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Analytica Chimica Acta (v.685, #1)

Editorial Board (pp. iii).

Ion mobility spectrometry: A valuable tool for kinetic studies in enzymology by S. Armenta; M. Blanco (pp. 1-8).

The inherent characteristics of IMS such as reduced measurement time, in the seconds time scale, sensitivity and selectivity make this technique an ideal methodology for enzyme reaction monitoring. The capability of IMS in the determination of enzyme kinetics and inhibition studies by the analysis of substrate depletion and/or product formation using only a few microliters of solution has been successfully demonstrated on the example of acetylcholine hydrolysis catalyzed by acetylcholinesterase (AChE) and inhibited by neostigmine and galanthamine. Michaelis–Menten and Lineweaver–Burk plots were obtained for the enzyme catalyzed reaction with and without neostigmine and galanthamine inhibition at two different inhibitor concentrations. Typical plots of competitive inhibitors were obtained agreeing well with previous results published in the literature. IMS procedure provided a limit of detection for acetylcholine in the low ppm range, a precision of 4.8% and an analysis frequency of 40s, being those analytical characteristics appropriate to perform enzyme kinetic studies. IMS offers a new and efficient tool to study enzyme reactions either as a high throughput screening tool for hit discovery and lead development for drug discovery proposes or to indirectly perform enzymological studies.

Keywords: Ion mobility spectrometry; Enzyme kinetics; Inhibition; Acetylcholinesterase; Neostigmine; Galanthamine

Ion mobility spectrometry: A valuable tool for kinetic studies in enzymology by S. Armenta; M. Blanco (pp. 1-8).

Keywords: Ion mobility spectrometry; Enzyme kinetics; Inhibition; Acetylcholinesterase; Neostigmine; Galanthamine

A novel approach for the determination of paracetamol based on the reduction of N-acetyl- p-benzoquinoneimine formed on the electrochemically treated pencil graphite electrode by Ali Özcan; Yücel Şahin (pp. 9-14).

A novel approach was proposed for the simple and rapid electrochemical determination of paracetamol (PC) in the presence of uric acid in body fluids. The voltammetric determination of PC is based on the electrochemical reduction of N-acetyl-p-benzoquinoneimine formed simultaneously on the electrochemically treated pencil graphite (ETPG) electrode during the measurement. ETPG electrodes were prepared by the potential cycling between −0.3V and +2.0V in 0.1M H₃PO₄ solution. The electrochemical performance of the ETPG electrode was evaluated by adsorptive transfer stripping differential pulse voltammetry (ATSDPV). The resulting sensor showed good performance for the determination of PC in human blood serum samples with a linear range of 0.05–2.5μM and a highly reproducible response (RSD of 3.1%). The calculated detection limit was 2.5nM (S/N=3). The proposed method does not require any sample pretreatment, prevents the interference of uric acid and allows the determination of PC directly in blood serum samples.

Keywords: Paracetamol; Blood serum; Electrochemically treated pencil graphite electrode; Voltammetry; Uric acid

CYP450 biosensors based on screen-printed carbon electrodes for the determination of cocaine by Laura Asturias-Arribas; M. Asunción Alonso-Lomillo; Olga Domínguez-Renedo; M. Julia Arcos-Martínez (pp. 15-20).

A new electrochemical method has been described and characterized for the determination of cocaine using screen-printed biosensors. The enzyme cytochrome P450 was covalently attached to screen-printed carbon electrodes. Experimental design methodology has been performed to optimize the pH and the applied potential, both variables that have an influence on the chronoamperometric determination of the drug. This method showed a reproducibility of 3.56% ( n=4) related to the slopes of the calibration curves performed in the range from 19 up to 166nM. It has been probed the used of this kind of biosensors in the determination of cocaine in street samples, with an average capability of detection of 23.05±3.53nM ( n=3, α= β=0.05).

Keywords: Enzymatic biosensor; Screen-printed electrodes; Cocaine; Covalent attachment

Stripping voltammetric detection of mercury(II) based on a surface ion imprinting strategy in electropolymerized microporous poly(2-mercaptobenzothiazole) films modified glassy carbon electrode by Xu-Cheng Fu; Xing Chen; Zheng Guo; Cheng-Gen Xie; Ling-Tao Kong; Jin-Huai Liu; Xing-Jiu Huang (pp. 21-28).

This work reports a surface ion imprinting strategy in electropolymerized microporous poly(2-mercaptobenzothiazole) (MPMBT) films at the surface of glassy carbon electrode (GCE) for the electrochemical detection of Hg(II). The Hg(II)-imprinted MPMBT/GCE exhibits larger binding to functionalized capacity, faster binding kinetics and higher selectivity to template Hg(II) due to their high ratio of surface-imprinted sites, larger surface-to-volume ratios, the complete removal of Hg(II) templates and larger affinity to Hg(II). The square wave anodic stripping voltammetry (SW ASV) response of the Hg(II)-imprinted MPMBT/GCE to Hg(II) is ca. 3.0 and 5.9 times larger than that at the direct imprinted poly(2-mercaptobenzothiazole) modified GCE and non-imprinted MPMBT/GCE sensor, respectively; and the detection limit for Hg(II) is 0.1nM (which is well below the guideline value given by the World Health Organization). Excellent wide linear range (1.0–160.0nM) and good repeatability (relative standard deviation of 2.5%) were obtained for Hg(II). The interference experiments showed that mercury signal was not interfered in the presence of Pb(II), Cd(II), Zn(II), Cu(II) and Ag(I), respectively. These values, particularly the high sensitivity and excellent selectivity compared favorably with previously reported methods in the area of electrochemical Hg(II) detection, demonstrate the feasibility of using the prepared Hg(II)-imprinted MPMBT/GCE for efficient determination of Hg(II) in aqueous environmental samples.

Keywords: Surface ion imprinting; Square wave anodic stripping voltammetry; Electropolymerization; Microporous film; Mercury(II) detection

Selective pressurized liquid extraction of estrogenic compounds in soil and analysis by gas chromatography–mass spectrometry by Zulin Zhang; Stewart M. Rhind; Christine Kerr; Mark Osprey; Carol E. Kyle (pp. 29-35).

A selective pressurized liquid extraction (SPLE) method, followed by gas chromatography–mass spectrometry (GC–MS), for the simultaneous extraction and clean-up of estrone (E1), 17β-estradiol (E2), 17α-ethynylestradiol (EE2), estriol (E3) and bisphenol A (BPA) from soil samples is described. The on-line clean-up of soil by SPLE was achieved using different organic matter retainers, including silica, alumina and Florisil, the most effective being silica. Thus, different amounts of silica, in conjunction with different extraction solvents (acetone, ethyl acetate, isohexane and dichloromethane), either alone or in combination, were used to extract the target chemicals from spiked soil samples. It was shown that 3g silica resulted in satisfactory rates of recovery of target compounds and acetone:dichloromethane (1:3, v/v) was efficient in extracting and eluting estrogenic compounds for SPLE. Variables affecting the SPLE efficiency, including temperature and pressure were studied; the optimum parameters were 60°C and 1500psi, respectively. The limits of detection (LODs) of the proposed method were 0.02–0.37ngg⁻¹ for the different estrogenic chemicals studied. The outputs using the proposed method were linear over the range from 0.1 to 120ngg⁻¹ for E1, E2, EE2, 0.2–120ngg⁻¹ for E3, and 0.5–120ngg⁻¹ for BPA. The optimized method was further verified by performing spiking experiments in natural soil matrices; good rates of recovery and reproducibility were achieved for all selected compounds and the method was successfully applied to soil samples from Northeast Scotland, for the determination of the target compounds.

Keywords: Selective pressurized liquid extraction; Endocrine disrupting chemicals; Gas chromatography–mass spectrometry; Soil

Strategy of using microsome-based metabolite production to facilitate the identification of endogenous metabolites by liquid chromatography mass spectrometry by Melisa Clements; Liang Li (pp. 36-44).

One of the challenges in metabolomic profiling of complex biological samples is to identify new and unknown compounds. Typically, standards are used to help identify metabolites, yet standards cannot be purchased or readily synthesized for many unknowns. In this work we present a strategy of using human liver microsomes (HLM) to metabolize known endogenous human metabolites (substrates), producing potentially new metabolites that have yet to be documented. The metabolites produced by HLM can be tentatively identified based on the associated substrate structure, known metabolic processes, tandem mass spectrometry (MS/MS) fragmentation patterns and, if necessary, accurate mass measurements. Once identified, these metabolites can be used as references for identification of the same compounds in complex biological samples. As a proof of principle, a total of 9 metabolites have been identified from individual HLM incubations using 5 different substrates. Each metabolite was used as a standard. In the analysis of human urine sample by liquid chromatography MS/MS, four spectral matches were found from the 9 microsome-produced metabolite standards. Two of them have previously been documented as endogenous human metabolites, the third is an isomer of a microsome-metabolite and the fourth compound has not been previously reported and is also an isomer of a microsome-metabolite. This work illustrates the feasibility of using microsome-based metabolism to produce metabolites of endogenous human metabolites that can be used to facilitate the identification of unknowns in biological samples. Future work on improving the performance of this strategy is also discussed.

Keywords: Metabolome analysis; Metabolite identification; Endogenous metabolite; Human liver microsomes; LC–MS; Metabolomics

The use of high performance liquid chromatography–quadrupole time-of-flight mass spectrometry coupled to advanced data mining and chemometric tools for discrimination and classification of red wines according to their variety by Lukas Vaclavik; Ondrej Lacina; Jana Hajslova; Jerry Zweigenbaum (pp. 45-51).

In this study, the potential of high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (HPLC–QTOFMS) for metabolomic profiling of red wine samples was examined. Fifty one wines representing three varieties (Cabernet Sauvignon, Merlot, and Pinot Noir) of various geographical origins were sourced from the European and US retail market. To find compounds detected in analyzed samples, an automated compound (feature) extraction algorithm was employed for processing background subtracted single MS data. Stepwise reduction of the data dimensionality was followed by principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) which were employed to explore the structure of the data and construct classification models. The validated PLS-DA model based on data recorded in positive ionization mode enabled correct classification of 96% of samples. Determination of molecular formula and tentative identification of marker compound was carried out using accurate mass measurement of full single MS spectra. Additional information was obtained by correlating the fragments obtained by MS/MS accurate mass spectra using the QTOF with collision induced dissociation (CID) of precursor ions.

Keywords: Food metabolomics; Wine; Liquid chromatography–quadrupole-time-of-flight mass spectrometry; Multivariate data analysis

Ultra-sensitive DNA assay based on single-molecule detection coupled with fluorescent quantum dot-labeling and its application to determination of messenger RNA by Li Li; Xincang Li; Lu Li; Jinxing Wang; Wenrui Jin (pp. 52-57).

An ultra-sensitive single-molecule detection (SMD) method for quantification of DNA using total internal reflection fluorescence microscopy (TIRFM) coupled with fluorescent quantum dot (QD)-labeling was developed. In this method, the target DNA (tDNA) was captured by the capture DNA immobilized on the silanized coverslip blocked with ethanolamine and bovine serum albumin. Then, the QD-labeled probe DNA was hybridized to the tDNA. Ten fluorescent images of the QD-labeled sandwich DNA hybrids on the coverslip were taken by a high-sensitive CCD. The tDNA was quantified by counting the bright spots on the images using a calibration curve. The LOD of the method was 1×10⁻¹⁴molL⁻¹. Several key factors, including image acquirement, fluorescence probe, substrate preparation, noise elimination from solutions and glass coverslips, and nonspecific adsorption and binding of solution-phase detection probes were discussed in detail. The method could be applied to quantify messenger RNA (mRNA) in cells. In order to determine mRNA, double-stranded RNA-DNA hybrids consisting of mRNA and corresponding cDNA were synthesized from the cellular mRNA template using reverse transcription in the presence of reverse transcriptase. After removing the mRNA in the double-stranded hybrids using ribonuclease, cDNA was quantified using the SMD-based TIRFM. Osteopontin mRNA in decidual stromal cells was chosen as the model analyte.

Keywords: Abbreviations; BSA; bovine serum albumin; DEPC; diethypyrocarbonate; DNase; deoxyribonuclease; dNTP; deoxy-ribonucleoside triphosphate; DSC; decidual stromal cell; GOPS; 3-glycidoxypropyltrimethoxysilane; mRNA; messenger RNA; OPN; osteopontin; PBS; physiological buffer saline; pDNA; probe DNA; QD; quantum dot; QD-pDNA; QD-labeled probe DNA; R6G; rhodamine 6G; RNase; ribonuclease; rRNAsin; RNase-inhibitor; SA-QD; Streptavidin-coated QD; SDS; sodium dodecyl sulfate; SMD; single-molecule detection; tDNA; target DNA; TIRFM; total internal reflection fluorescence microscopySingle-molecule detection; Deoxyribonucleic acid determination; Messenger ribonucleic acid determination; Total internal reflection fluorescence microscopy

Optical ammonia gas sensor based on a porous silicon rugate filter coated with polymer-supported dye by Yunling Shang; Xiaobo Wang; Erchao Xu; Changlun Tong; Jianmin Wu (pp. 58-64).

An ammonia gas sensor chip was prepared by coating an electrochemically-etched porous Si rugate filter with a chitosan film that is crosslinked by glycidoxypropyltrimethoxysilane (GPTMS). The bromothylmol blue (BTB), a pH indicator, was loaded in the film as ammonia-sensing molecules. White light reflected from the porous Si has a narrow bandwidth spectrum with a peak at 610nm. Monitoring reflective optical intensity at the peak position allows for direct, real-time observation of changes in the concentration of ammonia gas in air samples. The reflective optical intensity decreased linearly with increasing concentrations of ammonia gas over the range of 0–100ppm. The lowest detection limit was 0.5ppm for ammonia gas. At optimum conditions, the full response time of the ammonia gas sensor was less than 15s. The sensor chip also exhibited a good long-term stability over 1 year. Therefore, the simple sensor design has potential application in miniaturized optical measurement for online ammonia gas detection.

Keywords: Ammonia gas; Sensor; Porous silicon rugate filter; Chitosan; Reflective spectrum

Optical ammonia gas sensor based on a porous silicon rugate filter coated with polymer-supported dye by Yunling Shang; Xiaobo Wang; Erchao Xu; Changlun Tong; Jianmin Wu (pp. 58-64).

Keywords: Ammonia gas; Sensor; Porous silicon rugate filter; Chitosan; Reflective spectrum

Development of direct competitive enzyme-linked aptamer assay for determination of dopamine in serum by Hoyoung Park; Insook Rhee Paeng (pp. 65-73).

A rapid and cost-effective screening method based on a competitive enzyme-linked aptamer assay (ELAA) for dopamine (DA) in serum has been optimized and validated. In this paper, we report advantageous sensitivity and specificity of aptamer assays as compared to the existing antibody based-immunoassays. The RNA aptamer (67 mer) was immobilized via site-directed immobilization with biotin both at the 3′-end on aptamer and at neutravidin plate. Various factors such as incubation temperature, divalent ion – Mg²⁺ ion and treatment of serum solution were evaluated for the performance of ELAA. The aptamer was incubated for 1h at 4°C in the assay buffer containing 5mM Mg²⁺ ion, and serum was diluted (1:9, serum:assay buffer) and filtrated through a 3kDa dialysis membrane to extract the proteins present in the serum. Assay was performed with 0.01μgmL⁻¹ of aptamer and 1.205×10⁻⁷M DA-HRP conjugate using the optimized method. A dose–response curve was constructed, and the limit of detection and a dynamic range for the DA were determined as 1.0×10⁻¹²M and four orders (1.0×10⁻⁷M to 5.0×10⁻¹¹M) of magnitude, respectively. The correlation diagram of the absorbance obtained both in buffer and in serum has shown a good agreement with the correlation coefficient ( R²=0.9872): Abs. (in serum)=0.9612×Abs. (in buffer)−0.0556. The cross-reactivity evaluation demonstrated that norepinephrine showed some cross-reactivity (3.68%) whereas 3-methoxytyramine, epinephrine, homovanillic acid and 3,4-dihydroxyphenylacetic acid showed almost no cross-reactivity (<1%). Percent recoveries of DA in serum were quite satisfactory (∼95%). This paper describes usefulness of the aptamer assay in monitoring DA in human serum.

Keywords: Aptamer assay; Dopamine; Serum; Catecholamine; Horseradish peroxidase

Development of direct competitive enzyme-linked aptamer assay for determination of dopamine in serum by Hoyoung Park; Insook Rhee Paeng (pp. 65-73).

Keywords: Aptamer assay; Dopamine; Serum; Catecholamine; Horseradish peroxidase

Sensitive and specific detection of potentially allergenic almond ( Prunus dulcis) in complex food matrices by Taqman^® real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay by Martin Röder; Stefan Vieths; Thomas Holzhauser (pp. 74-83).

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000mgkg⁻¹ almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200mgkg⁻¹.We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman^® probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000mgkg⁻¹ almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5mgkg⁻¹. Further, between 100 and 100,000mgkg⁻¹ spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman^® real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% ( n=5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and potentially quantitative almond detection. This PCR method detects almond at a level where severe allergic reactions should not be expected for the majority of the almond allergic individuals.

Keywords: Almond (; Prunus dulcis; ); Real-time polymerase chain reaction; Allergen labelling; Allergen detection; Enzyme-linked immunosorbent assay

Determination of the spectrum of low molecular mass organic acids in urine by capillary electrophoresis with contactless conductivity and ultraviolet photometric detection—An efficient tool for monitoring of inborn metabolic disorders by Petr Tůma; Eva Samcová; Karel Štulík (pp. 84-90).

A mixture of 29 organic acids (OAs) occurring in urine was analyzed by capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C⁴D) and UV photometric detection. The optimized analytical system involved a 100cm long polyacrylamide-coated capillary (50μm i.d.) and the background electrolyte of 20mM 2-morpholinoethanesulfonic acid (MES)/NaOH+10% (v/v) methanol, pH 6.0 (pH is related to the 20mM MES/NaOH buffer in water). The LOD values obtained by C⁴D for the OAs which do not absorb UV radiation range from 0.6μM (oxalic acid) to 6.8μM (pyruvic acid); those obtained by UV photometry for the remaining OAs range from 2.9μM (5-hydroxy-3-indoleacetic acid) to 10.2μM (uric acid). The repeatability of the procedure developed is characterized by the coefficients of variation, which vary between 0.3% (tartaric acid) and 0.6% (5-hydroxy-3-indoleacetic acid) for the migration time and between 1.3% (tartaric acid) and 3.5% (lactic acid) for the peak area. The procedure permitted quantitation of 20 OAs in a real urine sample and was applied to monitoring of the occurrence of the inborn metabolic fault of methylmalonic aciduria.

Keywords: Abbreviations; C; ⁴; D; capacitively coupled contactless conductivity detection; OA; organic acid; IEM; inborn errors of metabolism; Crea; creatinineCapillary electrophoresis; Contactless conductivity detection; Photometric detection; Organic acids; Inborn errors of metabolism; Urine

Automatic twin vessel recrystallizer. Effective purification of acetaminophen by successive automatic recrystallization and absolute determination of purity by DSC by Osamu Nara (pp. 91-102).

I describe an interchangeable twin vessel (J, N) automatic glass recrystallizer that eliminates the time-consuming recovery and recycling of crystals for repeated recrystallization. The sample goes in the dissolution vessel J containing a magnetic stir-bar K; J is clamped to the upper joint H of recrystallizer body D. Empty crystallization vessel N is clamped to the lower joint M. Pure solvent is delivered to the dissolution vessel and the crystallization vessel via the head of the condenser A. Crystallization vessel is heated (P). The dissolution reservoir is stirred and heated by the solvent vapor (F). Continuous outflow of filtrate E out of J keeps N at a stable boiling temperature. This results in efficient dissolution, evaporation and separation of pure crystals Q. Pure solvent in the dissolution reservoir is recovered by suction. Empty dissolution and crystallization vessels are detached. Stirrer magnet is transferred to the crystallization vessel and the role of the vessels are then reversed. Evacuating mother liquor out of the upper twin vessel, the apparatus unit is ready for the next automatic recrystallization by refilling twin vessels with pure solvent. We show successive automatic recrystallization of acetaminophen from diethyl ether obtaining acetaminophen of higher melting temperatures than USP and JP reference standards by 8× automatic recrystallization, 96% yield at each stage. Also, I demonstrate a novel approach to the determination of absolute purity by combining the successive automatic recrystallization with differential scanning calorimetry (DSC) measurement requiring no reference standards. This involves the measurement of the criterial melting temperature T₀ corresponding to the 100% pure material and quantitative Δ T in DSC based on the van’t Hoff law of melting point depression. The purity of six commercial acetaminophen samples and reference standards and an eight times recrystallized product evaluated were 98.8mol%, 97.9mol%, 99.1mol%, 98.3mol%, 98.4mol%, 98.5mol% and 99.3mol% respectively.

Keywords: Interchangeable twin vessel automatic recrystallizer; Acetaminophen; Successive automatic recrystallization; Differential scanning calorimetry; Absolute purity determination; Criterial; T; ₀; determination

Automatic twin vessel recrystallizer. Effective purification of acetaminophen by successive automatic recrystallization and absolute determination of purity by DSC by Osamu Nara (pp. 91-102).

Investigation of polar stationary phases for the separation of sympathomimetic drugs with nano-liquid chromatography in hydrophilic interaction liquid chromatography mode by Zeineb Aturki; Giovanni D’Orazio; Anna Rocco; Kahina Si-Ahmed; Salvatore Fanali (pp. 103-110).

In this study, the retention and selectivity of a mixture of basic polar drugs were investigated in hydrophilic interaction chromatographic conditions (HILIC) using nano-liquid chromatography (nano-LC). Six sympathomimetic drugs including ephedrine, norephedrine, synephrine, epinephrine, norepinephrine and norphenylephrine were separated by changing experimental parameters such as stationary phase, acetonitrile (ACN) content, buffer pH and concentration, column temperature. Four polar stationary phases (i.e. cyano-, diol-, aminopropyl-silica and Luna HILIC, a cross-linked diol phase) were selected and packed into fused silica capillary columns of 100μm internal diameter (i.d.). Among the four stationary phases investigated a complete separation of the all studied compounds was achieved with aminopropyl silica and Luna HILIC stationary phases only. Best chromatographic results were obtained employing a mobile phase composed by ACN/water (92/8, v/v) containing 10mM ammonium formate buffer pH 3. The influence of the capillary temperature on the resolution of the polar basic drugs was investigated in the range between 10 and 50°C. Linear correlation of ln k vs. 1/ T was observed for all the columns; Δ H° values were negative with Luna HILIC and positive with aminopropyl- and diol-silica stationary phases, demonstrating that different mechanisms were involved in the separation.To compare the chromatographic performance of the different columns, Van Deemter curves were also investigated.

Keywords: Nano-liquid chromatography; Hydrophilic interaction liquid chromatography; Polar stationary phases; Sympathomimetic drugs

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Analytica Chimica Acta (v.685, #1)