESI–MS/MS method were determined by analysing FMOC-, butanol- and propyl chloroformate-derivatised lysine, β-aminobutyric acid, threonine and glutamic acid. The limits of detection for the derivatised amino acids (7.5–75 fmol) were as much as 2–60 times lower than those of the corresponding underivatised molecules. The best linearity was observed for amino acids derivatised with propyl chloroformate or butanol (r 2  = 0.996–0.999, range = 100–8500 nmol L−1). Propyl chloroformate was the best suited of the reagents tested for the analysis of amino acids with LC–MS/MS and was used for the analysis of amino acids in rat brain microdialysis samples.
Keywords: Derivatisation; Amino acids; Tandem mass spectrometry; Liquid chromatography; Microdialysis;

Positive mode electrospray ionization mass spectrometry of bisphosphonates using dicationic and tricationic ion-pairing agents by Molly M. Warnke; Zachary S. Breitbach; Edra Dodbiba; Jeffrey A. Crank; Tharanga Payagala; Pritesh Sharma; Eranda Wanigasekara; Xiaotong Zhang; Daniel W. Armstrong (232-237).
A general method for detecting bisphosphonate drugs by ESI-MS and LC–ESI-MS as positive ions has been developed. Bisphosphonates can have multiple negative charges in solution. Tricationic ion-pairing reagents were paired with bisphosphonates to form a positively charged complex. It was clear that this facile pairing method worked. However, an appreciable presence of −1 bisphosphonate species were observed in positive mode ESI-MS (i.e. as the +2 complex with tricationic reagents). This led to an extended investigation on the use of dicationic pairing agents. The use of dicationic reagents improved the detection sensitivity for all of the bisphosphonates. Tandem mass spectrometry also improved the limits of detection for most of the bisphosphonates using both the tricationic and dicationic pairing reagents. A tricationic reagent also was used as an ion-pairing reagent in chromatography experiments. Thus the addition of a single reagent produced benefits in that it increased chromatographic retention and enhanced the ESI-MS detection of bisphosphonates.
Keywords: Electrospray-ionization mass spectrometry; Bisphosphonates; Tricationic pairing reagent; Ion-pairing;

Mode-filtered light methane gas sensor based on cryptophane A by Suozhu Wu; Yan Zhang; Zhongping Li; Shaomin Shuang; Chuan Dong; Martin M.F. Choi (238-243).
A mode-filtered light sensor has been developed for methane (CH4) gas determination at ambient conditions. The proposed chemosensor consisted of an annular column which was constructed by inserting an optical fiber coated with a thin silicone cladding of cryptophane A into a fused-silica capillary. When CH4 was introduced to the sensor, selective inclusion of CH4 into the silicone layer would cause a change in the local refractive index of the cladding, resulting in the change of mode-filtered light that emanated from the fiber. Three detection windows were set alongside the capillary to propagate the light to a charge-coupled device (CCD). The changes of mode-filtered light on exposure to various concentrations of CH4 were thus simultaneously monitored. The mode-filtered light intensity decreased with the increase in concentration of CH4. The dynamic concentration range of the sensor for CH4 was 0.0–16.0% v/v with a detection limit of 0.15% v/v. The highest sensitivity was found at the channel furthest away from the excitation light source. The response time (t 95%) was about 5 min. The reproducibility was good with a relative standard deviation (RSD) of less than 7% from evaluating six cryptophane A-coated fibers. Oxygen, hydrogen and carbon dioxide showed very little interference on detection but interferences from dichloromethane and carbon tetrachloride were observed. The proposed mode-filtered light sensor has been successfully applied to determine CH4 samples and the accuracy was good. Our work offers a promising approach for CH4 detection.
Keywords: Mode-filtered light; Methane; Cryptophane A;

The aim of this study is to elaborate a simple and sensitive electrochemical immunoassay using ferrocenecarboxylic (Fc-COOH)-doped silica nanoparticles (SNPs) as an immobilized affinity support for cancer antigen 15-3 (CA 15-3) detection. The Fc-COOH-doped SNPs with redox-active were prepared by using a water-in-oil microemulsion method. The use of colloidal silica could prevent the leakage of Fc-COOH and were easily modified with trialkoxysilane reagents for covalent conjugation of CA 15-3 antibodies (anti-CA 15-3). The Fc-COOH-doped SNPs were characterized by X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). The fabrication process of the electrochemical immunosensor was demonstrated by using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques. Under optimal conditions, the developed immunosensor showed good linearity at the studied concentration range of 2.0–240 U mL−1 with a coefficient 0.9986 and a detection limit of 0.64 U mL−1 at S/N = 3.
Keywords: Amperometric immunosensor; Fc-COOH-doped silica nanoparticles; Cancer antigen 15-3;

Analysis of omeprazole and its main metabolites by liquid chromatography using hybrid micellar mobile phases by Maria Rambla-Alegre; Josep Esteve-Romero; Samuel Carda-Broch (250-256).
Omeprazole is a selective inhibitor of gastric acid secretion and is one of the most widely prescribed drugs internationally. A chromatographic procedure that uses micellar mobile phases of sodium dodecyl sulphate and propanol buffered at pH 7 and a C18 column is reported for the determination of omeprazole and its principal metabolites (omeprazole sulphone and hydroxyomeprazole) in urine and serum samples.In this work, direct injection and UV detection set at 305 nm was used. Omeprazole and its metabolites were eluted in less than 11 min with no interference by the protein band or endogenous compounds. Adequate resolution was obtained with a chemometric approach, in which the retention factor and shape of the chromatographic peaks were taken into account. The analytical parameters including linearity (r  > 0.9998), intra- and inter-day precision (RSD, %: 0.6–7.9 and 0.14–4.7, respectively) and robustness were studied in the validation of the method for the three compounds. The limits of detection and quantification were less than 6 and 25 ng mL−1, respectively. Recoveries in micellar medium, plasma and urine matrices were in the 98–102% range. Finally, the method was successfully applied to the determination of omeprazole and its metabolites in physiological samples. Omeprazole was also analysed in pharmaceutical formulations.
Keywords: Direct injection; Micellar liquid chromatography; Omeprazole; Metabolites; Urine; Serum; Pharmaceuticals;

Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry for metabonomics: Biomarker discovery for diabetes mellitus by Xiang Li; Zhiliang Xu; Xin Lu; Xuehui Yang; Peiyuan Yin; Hongwei Kong; Ying Yu; Guowang Xu (257-262).
Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC–TOFMS) coupled with pattern recognition methods was applied to analyze plasma from diabetic patients and healthy controls. After sample preparation and GC × GC–TOFMS analysis, collected data were transformed, the peak alignment between different chromatograms was performed to generate the metabolites’ peak table, then orthogonal signal correction filtered partial least-squares discriminant analysis (OSC-PLSDA) was carried out to model the data and discover metabolites with a significant concentration change in diabetic patients. With the method above, diabetic patients and healthy controls could be correctly distinguished based on the metabolic abnormity in plasma. Five potential biomarkers including glucose, 2-hydroxyisobutyric acid, linoleic acid, palmitic acid and phosphate were identified. It was found that elevated free fatty acids were essential pathophysiological factors in diabetes mellitus which reflected either the hyperglycemia or the deregulation of fatty acids metabolism. These potential biomarkers in plasma, e.g. palmitic acid, linoleic acid and 2-hydroxybutyric acid might be helpful in the diagnosis or further study of diabetes mellitus. This study shows the practicability and advantage of GC × GC–TOFMS coupled with data analysis and mining for metabonomics in biomarker discovery.
Keywords: Metabonomics; Metabolomics; GC × GC–TOFMS; Biomarker; Diabetes mellitus;

For olive oil production a metal hammer-decanter olive processing line was compared to a traditional metal hammer-press line, a discontinuous method which, if properly used, yields high-quality virgin olive oils. Galega, Carrasquenha and Cobrançosa olives (traditional Portuguese varieties) were studied. The analysis of the aroma compounds was performed after headspace-solid phase micro extraction. The analytical results obtained after comprehensive gas chromatography in tandem with time of flight mass spectrometry (GC × GC/ToFMS) for these three different olive oil varieties, from a single year harvest and processed with two different extraction technologies, were compared using statistical image treatment, by means of ImageJ software, for fingerprint recognitions and compared with principal component analysis when the area data of each chromatographic spot of the contour plots were considered. The differences used to classify the olive oils studied under different groups after principal component analysis were observed independently of the treatment used (peak areas or the sum of the pixels counts). When the individual peak areas were considered, more then 75.7% of the total variance is explained by the first two principal components while in the case where the data were subjected to image treatment 84.0% of the total variance is explained by the first two principal components. In both cases the first and second principal components present eigenvalues higher then 1.0. Fingerprint image monitoring of the aroma compounds of the olive oil allowed a rapid differentiation of the three varieties studied as well as the extraction methods used. The volatile compounds responsible for their characterization were tentatively identified in a bi-dimensional polar/non-polar column set in the GC × GC/Tof-MS apparatus. This methodology allowed the reduction of the number of compounds needed for matrices characterization, preserving the efficiency of the discrimination, when compared with the traditional methods where the identification of all peaks is needed.
Keywords: Comprehensive two-dimensional gas chromatography; Olive oil; Volatile organic compounds; Solid phase micro extraction; Fingerprinting; Image treatment;

Development of a validated liquid chromatographic method for the quality control of Prunellae Spica: Determination of triterpenic acids by Mi Kyoung Lee; Young Min Ahn; Kang Ro Lee; Jee H. Jung; Ok-Sang Jung; Jongki Hong (271-277).
A simple and rapid reversed-phase HPLC-UV method was developed for the determination of triterpenic acids in the crude extract of Prunellae Spica. Five triterpenic acids were extracted and isolated from P. Spica as marker compounds for use in the quality control of herbal medicines. Various solvent extraction techniques were evaluated, and the greatest efficiency was observed with sonication in 100% ethanol. Elemental compositions of the five marker compounds were determined by high-resolution mass spectroscopy. The dynamic range of the HPLC-UV method depended on the specific analyte, and acceptable quantitation was obtained between 10 and 250 μg mL−1 for oleanolic acid, between 10 and 300 μg mL−1 for ursolic acid, between 3 and 75 μg mL−1 for 2α,3α,24-trihydroxyolean-12en-28oic acid, between 5 and 100 μg mL−1 for euscaphic acid, and between 5 and 100 μg mL−1 for 2α,3α-dihydroxyurs-12en-28oic acid. The method was deemed satisfactory by inter- and intra-day validation and exhibited both high accuracy and precision (relative standard deviation <9.4%). Overall limits of quantitation and detection were approximately 0.5–2.5 μg mL−1 at a signal-to-noise ratio (S/N) of 3 and were about 3.0–10.0 μg mL−1 at a S/N of 10. In addition, principal component analysis (PCA) was performed on the analytical data of 15 different P. Spica samples in order to classify samples collected from different regions.
Keywords: Prunellae Spica; Quality control; Triterpenic acids; Method validation; Principal component analysis;

A reliable and sensitive competitive real-time immuno-PCR (RT-IPCR) assay for the determination of anthracene (AN) was developed. 9-Anthracenebutanoic acid, γ-oxo-(ANA) was synthesized as the hapten of AN. Mixed anhydride reaction (MAR) was used to couple the ANA to ovalbumin (OVA) to form artificial coating antigen. Active ester method (AEM) was used to couple the ANA to bovine serum albumin (BSA) to form artificial immune antigen. Male New Zealand white rabbits were immunized with immune antigen to obtain polyclonal antibodies (pAbs), with which, a novel RT-IPCR assay for determination of AN was described. Under optimized assay conditions, AN can be determined in the concentration range from 1 fg mL−1 to 100 pg mL−1 with a detection limit of 0.5 fg mL−1. The cross-reactivities of the anti-AN antibody to seven structurally related compounds were below 15%. Environmental water samples were successfully analyzed, showing a good accuracy and suitability to analyze AN in field samples. Recovery was between 93.3% and 120.0% and would be acceptable for use in an on-site field test to provide rapid, semiquantitative, and reliable test results for making environmental decisions.
Keywords: Anthracene; Antigen; Polyclonal antibody; Real-time immuno-polymerase chain reaction assay;

High resolution 1H NMR spectroscopy has been employed as a versatile and rapid method to analyze the polar fraction of extra virgin olive oils containing various classes of phenolic compounds. The strategy for identification of phenolic compounds is based on the NMR chemical shifts of a large number of model compounds assigned by using two-dimensional (2D) NMR spectroscopy. Furthermore, 2D NMR was applied to phenolic extracts in an attempt to discover additional phenolic compounds. The 1H NMR methodology was successful in detecting simple phenols, such as p-coumaric acid, vanillic acid, homovanillyl alcohol, vanillin, free tyrosol, and free hydroxytyrosol, the flavonols apigenin and luteolin, the lignans (+) pinoresinol, (+) 1-acetoxypinoresinol and syringaresinol, two isomers of the aldehydic form of oleuropein and ligstroside, the dialdehydic form of oleuropein and ligstroside lacking a carboxymethyl group, and finally total hydroxytyrosol and total tyrosol reflecting the total amounts of free and esterified hydroxytyrol and tyrosol, respectively. The absolute amount of each phenolic constituent was determined in the polar fraction by using anhydrous 1,3,5-triazine as an internal standard.
Keywords: Olive oil; Phenolic compounds; 1H nuclear magnetic resonance spectroscopy;

Analysis of EU priority polycyclic aromatic hydrocarbons in food supplements using high performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector by Sophie Danyi; François Brose; Catherine Brasseur; Yves-Jacques Schneider; Yvan Larondelle; Luc Pussemier; Johan Robbens; Sarah De Saeger; Guy Maghuin-Rogister; Marie-Louise Scippo (293-299).
High performance liquid chromatography coupled to an ultraviolet, diode array or fluorescence detector (HPLC/UV-FLD) has been used to set up a method to detect the 15(+1) EU priority polycyclic aromatic hydrocarbons (PAHs) in food supplements covering the categories of dried plants and plant extracts excluding oily products. A mini validation was performed and the following parameters have been determined: limit of detection, limit of quantification, precision, recovery and linearity. They were in close agreement with quality criteria described in the Commission Regulation (EC) No 333/2007 concerning the PAH benzo[a]pyrene in foodstuffs, except the not fluorescent cyclopenta[c,d]pyrene for which the UV detection leads to a higher limit of detection. Analysis of twenty commercial food supplements covering mainly the class of dried plants was performed to evaluate their PAHs contamination levels and to test the applicability of the method to various plant matrices. Fifty percent of analyzed samples showed concentration exceeding 2 μg kg−1 for one or more PAHs.
Keywords: Polycyclic aromatic hydrocarbons; High performance liquid chromatography; Fluorescence detection; Food supplement; Analytical procedure validation;