Analytical and Bioanalytical Chemistry

An isotope-dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) measurement procedure was developed to accurately quantify amino acid concentrations in National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 2389a-amino acids in 0.1 mol/L hydrochloric acid. Seventeen amino acids were quantified using selected reaction monitoring on a triple quadrupole mass spectrometer. LC-MS/MS results were compared to gravimetric measurements from the preparation of SRM 2389a-a reference material developed at NIST and intended for use in intra-laboratory calibrations and quality control. Quantitative mass spectrometry results and gravimetric values were statistically combined into NIST-certified mass fraction values with associated uncertainty estimates. Coefficients of variation (CV) for the repeatability of the LC-MS/MS measurements among amino acids ranged from 0.33% to 2.7% with an average CV of 1.2%. Average relative expanded uncertainty of the certified values including Types A and B uncertainties was 3.5%. Mean accuracy of the LC-MS/MS measurements with gravimetric preparation values agreed to within |1.1|% for all amino acids. NIST SRM 2389a will be available for characterization of routine methods for amino acid analysis and serves as a standard for higher-order measurement traceability. This is the first time an ID LC-MS/MS methodology has been applied for quantifying amino acids in a NIST SRM material.

Procedures for estimating the measurement uncertainty for the acidity constant Ka (or the pKa value) in different media (I=0 and I=0.1 mol L(-1) KCl), as determined by potentiometric titration, are presented. The uncertainty budgets (the relative contributions of the different input quantities to the uncertainty in the result) of the pKa (I=0) and pKa (I=0.1 mol L(-1) KCl) values are compared. Unlike the values themselves, the uncertainties and uncertainty budgets of the values are comparable. The uncertainty estimation procedures are based on mathematical models of pKa measurement and involve the identification and quantification of individual uncertainty sources according to the ISO GUM approach. The mathematical model involves 52 and 48 input parameters for pKa (I=0) and pKa (I=0.1 mol L(-1) KCl), respectively. The relative importance of each source of uncertainty is discussed. In both cases, the main contributors to the uncertainty budget are the uncertainty components due to the hydrogen ion concentration/activity measurement, which provide 63.7% (for pKa (I=0)) and 89.3% (for pKa (I=0.1 mol L(-1) KCl)) of the uncertainty. The remaining uncertainty contributions arise mostly from the limited purity of the acid. From this work, it is clear that the uncertainties of the pKa (I=0.1 mol L(-1) KCl) values tend to be lower than those of the pKa (I=0) values. The main reasons for this are that: (1) the uncertainty due to the residual liquid junction potential is nominally absent in the case of pKa (I=0.1 mol L(-1) KCl) due to the similarly high concentrations of background electrolyte in the calibration solutions and measured solution; (2) the electrode system is more stable in solutions containing the 0.1 mol L(-1) KCl background electrolyte and so the readings obtained in these solutions are more stable.

The fabrication of novel iron-doped barium strontium titanate thin films by means of radio frequency (RF) magnetron co-sputtering is shown. Investigations of the elemental composition and the dopant distribution in the thin films obtained by X-ray photoelectron spectroscopy, Rutherford backscattering spectrometry, and time-of-flight secondary ion mass spectroscopy reveal a homogeneous dopant concentration throughout the thin film. The incorporation of the iron dopant and the temperature-dependent evolution of the crystal structure and morphology are analyzed by electron paramagnetic resonance spectroscopy, X-ray diffraction, Raman spectroscopy, atomic force microscopy, and scanning electron microscopy. In summary, these results emphasize the RF magnetron co-sputter process as a versatile way to fabricate doped thin films. Figure Cross section of the RF magnetron co-sputter setup and the X-ray phototelectron spectroscopy iron spectrum of a co-sputtered iron doped Barium strontium titanate thin film

A novel inexpensive 0.6 MPa (84 psi) flow system based on a low-cost diaphragm pump has been developed. The unfavourable strong pulsation of the pump has been overcome by using highly flexible silicone tubing as a pulse suppression coil. This results in a smooth pulse-free continuous flow of 100 mL min(-1) in circulation. This flow rate is much too high for a flow-injection system; however, with a restrictor capillary the flow rate required can be tapped off down to a range of 0.1-50 mL min(-1). By employing diaphragm pumps in an analytical flow system the pressure gap between HPLC pumps (2-40 MPa) and peristaltic pumps (<0.2 MPa), mainly used in FIA systems, can be filled. Due to the higher pressure delivered by diaphragm pumps relative to peristaltic pumps, the new flow system can be applied to on-line sample pre-concentration and matrix separation in flame AAS and ICP-OES by using standard HPLC pre-columns or small ion-exchange columns, respectively. In this way, very low detection limits in flame AAS have been reached (Cd 0.07 micro g L(-1), Cu 0.05 micro g L(-1), Co 0.9 micro g L(-1), Ni 0.8 micro g L(-1), Mn 0.7 micro g L(-1), Pb 0.8 micro g L(-1) and Tl 0.2 micro g L(-1)).

The adsorption of the aromatic molecule hexabenzobenzene (coronene) on an HOPG(0001) surface was investigated under UHV conditions by means of variable temperature scanning tunneling microscopy (STM) and spectroscopy (STS). Imaging on a mesoscopic scale showed a distribution of coronene islands. These islands are mobile on the surface and can be pinned at step-edges. Zooming in on areas apart from the islands reveals an hexagonal arrangement of coronene molecules in a closed layer. Submolecular resolved molecules consist of bright spots with varying intensity. This variation in intensity is explained with the commensurability of the adlayer. STS investigations were performed for various tip-sample distances, adjusted by the tunneling current setpoint. A gap can be seen for every setpoint, but its width is dependent on the setpoint. The gap for the largest tip-sample distance and therefore the smallest tip-sample interaction is compared with the theoretical value.

We report on the first study of the cleaved (001) topaz surface and the characterization of the chemical composition and atomic arrangement of the surface. We conclude that there is strong evidence for a hydroxyl group termination appropriate for further chemical reactions. The surface itself is easily accessible, atomically flat and suitable for potential technological applications.

This pilot study was performed to study the main metabolic reactions of four synthetic cannabinoids: JWH-015, JWH-098, JWH-251, and JWH-307 in order to setup a screening method for the detection of main metabolites in biological fluids. In silico prediction of main metabolic reactions was performed using MetaSite™ software. To evaluate the agreement between software prediction and experimental reactions, we performed in vitro experiments on the same JWHs using rat liver slices. The obtained samples were analyzed by liquid chromatography-quadrupole time-of-flight and the identification of metabolites was executed using Mass-MetaSite™ software that automatically assigned the metabolite structures to the peaks detected based on their accurate masses and fragmentation. A comparison between the experimental findings and the in silico metabolism prediction using MetaSite™ software showed a good accordance between experimental and in silico data. Thus, the use of in silico metabolism prediction might represent a useful tool for the forensic and clinical toxicologist to identify possible main biomarkers for synthetic cannabinoids in biological fluids, especially urine, following their administration. Figure JWH-098: Most probable predicted sites of metabolism and main metabolites formed in vitro

The in vitro microsomal metabolism of JWH-015, a ligand that exhibits a high binding affinity at the peripheral cannabinoid receptor CB(2), has been studied. A total of 22 metabolites were identified and structurally characterized. The metabolites are products of: 1) monohydroxylation on the naphthalene ring (m/z 344, M20 and M21), indole ring (m/z 344, M17 and M18), or the N-alkyl group (m/z 344, M14); 2) arene oxidation leading to dihydrodiols (m/z 362, M12 and M15); 3) dihydroxylation on the naphthalene ring (m/z 360, M7) or indole ring (m/z 360, M13), resulting from a combination of monohydroxylations on both the naphthalene and indole rings (m/z 360, M16), or a combination of monohydroxylations on the naphthalene ring and on the N-propyl group (m/z 360, M9); 4) trihydroxylation (m/z 378, M1, M3, M4, M6, and M10); 5) N-dealkylation (m/z 286, M19); 6) N-dealkylation and monohydroxylation on the naphthalene ring (m/z 302, M11); 7) N-dealkylation and dihydrodiol formation from arene oxidation (m/z 320, M2 and M5); 8) dehydrogenation after monohydroxylation on the N-alkyl group (m/z 326, M22); 9) dehydrogenation and monohydroxylation on the indole ring (m/z 342, M8).

Analysis of drugs in hair is often used as a routine method to obtain detailed information about drug ingestion. However, few studies have been conducted on deposition of synthetic cannabinoids and metabolites in hair. The first purpose of this study was to establish and validate an analytical method for detection of JWH-018, JWH-073, and their metabolites in hair, by use of UHPLC-MS-MS, for forensic application. The second purpose was to investigate the distribution of synthetic cannabinoids metabolites in hair and the effect of hair pigmentation, by use of an animal model. For this, JWH-073 was chosen as a representative synthetic cannabinoid. Finally, the developed method was applied to hair samples from 18 individuals suspected of synthetic cannabinoids use. JWH-018, JWH-073, and their metabolites were extracted from hair with methanol. The extract was then filtered and analyzed by UHPLC-MS-MS with an electrospray ion source in positive-ionization mode. Validation proved the method was selective, sensitive, accurate, and precise, with acceptable linearity within the calibration ranges. No significant variations were observed when different sources of both human and rat hair were used. The animal study demonstrated that JWH-073 N-COOH M was the major metabolite of JWH-073 in rat hair, and hair pigmentation did not have a significant effect on incorporation of JWH-073 and its metabolites into hair. In the analysis of 18 authentic hair samples, only JWH-018, JWH-018 N-5-OH M, and JWH-073 were detected, with wide variation in concentrations.

A potent synthetic cannabinoid receptor agonist, JHW-018, was recently detected as one of the most prominent active agents in abusively used incenses such as Spice and other herbal blends. The high pharmacological and addictive potency of JWH-018 highlights the importance of elucidating the metabolism of JWH-018, without which a meaningful insight into its pharmacokinetics and its toxicity would not be possible. In the present study, the cytochrome P450 phase I metabolites of JWH-018 were investigated, after in vitro incubation of the drug with human liver microsomes, followed by liquid chromatography-tandem mass spectrometry analysis. This revealed monohydroxylation of the naphthalene ring system, the indole moiety, and the alkyl side chain. In addition, observations were made of dihydroxylation of the naphthalene ring system, and the indole moiety, or as result of a combination of monohydroxylations of both the naphthalene ring system and the indole moiety or the alkyl side chain, or a combination of monohydroxylations of both the indole ring system and the alkyl side chain. There is also evidence of trihydroxylation at different locations of the hydroxyl groups in the molecule. Furthermore, dehydration of the alkyl side chain, in combination with both monohydroxylation and dihydroxylation as well as arene oxidation of the naphthalene ring system, combined with both monohydroxylation and dihydroxylation at different sites of oxidation were found. N-dealkylation also in combination with both monohydroxylation and dihydrodiol formation of the N-dealkylated metabolite was detected. Finally, a metabolite was found carboxylated at the alkyl side chain.

The application of near-infrared (NIR) dyes (λ em > 750 nm) to the analysis of biological samples shows much promise, because the long emission wavelengths of such dyes allow interferences from biomolecule matrices to be minimized. In this paper, a novel NIR dye, 5,5′-dicarboxy-1,1′-disulfobutyl-3,3,3′,3′-tetramethylindotricarbocyanine (DCDSTCY) has been developed for the spectrophotometric determination of total protein in serum. Under acidic conditions, the binding of DCDSTCY to proteins caused a new peak at 878 nm, the height of which was proportional to the concentration of protein. The linear range of the method was found to be 0.04–0.5 μg mL−1 for bovine serum albumin (BSA) and human serum albumin (HSA), and detection limits of 5 ng mL−1 were obtained for these substances. The maximum binding number of BSA with DCDSTCY was measured to be 133. The method proposed here has been applied to the quantitation of total protein in serum, and recoveries of 96.6–104% were achieved. Figure Near-infrared probe for protein determination

1,1,3,3-Tetramethylguanidine (TMG), methanol and carbon dioxide were investigated as switchable polarity solvents (SPS) in the simultaneous derivatization and extraction of triacylglycerols for the gas chromatographic (GC) characterization of olive oil. Three commercial olive oils were used as test samples. Results of the developed method did not differ statistically from those provided by reference derivatization procedures. The transesterification reaction was carried out under a very mild condition, one step and in situ, and no particular matrix interferences were evidenced. The method represented the first example of the use of a switchable polarity mixture for the preparation of methyl ester derivatives of fatty acids (FAME).

A novel luminescent lifetime optrode is presented for the detection of gaseous and dissolved oxygen. The optrode utilises ruthenium (4,7-diphenyl-1,10-phenanthroline)(3)Cl(2) as the sensing fluorophore immobilised in a hydrophobic ormosil matrix. The ormosil matrix is synthesised at room temperature from octyltriethoxysilane and methyltriethoxysilane precursors. Investigations of different ormosils were conducted and the most effective one was selected for optrode production. Optrodes were tested for responses to gaseous and dissolved oxygen. Their responses were modelled using traditional two-site or two-exponential methods and feed-forward artificial neural networks. Comparison of the two modelling methodologies is presented and further improvements in modelling and ormosil design are suggested.

A new lanthanide-sensitized luminescence system: europium-sparfloxacin-1,10-phenanthroline-sodium dodecyl sulfate has been discovered. The spectrofluorimetric properties of the system were studied. The effect of experimental conditions on the fluorescence intensity was defined. Under the optimum conditions, the fluorescence intensity of the system is a linear function of the concentration of europium in the range 5.0 x 10(-9) - 1.0 x 10(-6) mol L(-1) and the detection limit is 1.0 x 10(-10) mol L(-1). The system was used for the determination of trace amounts of europium in rare earth samples with satisfactory results.

The capillary electrophoretic separation was accomplished for Fe(II) and Ni(II) precomplexed with 1,10-phenanthroline (phen) in 2 M n-butyric acid/ n-butyrate buffer at pH 4.5 with direct UV detection at 260 nm. The applied voltage was 5 kV. The high concentration buffer of the n-butyrate resulted in a similar separation mechanism to that of ion-pair reversed-phase high-performance liquid chromatography. The separation would be due to the hydrophobic interaction between the ionic associates, [Fe(phen)(3)]( n-butyrate)(+) and [Ni(phen)(3)]( n-butyrate)(+), with the n-butyrate ion and n-butyric acid as background electrolyte. Linear calibration ranges were obtained for Fe(II) and Ni(II) from 100 to 500 ng ml(-1). The relative standard deviations ( n=10) for 3 g mL(-1) Fe(II) and Ni(II) were 0.090 and 0.086, respectively. Detection limits ( S/ N=3) for Fe(II) and Ni(II) were 20 ng mL(-1). The method was applied to the determination of nickel in aluminium and duralumin alloys.

A solvent-soluble membrane filter is proposed for the simple and rapid preconcentration and spectrophotometric determination of ascorbic acid based on the reduction of 1, 10-phenanthroline (phen)-iron (III), which is collected on a nitrocellulose membrane filter as an ion-associate of the cationic complex of tri,phen-iron (II) [ferroin, Fe(phen) 32+] with an anionic surfactant (of dodecyl sulfate). The ion-associate collected is dissolved in a small volume of 2-methoxyethanol together with the filter. The colour intensity is measured at 510 nm against the reagent blank and is proportional to the content of ascorbic acid in the range 2.5–50 μg ascorbic acid in 5 ml of solvent with excellent reproducibility (RSD 3.2% for 200 μg 1−1 ascorbic acid), the enrichment factor achieves 100-fold and detection limits better than 2.0 μg 1−1 can be obtained. Diverse components of organic and inorganic compounds normally present in fruits, vegetable, beverages and urine do not interfere. The recoveries of the ascorbic acid added to the samples are quantitative.

Three new polymeric chiral stationary phases were synthesized based on (1S,2S)-1,2-bis(2,4,6-trimethylphenyl)ethylenediamine, (1S,2S)-1,2-bis(2-chlorophenyl)ethylenediamine, and (1S,2S)-1,2-di-1-naphthylethylenediamine via a simple free-radical-initiated polymerization in solution. These monomers are structurally related to (1S,2S)-1,2-diphenylethylenediamine which is the chiral monomer used for the commercial P-CAP-DP polymeric chiral stationary phase (CSP). The performance of these three new chiral stationary phases were evaluated in normal phase high-performance liquid chromatography (HPLC) and supercritical fluid chromatography and the results were compared with those of the P-CAP-DP column. All three new phases showed enantioselectivity for a large number of racemates with a variety of functional groups, including amines, amides, alcohols, amino acids, esters, imines, thiols, and sulfoxides. In normal phase, 68 compounds were separated with 28 baseline separations (Rs ≥ 1.5) and in SFC, 65 compounds were separated with 24 baseline separations. In total 72 out of 100 racemates were separated by these CSPs with 37 baseline separations. Complimentary separation capabilities were observed for many analytes. The new polymeric CSPs showed similar or better enantioselectivities compared with the commercial column in both HPLC and SFC. However, faster separations were achieved on the new stationary phases. Also, it was shown that these polymeric stationary phases have good sample loading capacities while maintaining enantioselectivity. Figure Faster separations on new polymeric chiral stationary phases in HPLC

Doped organically modified silica nanoparticles (ORMOSIL NPs) with luminescent molecules represent a potent approach to signal amplification in biomolecule labeling. Herein, we report the synthesis of new ORMOSIL NPs incorporating thermochemiluminescent (TCL) 1,2-dioxetane derivatives to prepare TCL labels for ultrasensitive immunoassay, displaying a detectability comparable to those offered by other conventional luminescence-based systems. Amino-functionalized ORMOSIL NPs were synthesized for inclusion of acridine-containing 1,2-dioxetane derivatives with a fluorescence energy acceptor. The doped ORMOSIL NPs were further functionalized with biotin for binding to streptavidin-labeled species to be used as universal detection reagents for immunoassays. A quantitative non-competitive immunoassay for streptavidin has been developed by immobilizing anti-streptavidin antibody to capture streptavidin, then the antibody-bound streptavidin was detected by the biotinylated TCL ORMOSIL NPs. The analytical performance was similar to that obtained by chemiluminescent (CL) detection using horseradish peroxidase (HRP) as label, being the limits of detection 2.5-3.8 and 0.8 ng mL(-1) for TCL and CL detection, respectively. In addition, since the TCL emission is simply initiated by thermolysis of the label, chemical reagents were not required, thus allowing reagentless detection with a simplification of the analytical protocols. A compact mini dark box device based on the use of a cooled charge-coupled device (CCD) and a miniaturized heater has been developed and used to quantify the light emission after heat decomposition of the label at a temperature of 90-120 °C. These characteristics make TCL-doped ORMOSIL NPs ideal universal nanoprobes for ultrasensitive bioassays such as immuno- and DNA-based assay.

The transfer mechanism of an amphoteric rhodamine, sulforhodamine 101 (SR101), across the polarized water/1,2-dichloroethane (DCE) interface was investigated using cyclic voltammetry, differential voltfluorometry and potential-modulated fluorescence (PMF) spectroscopy. The voltammetric response for the ion transfer of SR101 monoanion from water to DCE was observed as the diffusion-controlled transfer process. An unusual voltammetric response was found at 0.15 V more negative than the formal transfer potential of SR101(-) (deltaW(O)phi degrees') in the cyclic voltammogram and voltfluorogram. The frequency dependence of the PMF responses confirmed the presence of the adsorption processes at negative potentials. In addition, a further transient adsorption step was uncovered at deltaW(O)phi degrees'. The interfacial mechanism of SR101 is discussed by comparing the results obtained from each technique.

Dear Sir,Recently, Spichtig et al. [1] published in this journal aTechnical Note regarding the analysis of sialic acids frominfant formulas using 1,2-diamino-4,5-methylenedioxyben-zene derivatives, a method previously proposed by us in2007 [2]. In this Technical Note, Spichtig et al. stated thatlactose does not interfere with the analysis, conversely towhat we hypothesized in our article. Although in the workreported in their Technical Note they did not include infantformulas or milk, in workpublished in 2010 [3]they appliedthe proposed method to several dairy products, includinginfant formulas. In the most recent work, they used com-mercial lactose and quantified fetuin sialic acids when thisprotein was in lactose solution (no concentration reported)and in aqueous solution. They reported an increase of30 μg/100 mg in the former solution when comparedwith the latter, which they attributed to a certain amountof sialyllactose present in the commercial lactose be-cause of its dairy origin. This residual sialic acid in lactosewas quantified (6.5-24.7 μg/100 mg), and the concentrationdiffered withthe supplierand was consideredverylow bytheauthors.Regarding this controversial topic, and in response to theaforementioned article, we would like to clarify our previousreport.First,weobservedaproblemintheaccuratequantitationof sialic acids from infant formulas and proposed a way toeffectively resolve this via an additional purification step. Wesuggested that lactose could be a possible reason for the ob-served phenomenon. In fact, in a series of recent tests in ourlaboratory, when commercial lactose (from Sigma) was spikedwith different amounts of N-acetylneuraminic acid (Neu5Ac)and quantified using the method reported the recovery in-creased as the amount of Neu5Ac added increased: 5.15 μgwas recovered when 5 μg was added, but 19.55 μg wasrecoveredwhen15μg was added. This represents a matrixeffect caused by lactose that produces a systematic error pro-portional to the concentration of Neu5Ac. So, we do not agreewith the statement in the abstract of the technical note thatlactose does not interfere with the analysis and we stronglyrecommend the purification step if accurate quantitation ofsialic acids in infant formulas is required.References

A sensitive and accurate method utilizing ultrahigh performance liquid chromatography (U-HPLC) coupled to high resolution mass spectrometry based on orbitrap technology (orbitrapMS) for the analysis of nine 3-chloropropane-1,2-diol (3-MCPD) diesters in vegetable oils was developed. To remove the interfering triacylglycerols that induce strong matrix effects, a clean-up step on silica gel column was used. The quantitative analysis was performed with the use of deuterium-labeled internal standards. The lowest calibration levels estimated for the respective analytes ranged from 2 to 5 μg kg(-1). Good recovery values (89-120%) and repeatability (RSD 5-9%) was obtained at spiking levels of 2 and 10 mg kg(-1). As an alternative, a novel ambient desorption ionization technique, direct analysis in real time (DART), hyphenated with orbitrapMS, was employed for no separation, high-throughput, semi-quantitative screening of 3-MCPD diesters in samples obtained by chromatographic fractionation. Additionally, the levels of 3-MCPD diesters measured in reallife vegetable oil samples (palm oil, sunflower oil, rapeseed oil) using both methods are reported. Relatively good agreement of the data generated by U-HPLC-orbitrapMS and DART-orbitrapMS were observed. With regard to a low ionization yield achieved for 3-MCPD monoesters, the methods presented in this paper were not yet applicable for the analysis of these contaminants at the naturally occurring levels.

The fatty acid esters of 3-(N-phenylamino)propane-1,2-diol (PAP) are biomarkers of toxic oil batches that caused toxic oil syndrome (TOS), an intoxication that caused over 400 deaths and affected 20,000 people in Spain in 1981. PAP esters are converted into PAP by human pancreatic lipase. The in vivo biotransformation of PAP in two mouse strains generated potentially toxic metabolites. Here we report an enzyme-linked immunosorbent assay (ELISA) for PAP detection incorporating antibodies generated using PAP-hapten derivatives 1 and 2. The immunizing haptens were designed to recognize the phenylamino and hydroxymethylene moieties of the PAP structure. The antisera raised against 1-HCH showed greater affinity for free PAP, as demonstrated in competitive experiments using either 1-BSA or 2-BSA as coating antigens. The developed ELISA detects PAP at a threshold of 130 μg L−1 and can be used over a wide range of pH and ionic strength values. The assay can be applied to human urine samples, after a simple treatment method, with good recovery according to the correlation obtained when analyzing blind spiked urine samples. Figure Development of an ELISA for PAP in human urine

In 2007, Martin et al. developed a method for the analysis of sialic acids by HPLC following 1,2-diamino-4,5-methylenedioxybenzene (DMB) derivatisation (Martín et al., Anal Bioanal Chem 387:2943-2949, 2007). Within the article, the authors noted that lactose interfered with the analysis, giving erroneously high results when lactose-containing products were analysed. Such an observation is important when analysing milk-based products, yet was contradictory to the observations of Nakamura et al. (Chem Pharm Bull 35(2):687-692, 1987) who demonstrated that DMB was specific for α-keto acids and did not react with simple sugars such as glucose or lactose. In order to clarify the situation, this phenomenon was investigated and it was confirmed that lactose does not interfere with the analysis. However, it was found that most commercial preparations of lactose do contain small amounts of sialic acids, either as the free monosaccharide or bound to lactose in the form of 3'- and 6'-sialyllactose.

We have developed a highly selective and sensitive method using gas chromatography-mass spectrometry with negative chemical ionization for measuring 3-chloropropane-1,2-diol (3-MCPD) in rat blood and urine. Samples were adsorbed on silica gel, extracted with ethyl acetate, and derivatized by chemical derivatization with heptafluorobutyric acid anhydride. For quantification, matrix-based calibration curves and 3-MCPD-d (5), as an isotope-labeled internal standard, were used. The relative recoveries of 3-MCPD were between 80 and 110% in most cases and the relative standard deviations were typically less than 10%, with some exceptions. The limit of quantification of the method was found to be about 2 ng/mL. In conclusion, a valuable, robust, and sensitive method for detection of 3-MCPD is now available for biokinetics studies.

The previously introduced technique of potential-modulated fluorescence (PMF) spectroscopy was used to study the potential-induced fluorescence change of some different dyes at the polarized 1,2-dichloroethane (DCE)/water (W) interface. A zwitterionic dye (POLARIC 488PPS) showed a PMF response similar to that for the previously studied dye (di-4-ANEPPS) with the same ionic state, and the PMF response was likewise explained by the potential-dependent reorientation of the dye at the DCE/W interface. Though a monocationic dye (POLARIC 488PM) showed no distinct PMF signal, a dicationic dye (di-2-ANEPEQ) showed two relatively weak but detectable PMF signals at lower and higher potential. It has thus been found that the ionic state of a potential-sensitive dye strongly influences the potential-induced reorientation of the dye at the interface and consequently its PMF response. These results support the reorientation/solvatochromic mechanism proposed for "slow" dyes but do not necessarily exclude the electrochromic mechanism proposed for "fast" dyes. PMF spectroscopy would provide useful information on the design of slow dyes for the measurement of the resting potential of cell membranes.

A silver-selective optode membrane incorporating hexathia-18-crown-6 for cation recognition and a lipophilic chromoionophore 1,2-benzo-3-octadecanoylimino-7-diethylaminophenoxazine for transduction has been prepared. The PVC membrane composition was optimized to result in the largest working concentration range. The response range of the proposed optode is 5.0 x 10(-9)-5.0 x 10(-5) mol L(-1) Ag(+) with a limit of detection of 1.0 x 10(-9) mol L(-1). The probe works well at pH 5.0 and revealed small ion interference and good selectivity, reproducibility and stability.

The spectrofluorometric behavior of a membrane potential-sensitive dye, 1-(3-sulfonatopropyl)-4-[β-{2-(di-N-butylamino)-6-naphthyl}vinyl]pyridinium betaine (di-4-ANEPPS), at the polarized 1,2-dichloroethane/water interface was studied by means of potential-modulated fluorescence (PMF) spectroscopy. The results, combined with those from cyclic and alternating current voltammetry, clearly suggested that the dye adsorbed at the interface underwent a reorientation with increasing the interfacial potential, giving a well-developed PMF response as well as a voltammetric response. In addition to the PMF response, another PMF response was observed by addition of dilauroyl phosphatidylcholine (DLPC). This additional response was well explained in terms of a reorientation of di-4-ANEPPS at the interface, which would be induced by the potential-dependent desorption of DLPC from the interface. Thus, the present study supported the reorientation/solvatochromic mechanism for the membrane potential-sensitive dye rather than the electrochromic mechanism. Figure Reaction model for the potential-induced reorientation of di-4-ANEPPS at the O/W interface in the presence of DLPC in the DCE phase.

Polyurethane foam (PUF) has been suggested as a solid polymeric reagent for determination of nitrite. The determination is based on the diazotization of end toluidine groups of PUF with nitrite in acidic medium followed by coupling of polymeric diazonium cation with 3-hydroxy-7,8-benzo-1,2,3,4-tetrahydroquinoline. The intensely colored polymeric azodye formed in this reaction can be used as a convenient analytic form for the determination of nitrite by diffuse reflectance spectroscopy (c min = 0.7 ng mL−1). The possibility of using a desktop scanner, digital camera, and computer data processing for the numerical evaluation of the color intensity of the polymeric azodye has been investigated. A scanner and digital camera can be used for determination of nitrite with the same sensitivity and reproducibility as with diffuse reflectance spectroscopy. The approach developed was applied for determination of nitrite in river water and human exhaled breath condensate.

The stereoisomers of 1,2,3,4-tetrahydroisoquinoline analogs were resolved for the first time by applying a polar ionic mobile phase on a quinine or a quinidine moiety fused with a chiral sulfonic acid-type chiral selector immobilized on silica [Chiralpak ZWIX(+)™ and Chiralpak ZWIX(-)™]. The effects of the nature and concentrations of the mobile phase components and additives and temperature on the retention and enantioseparation on the investigated chiral columns were studied. Experiments were performed in the temperature range 10-50 °C. Thermodynamic parameters were calculated from plots of ln α versus 1/T. The separations were generally enthalpy-controlled, but entropy-controlled separation was also observed below 30 °C. The enantiomer elution order was determined in some cases and was observed to be opposite on the ZWIX(+)™ and ZWIX(-)™ columns. Our results contribute to a better understanding of the enantiorecognition mechanism of chiral bases with chiral zwitterionic selectors.

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) library which contains over 5,600 spectra of 1,253 compounds relevant in clinical and forensic toxicology has been developed using a hybrid tandem mass spectrometer with a linear ion trap. Pure compound solutions-in some cases solutions made of tablets-were prepared and 1 to 2,000 ng of each compound were injected into the system using standard reversed-phase analytical columns with gradient elution. To obtain maximum mass spectral information enhanced product ion spectra were acquired with positive and/or negative ionization at low, medium, and high collision energies and additionally applying collision energy spread. In this mode, all product ions generated by the different collision energies are trapped in the linear ion trap prior to their detection. The applicability of the library for other types of hybrid tandem mass spectrometers with a linear ion trap of the same manufacturer as well as a standard triple-quadrupole tandem mass spectrometer has been investigated with a selection of compounds. The spectra of the developed library can be used to create methods for target analysis, either screening methods or quantitative procedures by generating transitions for multiple reaction monitoring. For those procedures, suitable transitions and convenient collision energies are selected from the library. It also has been utilized to identify compounds with a multi target screening approach for clinical and forensic toxicology with a standardized and automated system. The novel aspects compared to our former library produced with a standard triple-quadrupole mass spectrometer are the enlargement of the ESI-MS/MS library and the additional acquisition of spectra with collision energy spread.

A novel reaction of cyanide with 2,2-dihydroxy-1,3-indanedione in the presence of sodium carbonate is described. It is highly selective and sensitive, and suitable for the determination of hydrogen cyanide in the environment and free cyanide ions in water, blood, urine, serum, etc. As little as 1.25x10(-7) mol x L(-1) CN(-) (3.25x10(-9) g x mL(-1) cyanide) can be determined by use of this reaction. The color system obeys Beer's law in the range 10 ng x mL(-1) to 1.0 microg x mL(-1) at 510 nm. The molar absorptivity was 8.0x10(4) L x mol(-1) x cm(-1) for a solution of concentration 0.2 microg x mL(-1). All other important analytical properties of the reaction have been studied. It is proposed that the purple color produced under these reaction conditions is that of 2-cyano-1,2,3-trihydroxy-2 H indene.

1,3-Butadiene and acrylonitrile are important industrial chemicals that have a high production volume and are ubiquitous environmental pollutants. The urinary mercapturic acids of 1,3-butadiene and acrylonitrile-N-acetyl-S-(3,4-dihydroxybutyl)cysteine (DHBMA) and MHBMA (an isomeric mixture of N-acetyl-S-((1-hydroxymethyl)-2-propenyl)cysteine and N-acetyl-S-((2-hydroxymethyl)-3-propenyl)cysteine) for the former and N-acetyl-S-2-cyanoethylcysteine (CEMA) for the latter-are specific biomarkers for the determination of individual internal exposure to these chemicals. We have developed and validated a fast, specific, and very sensitive method for the simultaneous determination of DHBMA, MHBMA, and CEMA in human urine using an automated multidimensional LC/MS/MS method that requires no additional sample preparation. Analytes are stripped from urinary matrix by online extraction on a restricted access material, transferred to the analytical column, and subsequently determined by tandem mass spectrometry using labeled internal standards. The limits of quantification (LOQs) for DHBMA, MHBMA, and CEMA were 10 microg/L, 2 microg/L, and 1 microg/L urine, respectively, and were sufficient to quantify the background exposure of the general population. Precision within series and between series for all analytes ranged from 5.4 to 9.9%; mean accuracy was between 95 and 115%. We applied the method on spot urine samples from 210 subjects from the general population with no occupational exposure to 1,3-butadiene or acrylonitrile. A background exposure of the general population to acrylonitrile was discovered that is basically influenced by individual exposure to passive smoke as well as active smoking habits. Smokers showed a significantly higher excretion of MHBMA, whereas DHBMA levels did not differ significantly. Owing to its automation, our method is well suited for application in occupational or environmental studies.

A new, simple, rapid, and sensitive spectrophotometric method has been developed for the determination of manganese in sewage. The method is based on the reaction of manganese with derivatives of 1,3,3-trimethyl-2-[3-(1,3,3-trimethyl-1,3- H-indol-2-ylidene)propenyl]-3 H-indolium to form a colored ion associate with a sensitive absorption maximum at 560 nm. The appropriate reaction conditions have been established: pH 8.5-10.0, 1.25-2.3 x 10(-3) mol L(-1) 1-nitroso-2-naphthol, and 1.6-2.4 x 10(-4) mol L(-1) dye reagent. Beer's law is obeyed for manganese concentrations up to 4.2 mg L(-1). The limit of detection is 0.01 mg L(-1) Mn(2+); the molar absorptivity of the ion associate was 7.5 x 10(4) L mol(-1) cm(-1). The effect of various foreign ions was examined. A reaction mechanism is suggested. The developed procedure was tested for determination of manganese in sewage with satisfactory precision and accuracy.

A rapid analytical method for sensitive determination of 1,3-dichloro-2-propanol (1,3-DCP) in river water has been developed. 1,3-DCP is extracted from water with ethyl acetate. After filtration through sodium sulfate the ethyl acetate phase is analyzed by gas chromatography-mass spectrometry. The method uses 1,3-DCP-d5 as internal standard. Different extraction solvents, concentrations of ammonium sulfate in the water samples, and the effect of humic acid were tested and their influence on the recovery of DCP has been evaluated. The method quantification limit was 0.1 microg L(-1). For spiked water samples (0-5.2 microg L(-1), n=21) a repeatability coefficient of variation of 5.4% was obtained. The average recovery rate of 1,3-DCP was 105+/-3% (n=21). Stability tests, which were carried out with Danube river water, led to an estimated 1,3-DCP degradation rate of 0.008+/-0.0008 day(-1) at 6 degrees C.

The absorption spectra of 4f electron transitions of the systems of neodymium and erbium with 2-isobutylformyl-1,3-dione-indane and TX-100 have been studied by normal and derivative spectrophotometry. Their molar absorptivities at the maximum absorption bands are about 7.2 (at 571 nm) times greater for neodymium and 13.1 (at 519 nm) times greater for erbium than those in the absence of the complexing agents. Use of second derivative spectra both eliminates the interference from other rare earths and increases the sensitivity from neodymium and erbium. Beer's law is obeyed from 0 approximately 20 microg/ml for neodymium and 0 approximately 25 microg/ml for erbium. The relative standard deviations are 1.2% and 1.6% for 5.0 microg/ml of neodymium and 8.0 microg/ml of erbium, respectively. The detection limits (signal-to-noise ratio=2) are 0.14 microg/ml and 0.20 microg/ml. A method for the direct determination of neodymium and erbium in rare earth mixtures with good accuracy and selectivity is proposed.

Novel and selective microbial amperometric biosensors that use Gluconobacter oxydans cells to monitor the bacterial bioconversion of glycerol (Gly) to 1,3-propanediol (1,3-PD) are described. Two different mediators, ferricyanide and flexible polyvinylimidazole osmium functionalized polymer (Os-polymer), were employed to prepare two different microbial biosensors, both of which gave high detection performance. The good operational stabilities of both types of biosensor were underlined by the ability to detect 1,3-PD throughout 140 h of continuous operation. Both microbial biosensor systems showed excellent selectivity for 1,3-PD in the presence of a high excess of glycerol [selectivity ratios (1,3-PD/Gly) of 118 or 245 for the ferricyanide and Os-polymer systems, respectively]. Further, the robustness of each microbial biosensor was highlighted by the high reliability of 1,3-PD detection achieved (average RSD of standards <2%, and well below 4% for samples). The biosensor implementing the Os-polymer mediator exhibited high selectivity towards 1,3-PD detection and allowed moderate sample throughput (up to 12 h−1) when integrated into a flow system. This system was used to monitor the concentration of 1,3-PD during a real bioprocess. Results from biosensor assays of 1,3-PD in bioprocess samples taken throughout the fermentation were in a very good agreement with results obtained from reference HPLC assays (R 2 = 0.999).

Organophosphate triesters tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate are widely used flame retardants (FRs) present in many products common to human environments, yet understanding of human exposure and health effects of these compounds is limited. Monitoring urinary metabolites as biomarkers of exposure can be a valuable aid for improving this understanding; however, no previously published method exists for the analysis of the primary TDCPP metabolite, bis(1,3-dichloro-2-propyl) phosphate (BDCPP), in human urine. Here, we present a method to extract the metabolites BDCPP and diphenyl phosphate (DPP) in human urine using mixed-mode anion exchange solid phase extraction and mass-labeled internal standards with analysis by atmospheric pressure chemical ionization liquid chromatography tandem mass spectrometry. The method detection limit was 8 pg mL−1 urine for BDCPP and 204 pg mL−1 for DPP. Recoveries of analytes spiked into urine ranged from 82 ± 10% to 91 ± 4% for BDCPP and from 72 ± 12% to 76 ± 8% for DPP. Analysis of a small number of urine samples (n = 9) randomly collected from non-occupationally exposed adults revealed the presence of both BDCPP and DPP in all samples. Non-normalized urinary concentrations ranged from 46–1,662 pg BDCPP mL−1 to 287–7,443 pg DPP mL−1, with geometric means of 147 pg BDCPP mL−1 and 1,074 pg DPP mL−1. Levels of DPP were higher than those of BDCPP in 89% of samples. The presented method is simple and sufficiently sensitive to detect these FR metabolites in humans and may be applied to future studies to increase our understanding of exposure to and potential health effects from FRs. Figure The flame retardant TDCPP is metabolized to BDCPP and detected in human urine.

The headspace solid-phase micro-extraction technique with on-fibre derivatisation followed by gas chromatography-tandem mass spectrometry has been evaluated for the analysis of 1,3-dichloro-2-propanol in water. An asymmetric factorial design has been performed to study the influence of five experimental factors: extraction time and temperature, derivatisation time and temperature and pH. The best extraction performance is achieved in the headspace mode, with 5 mL stirred water samples (pH 4) containing 1.3 g of NaCl, equilibrated for 30 min at 25 degrees C, using divinylbenzene-carboxen-polydimethylsiloxane as the fibre coating. On-fibre derivatisation has been used for the first time with 50 microL of bis(trimethylsilyl)trifluoroacetamide at 25 degrees C during 15 min, leading to effective yields. The proposed method provides high sensitivity, good linearity and repeatability (relative standard deviation of 5.1% for 10 ng mL(-1) and n = 5). The limits of detection and quantification were 0.4 and 1.4 ng mL(-1), respectively. Analytical recoveries obtained for different water samples were approx. 100%.

A new, simple, rapid, and sensitive spectrophotometric method has been developed for the determination of nitrophenols [picric acid (PA); dinitrophenols (DNP)] in wastewater samples. The method is based on the reaction of nitrophenols with 2-[(E)-2-(4-diethylaminophenyl)-1-ethenyl]-1,3,3-trimethyl-3 H-indolium chloride reagent to form the colored ion associates, which are extracted by organic solvents. The molar absorptivity of the ion associates of PA with the investigated reagent ranges from 8.3x10(4) to 11.3 x 10(4) L mol(-1) cm(-1), depending on the extractant. Because only PA is extracted in an acidic medium with the investigated reagent, but both PA and DNP are extracted in an alkaline medium, it is possible to determine both substances in a mixture. Appropriate reaction conditions have been established. The absorbance of the colored extracts obeys Beer's law in the range of 0.04-4.58 mg L(-1) PA, 1.0-18.4 mg L(-1) 2,4-DNP and 1.2-14.7 mg L(-1) 2,6-DNP, respectively. The limit of detections, calculated from a blank test (n=10; P=0.95), are 0.05 mg L(-1) PA, 0.9 mg L(-1) (2,4-DNP), and 1.1 mg L(-1) (2,6-DNP), respectively.

The infrared absorption spectra of 2-[4-( N -dodecanoylamino)phenyl]-5-(4-nitrophenyl)-1,3,4-oxadiazole (also denoted AF51) in solution and in the solid state as well as the solid-state Raman spectrum of the powdered compound are compared with the infrared linear dichroic (LD) spectra recorded at two orthogonal polarizations. The IR-LD spectra were measured in an anisotropic solvent-the nematic liquid crystal ZLI-1695 (Merck). The solvent spectrum taken under precisely the same experimental conditions and polarized radiation azimuths was subtracted from the polarized sample spectra in order to achieve pure oriented spectra of the compound studied. These spectra were further processed using a stepwise reduction procedure that allows for assignment of vibrational modes having mutually orthogonal dipole moments.

Two novel N-(2-mercapto-1,3,4-thiadiazol-5-yl)-N'-(4-substituted-arylacetyl) urea compounds have been synthesized, characterized by NMR and MS, and used as self-assembly reagents to form self-assembled monolayers (SAMs) on Pt electrodes. The modified electrodes were characterized by electrochemical methods. The electrochemical behavior of p-benzenediol at the SAMs electrodes was investigated. It was found that the electrochemical response to p-benzenediol is controlled by diffusion and can be electrocatalyzed to obtain more symmetrical redox peaks and higher voltammetric current response at the SAMs electrodes, with a peak separation of 80 mV. For p-benzenediol the process at the SAMs electrodes is quasi-reversible with a rate constant of 0.6742 s-1. The SAMs electrodes have been used to determine p-benzenediol by differential pulse voltammetry. The peak current was linear for concentrations of p-benzenediol in the range 1x10(-7)-5x10(-4) mol L-1 and the detection limit was 4.0x10(-8) mol L-1. The SAMs electrodes were used to determine p-benzenediol in real photographic developer and in a synthetic waste water sample; the standard addition recovery was in the range 96.6-100.4%.

The far infrared spectra of (100), (010), and (001)-oriented RDX single crystals were measured as the crystal was rotated about the axis perpendicular to the polarization plane of the incident radiation. Absorption measurements were taken at temperatures of both 20 K and 295 K for all rotations using terahertz time-domain spectroscopy. A number of discrete absorptions were found ranging from 10-100 cm(-1) (0.3-3 THz). The absorptions are highly dependent on the orientation of the terahertz polarization with respect to crystallographic axes.

Quantitative 1H NMR spectroscopy was used to study chemical equilibria and reaction kinetics of both the formation and decomposition of 1,3,5-trioxane in aqueous formaldehyde solutions. The reaction was homogeneously catalyzed with up to 0.10 g g(-1) sulfuric acid at temperatures between 360 and 383 K so that most of the experiments had to be carried out pressurized. The studied mixtures were complex due to the formation of methylene glycol and poly(oxymethylene) glycols in aqueous formaldehyde and the presence of considerable amounts of ionized species. Most common internal standards are decomposed by the hot sulfuric acid and external standards were not applicable using the flow NMR probe or pressurizable NMR sample tubes. Therefore, for the quantification of the small trioxane signals, a novel procedure was applied, in which electronically generated NMR signals were used as highly stable Virtual References (VR). The NMR decoupler channel with wave-form generator was used as the source of the reference signal, which was irradiated into the probe using the lock coil. Details on the experimental procedure are presented. It is shown that the presented method yields reliable quantitative reaction data for the complex studied mixtures.

1,3,5,7-Tetramethyl-8-(N-hydroxysuccinimidyl butyric ester)difluoroboradiaza-s-indacene (TMBB-Su), a new BODIPY-based fluorescent probe, was designed and synthesized for the labeling of amino compounds. It was used as a pre-column derivatizing reagent for determination of amino acid neurotransmitters by high-performance liquid chromatography (HPLC). The fluorescence quantum yield in acetonitrile increased from 0.84 to 0.95 when it reacted with amino acid neurotransmitters. Derivatization of TMBB-Su with seven amino acid neurotransmitters was completed within 30 min at 25 °C in 24.0 mmol L(-1) pH 7.8 boric acid buffer. The separation was performed on a C(18) column with methanol-water-buffer 55:35:10 (v/v) as mobile phase (buffer: 0.10 mol L(-1) H(3)Cit-0.10 mol L(-1) NaOH). Interference from the other concomitant amino acids was eliminated successfully by means of pH gradient elution. With fluorescence detection at 494 and 504 nm for excitation and emission, respectively, the limits of detection (signal-to-noise ratio = 3) were from 2.1 to 12.0 nmol L(-1). The proposed method has been used to determine amino acid neurotransmitters in the cerebral cortex of mice with cerebral ischemia at the convalescence stage with satisfactory recoveries varying from 94.9 to 105.2%.

To study the relationship between amounts of nitric oxide (NO) in blood and the development of ischemic cardio-cerebrovascular diseases, trace NO in human blood serum has, for the first time, been determined by use of a 1,3,5,7-tetramethyl-8-(3',4'-diaminophenyl)difluoroboradiaza-s-indacene (TMDABODIPY)-based HPLC method. The proposed method is simple, rapid, and efficient, owing to its high sensitivity and good selectivity for NO. TMDABODIPY and its NO derivative are separated to baseline in 4 min, with simple separation conditions, on a C(18) column eluted with 50 mmol L(-1) ethanolamine in methanol. The derivative is detected by fluorescence at an emission wavelength of 507 nm with excitation at 498 nm. The response is a linear function of concentration in the range 0.8-800 nmol L(-1) NO. The detection limit can reach 2 x 10(-11) mol L(-1) (signal-to-noise ratio=3). The method has been used to detect NO in the serum of patients with five kinds of ischemic cardio-cerebrovascular disease and two diseases closely connected with ischemic cardio-cerebrovascular diseases. Recoveries of NO from spiked serum samples were between 96.58 and 105.71% and concentrations of NO observed in real samples were at 10(-7) mol L(-1) levels. Our studies indicate that the proposed TMDABODIPY-based HPLC technique can be developed into a sensitive and new method for clinical assay and pathology research.