Analytica Chimica Acta (v.531, #1)

Contents (v-vi).

A label-free amperometric immunosensor for fast and sensitive assay of Japanese B encephalitis vaccine is presented. Antiserum of Japanese B encephalitis were immobilized on bilayer nano-Au/o-phenylenediamine polymer film with deposited Prussian blue as an electronic mediator on the Pt electrode. The electrochemical behavior of the biosensor was studied with Fe2+/3+ as probe on the Pt surface using cyclic voltammetry technique. The variation of amperometric response to the concentration of Japanese B encephalitis vaccine, the target antigen, was evaluated by cyclic voltammetry in PBS. The immunosensor showed a specific response to Japanese B encephalitis vaccine in the range 1.1 × 10−8 to 1.9 × 10−6  lg pfu/ml (pfu/ml is plaque forming unit and lg is common logarithm) with a detection limit of 6 × 10−9  lg pfu/ml. The correlation coefficient is 0.9955. The incubation time, incubation temperature, pH, reproducibility and stability of the immunosensor were also studied. The present work supplied a promising test method for biological products.
Keywords: Label-free; Amperometric; Japanese B encephalitis vaccine; Immunosensor;

Fluorescence-based assay with enzyme amplification on a micro-flow immunosensor chip for monitoring coplanar polychlorinated biphenyls by Tatsuro Endo; Akira Okuyama; Yasutaka Matsubara; Kazuto Nishi; Masaaki Kobayashi; Shouhei Yamamura; Yasutaka Morita; Yuzuru Takamura; Haruki Mizukami; Eiichi Tamiya (7-13).
Detection of pollutants is of significant importance for environmental protection. However, conventional monitoring methods are often time-consuming, and require expensive equipments. Biosensors based on enzyme linked immunosorbent assay (ELISA) provide an alternative method to conventional ones. In this research, the reduction in the size of ELISA utilizing micro-chemical reaction is described in a micro-flow immunosensor chip. The immunosensor chips were fabricated by micro-electromechanical system (MEMS) technology. The quantitative determination of coplanar polychlorinated biphenyls (Co-PCBs) was performed by using a micro-flow immunosensor chip. Polystyrene beads were used as the solid substrate for the immobilization of Co-PCB antibody. The antibody-immobilized beads were introduced into the flow channel. As a competitive ELISA, sample solution mixed with horseradish peroxidase (HRP) conjugated antigen, and non-HRP conjugated antigen was allowed to react in the flow channel. After the antigen–antibody reaction, addition of phosphate buffer solution containing hydrogen peroxide and the fluorogenic substrate produced a fluorescent dye, which was monitored with the resulting change in the fluorescence intensity. By using our micro-flow immunosensor chip, it was possible to determine the sensing range of Co-PCB derivatives up to 0.1 ppt in 30 s. This immunosensor chip had a wide linear range for Co-PCB detection from 0.1 pg/ml to 1.0 μg/ml. The regression analysis provided the correlation coefficients of r  = 0.982−0.964 with good reproducibility and precision. In a series of five measurements with immunosensor chips prepared with a new batch of antibody-immobilized polystyrene beads, a relative standard deviation of 21.3% was obtained. Our immunosensor chip design reported here has the potential to be implemented to several different detection methodologies for numerous analytes.
Keywords: Microfluidics; Biosensor; ELISA; Coplanar polychlorinated biphenyls (Co-PCB); Environmental monitoring;

Traditionally, the effects of electrical double layer on pressure-driven flow in microchannels were modeled by using the Poisson–Boltzmann equation and the fluid momentum equation with a flow-induced body force term. Such a model, however, usually underestimate the electrical double layer effects on the flow. In this study, a theoretical model of the electrical double layer field is developed to provide a better understanding of the electrical double layer effects. The electrical potential and ionic concentration distribution in dilute solutions in small microchannels are investigated by numerically solving this new model. This newly developed model predicted the deficit of counter-ions in the bulk liquid region due to the accumulation of counter-ions in the EDL region, and the surplus of co-ions in the bulk liquid region due to rejection of the co-ions in the EDL region. The presence of the net charges in the bulk liquid region is responsible for the strong electroviscous effects in dilute solutions in small microchannels.
Keywords: Electrical double layer; Pressure-driven flow; Microchannels;

An automatic system that performs two analytical procedures, allowing the evaluation of the relative antioxidant capacity of wine samples, was developed. Automation was carried out using a sequential injection analysis (SIA) system that allowed, thanks to its versatility, the development of two methodologies. One is based on the decolorization assay of the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical, using a spectrophotometric detector. A second methodology allowed the evaluation of the hydrogen peroxide scavenging activity by measuring the oxidation of homovanylic acid (HVA) to its fluorescent dimer, using a fluorescent detector.The developed automatic methodologies were evaluated using trolox as standard and subsequently using other antioxidant substances as gallic acid, caffeic acid, ascorbic acid, catechin and taxifolin which are abundant in wine and whose antioxidant activities were compared to that shown by trolox. The spectrophotometric and fluorimetric assays showed linearity intervals between 0.001 and 0.01 mM, and 0.001 and 0.008 mM of trolox, respectively.The evaluation of the antioxidant power of 20 white and red wine samples, from different Portuguese wine producing regions, was carried out sequentially, in the automatic system. The results were expressed in trolox equivalent antioxidant capacity (TEAC) and presented, for the ABTS and hydrogen peroxide scavenging activity methodologies, detection limits of 8.4 × 10−7 and 1.4 × 10−4  mM and relative standard deviation (R.S.D. (%)) in the range 0.6–2.4 and 1–1.8, respectively.
Keywords: Wines; Antioxidant potential; Hydrogen peroxide scavenging activity; ABTS;

Synthesis and evaluation of molecularly imprinted polymers for organotin compounds: a screening method for tributyltin detection in seawater by Mercedes Gallego-Gallegos; Riansares Muñoz-Olivas; Antonio Martin-Esteban; Carmen Cámara (33-39).
The environmental impact of some organotin compounds (OTC) has given particulate importance to analytical studies. This paper reports the first attempt to apply the emerging molecular imprinting technology to this field. Several imprinted polymers have been synthesised by the non-covalent free radical approach using sodium methacrylate (NaMA) or 4-vinylpyridine (4-VP) as monomers in the presence of TBT as template molecule in three different polymerisation media (toluene, acetonitrile and methanol/water). The ability of the polymers synthesised to retain and distinguish TBT from its degradation products has been evaluated and optimized. The results clearly showed the presence of cavities within the polymeric matrix allowing specific recognition of TBT. Cross-reactivity from other Sn species (monobutyltin (MBT), dibutyltin (DBT) and inorganic Sn) has also been evaluated. Rapid and direct differentiation of TBT from its main degradation products in seawater was achieved. The analytical characteristics included linearity (0.05–50 μg l−1), a pre-concentration factor of 150, and a quantification limit of 0.04 μg l−1 for 1 l.
Keywords: Molecular imprinting technology; Organotin compounds; Seawater analysis;

Formaldehyde, HCHO, is one of the important causal agents of sick-building syndrome. It is also an important product of ambient air photochemistry. We report here a portable instrument capable of a 0.08 ppbv limit of detection (LOD) and a time resolution of 5 min that is useful for both indoor and ambient air applications. The detection is based on efficient gas collection and chromogenic reaction with 3-methyl-2-benzothiazolone hydrazone (MBTH) through a pair of alternately sampling small-bore porous-membrane tube diffusion scrubbers (DS). The chemistry is well established, requires no special reagent preparation or elevated reaction temperatures and permits the use of inexpensive light emitting diode (LED)-based detectors without need for long path cells. Stopped flow alternate sampling allows an HCHO collection performance, an order of magnitude better than any previous system with high throughput and high sensitivity. Results for indoor and ambient air analyses are presented.
Keywords: Flow analysis; Formaldehyde; Diffusion scrubber; Spectrophotometry; Stopped-flow; Portable instrument;

Measurement of gaseous and aqueous trace formaldehyde by Jianzhong Li; Purnendu K. Dasgupta; Winston Luke (51-68).
The measurement of atmospheric formaldehyde has been of interest to the corresponding author for the last two decades. The initial approach based on the Hantzsch reaction with 2,4-pentanedione (PD) and ammonium acetate was later abandoned in favor of 1,3-cyclohexanedione (CHD) as the β-diketone because of an order of magnitude better limits of detection (LOD). Subsequently, it was discovered that at very high H2O2 to HCHO ratios, the CHD chemistry has a perceptible positive interference from H2O2. In this paper, we review techniques for the measurement of HCHO that have appeared since an account of a CHD-based instrument to measure HCHO was last published, outline our rationale to return to PD-based chemistry, and show that nearly equivalent LODs (16 nM aqueous, 3 pmol, 70 pptv gaseous) can be obtained with reagent components that are separated rather than a single mixed reagent in combination with a fluorescence detector that utilizes multiple high intensity light emitting diodes (LEDs) for excitation without the benefit of a Teflon AF based waveguide, the use of which has become more difficult due to legal restrictions. We present design and construction details of both a ground-based instrument and an instrument intended for aircraft use and provide illustrative field data for both.
Keywords: Formaldehyde; Light emitting diodes; Fluorescence; Diffusion scrubber;

Three pairs of ginsenoside isomers (Rg2 and Rg3, Rg1 and F11 as well as Rd and Re) were differentiated and identified through accurate mass measurement of mass spectrometry (MS) and MS–MS. [M  + Li]+ and [M  − H] ions were detected in full-scan MS analyses and selected for the MS–MS experiments using positive and negative ion electrospray ionizations (ESI), respectively. The structures of aglycone and α- and β-saccharide sugars in various ginsenosides were determined from the spectrum interpretation and accurate mass measurement. Z and C type ions were predominantly observed in the MS–MS spectra of [M  + Li]+ ions, while Y type ions were the most abundant ions in the spectra obtained from the negative ion mode analysis. Furthermore, X and A ions resulted from cross-ring cleavage on the sugar directly connected to aglycone were detected in both positive and negative ion spectra, which provided the site information of the saccharide chains. The obtained MS–MS profiles were used for the structural confirmation of ginsenoside Rg2 collected from column chromatography separation of a Chinese Panax ginseng extract. The ESI–MS data with accurate mass assignment suggested that a co-eluted ginsenoside also existed in the sample fraction. The interpretation of its MS–MS spectrum and fragmentation pathways allowed the detection of the ginsenoside Rf, differentiating from its isomers Rg1 and F11.
Keywords: Ginsenosides; Structural differentiation; MS–MS; Panax ginseng;

The high throughput analysis of N-methyl carbamate pesticides in wine and juice by electrospray ionization liquid chromatography tandem mass spectrometry with direct sample injection into a short column by Tomomi Goto; Yuko Ito; Hisao Oka; Isao Saito; Hiroshi Matsumoto; Hideo Sugiyama; Chiyoji Ohkubo; Hiroyuki Nakazawa; Hisamitsu Nagase (79-86).
We developed a new analysis method for the N-methyl carbamate pesticides in juice and wine. The juice and wine were diluted with ultra pure water, and determined by electrospray ionization tandem mass spectrometry (ESI LC/MS/MS) with direct sample injection into a short column. The new method, including sample preparation and determination, is simple and rapid, and allows simultaneous determination of nine N-methyl carbamate pesticides in juice and wine within analysis time that is much shorter as compared with the traditional method. The average recoveries from juice and wine fortified at the level of 0.1 ppm ranged from 59.6 to 126.7% with the coefficients of variation ranging from 0.4 to 5.1% for intra-day (n  = 5 × 3 days) and from 0.5 to 22.6% for inter-day (n  = 15). At the fortified level of 0.5 ppm, the recoveries ranged from 69.3 to 127.2% with the coefficients of variation ranging from 0.4 to 6.9% for intra-day (n  = 5 × 3 days) and from 0.5 to 22.6% for inter-day (n  = 15). The method is considered to be satisfactory for the monitoring of the carbamate pesticides residues in juice and wine, suggesting that the present method is applicable to other pesticide residues in foods.
Keywords: N-methyl carbamate pesticides; Wine; Juice; ESI LC/MS/MS;

Application of a liquid chromatography tandem mass spectrometry method to the analysis of water-soluble vitamins in Italian pasta by Andrea Leporati; Dante Catellani; Michele Suman; Roberta Andreoli; Paola Manini; Wilfried M.A. Niessen (87-95).
A sensitive and selective liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the determination of several water-soluble vitamins, namely vitamins B1, B2, B6 (pyridoxine, pyridoxal, and pyridoxamine), and PP (nicotinamide and nicotinic acid), pantothenic acid, and folic acid was developed and validated. The analytes were characterized by means of their electrospray (ESI) and atmospheric pressure chemical ionization (APCI) mass spectra. In general, the positive ion spectra were 100- to 1000-fold more intense than the corresponding negative ion ones. Chromatography of water-soluble vitamins was obtained by using a reversed-phase C16 Amide (15 cm, 5 μm) column and a mobile phase made of ammonium formate buffer (20 mM, pH 3.75)/methanol under gradient elution conditions. Linearity of the MS response was observed over three to four orders for both ESI and APCI, and limits of detection were in the low μg/l range for both the ionization techniques. In particular, the sensitivity of ESI was about two- to five-fold higher for all vitamins except PP vitamers, for which APCI produced a better response. Precision calculated at two concentration levels (0.05 and 1.0 mg/l) was within 0.2–7.4% for all intra- and inter-day determinations and for all analytes. The LC–ESI-MS/MS method was applied to the quantitative analysis of the natural content of vitamins in typical Italian pasta samples, as well as in fortified pasta samples produced for the US market.
Keywords: Water-soluble vitamins; Pyridoxine; Pyridoxal; Pyridoxamine; Nicotinamide; Nicotinic acid; Pantothenic acid; Folic acid; LC–MS/MS; Italian pasta; Fortified food;

Various sampling techniques including flash evaporation (FE), headspace solid-phase microextraction (HS-SPME) and steam distillation (SD) were compared for the gas chromatography–mass spectrometry of volatile constituents present in Houttuynia cordata Thunb (HCT). 2-Undecanone (22.21%) and houttuynum (7.23%) were predominant components of HCT samples obtained by HS-SPME whereas those levels were 3.95 and 3.60% in the same samples by FE and 25.93 and 6.60% in those by SD, respectively. SPME with polydimethylsiloxane (PDMS) fibre was more selective and particularly efficient for the isolation of biologically active compounds and afforded a higher yield of total compounds than FE and SD. A total of 60 compounds were detected in SPME extracts. While in FE and SD extracts, the detected compounds were 41 and 51, respectively. The total amount of compounds isolated by SPME was much larger than that isolated by FE or SD. Some minor constituents were isolated by SPME, but not by SD and FE. This carries great significance because of the importance of the oil volatiles to clinical therapy. HS-SPME is a powerful tool for determining the volatile constitutes present in the TCMs.
Keywords: Headspace solid-phase microextraction; Flash evaporation; Stream distillation; Volatile compounds; Houttuynia cordata Thunb;

High speed liquid chromatography for in-process control of rifabutin by Domingo Blanco Gomis; Noé Sánchez Núñez; Elena Andrés García; Pilar Arias Abrodo; Miguel Bayod Jasanada; Mª. Dolores Gutiérrez Álvarez (105-110).
A fast HPLC method has been developed for simultaneous determination of rifabutin and its synthesis precursors. The analytes are separated in 1.8 min by means of a Kromasil 100 C18 column (50 mm × 2.1 mm i.d., 3.5 μm) at 30 °C. The mobile phase (A: 5 mM KH2PO4 adjusted to pH 6.5 with KOH; B: acetonitrile) was pumped at a flow rate of 0.4 ml min−1 according to the fast gradient mode: 0 min, 58% B; 0–0.4 min, 95% B. Detection was by ultraviolet absorbance at 275 nm. The method was validated in accordance with the International Conference on Harmonisation (ICH) guidelines and good accuracy, intermediate precision (≤4.6%) and linearity in the range 5–50 mg l−1 were observed for all compounds. This method is sensitive (limits of detection ranged between 0.1 and 0.3 mg l−1) and selective to quantify rifabutin and its synthesis precursors and could be used for in-process control.
Keywords: High speed HPLC; In-process control; Rifabutin;

Room temperature acid sonication ICP-MS multielemental analysis of milk by Patricia Cava-Montesinos; M. Luisa Cervera; Agustín Pastor; Miguel de la Guardia (111-123).
Room temperature acid sonication of milk samples is proposed as a fast alternative methodology for the determination of the total content of 45 elements (Li, Al, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Rb, Sr, Y, Mo, Ag, Cd, In, Sn, U, Sb, Te, Cs, Ba, Hg, Pb, Bi, Th, La, Ce, Pr, Nd, Sm, Eu, Gd, Dy, Ho, Er, Tm, Yb, Lu, Hf and Ta) in milk by inductively coupled plasma mass spectrometry (ICP-MS). The aforementioned procedure involves a 10 min sample pre-treatment. Measurements were made in quantitative and semiquantitative (Totalquant®) modes of analysis using Rh as internal standard and Be, Ge, Tb and Re for internal calibration of the equipment in the semiquantitative mode. The selected isotopes were in general the most abundant ones of each element, except in cases where polyatomic or isobaric interferences were detected. Results of total concentrations in 10 liquid and 11 powdered commercially available milk samples were presented. Method validation was performed by measuring a SRM NIST-1549 non-fat milk powder and through the use of recovery experiments. Additionally, the proposed methodology was compared with a method based on a previous microwave-assisted digestion of samples and a direct analysis of 1:4 diluted samples.
Keywords: Milk; Inductively coupled plasma mass spectrometry; Sonication; Microwave-assisted digestion; Semiquantitative mode;

Electrothermal atomic absorption spectrometric determination of germanium in soils using ultrasound-assisted leaching by I. López-García; N. Campillo; I. Arnau-Jerez; M. Hernández-Córdoba (125-129).
A procedure for determining germanium in soil samples using electrothermal atomic absorption spectrometry is discussed. The analyte is leached from the solid sample by the addition of 1 ml of concentrated hydrofluoric acid to 10–300 mg of sample, and the mixture is then submitted to a 10 min ultrasonic treatment. After adding 0.4 g boric acid and 3 ml concentrated hydrochloric acid, germanium is extracted into 1 ml chloroform and back-extracted into an aqueous phase containing (0.05%, w/v) nickel nitrate. Ten micro liter of aqueous phase are introduced into the atomizer and the analytical signal from germanium is obtained using a fast-heating cycle. The detection limit, calculated using three times the standard error of estimate (s y/x ) of the calibration graph, is 0.015 μg g−1. The reliability of the procedure is verified by analyzing several certified reference materials.
Keywords: Germanium; Electrothermal atomic absorption spectrometry; Ultrasonic treatment; Soils; Sediments;

Uncertainty assessment from robustness testing applied on an LC assay for R-timolol and other related substances in S-timolol maleate by R.D. Marini; B. Boulanger; Y. Vander Heyden; P. Chiap; J. Crommen; Ph. Hubert (131-140).
The robustness testing of a normal-phase liquid chromatographic (LC) method for the determination of R-timolol and other related substances in S-timolol maleate was performed applying a two-level Plackett–Burman design. Two qualitative and five quantitative factors were examined. Two types of responses were considered, qualitative, i.e. chromatographic performance criteria, and quantitative ones. The latter were taken into account to determine if the analytical procedure was robust. The quantitative responses were the contents of R-timolol in two S-timolol maleate samples. Even though some significant factor effects were observed on the qualitative responses, the R-timolol contents were not significantly different from those observed at nominal conditions, which demonstrated the robustness of the procedure.Since the experiments of the Plackett–Burman design can be assimilated to laboratories in an interlaboratory study, uncertainty can be evaluated using the robustness test data. The robustness test was set-up in such a way that the required variances could be estimated. It was shown that the robustness set-up allows estimating the reproducibility uncertainty without performing an interlaboratory study.
Keywords: Robustness testing; Liquid chromatography (LC); Timolol maleate; Quantitative responses; Uncertainty assessment;

Reassessment of pH reference values with improved methodology for the evaluation of ionic strength by M.J. Guiomar H.M. Lito; M. Filomena G.F.C. Camões (141-146).
The conflict between pH as empirical number in routine control and the pH value regarded as conveying some information concerning the effective concentration or activity of hydrogen ions, a H, has caused much confusion.There are, however, reasons to conclude that the overwhelming amount of thermodynamic data is not sufficiently accurate—either due to ignorance of metrological concepts or due to insufficiently specified measurement processes of fundamental chemical quantities pH.The commonly used seven reference buffer solutions to which primary pH values have been conventional assigned, represent a selection out of a more extensive list, recommended by NBS (now NIST) in 1962. From then onwards conventions concerning the Debye–Hückel model of electrolyte solutions and ionic strength have been revised and the pH(S) values reassessed in conformity but only for these seven reference buffer solutions. The others have, so far remained unchanged, locking harmonisation of the conventionally assigned pH(S) values.In this work, ionic strength is calculated through complete equations derived from the acidity constants. Concentrations of the various species involved in the conventional assignment of pH and their corresponding activity coefficients are therefore, more rigorously known. The process proves particularly useful for poliprotic acids with overlapping acidity constants, where the ratio is less than 103.As a consequence, conventionally assigned pH values of reference buffer solutions are recalculated and corrections are introduced as appropriate.
Keywords: Primary pH values; Ionic strength; Acidity constants; Species distribution; Activity coefficients;

The binding constants of 1-alkanols with α-cyclodextrin (α-CD) were determined by indirect competitive potentiometry, although potentiometry is usually inapplicable to nonionic compounds. This novel method utilizes competitive binding of 1-alkanol and octyltrimethylammonium bromide (OTAB) to α-CD in aqueous media, where the concentration of free OTAB depending on the competitive binding is monitored with an OTAB-selective electrode. Therefore, the concentration of 1-alkanol is indirectly estimated from the observed electromotive force. The binding constants of 1-propapnol, 1-butanol, 1-hexanol, and 1-octanol with α-CD, obtained by this method, are close to the literature values. The implications and limitations of this indirect competitive potentiometry were discussed.
Keywords: Indirect competitive potentiometry; Binding constant; 1-Alkanol; α-Cyclodextrin;

Non-linear absorption spectral data obtained from ternary mixtures of analytes are analyzed by using a linear model, iterative target transformation factor analysis (ITTFA). The use of transformed original variables is used to correct non-linearities in the original data. Absorbance below a certain limit (k) is described as linear and above this limit as non-linear. The extension of the regressor variables is the squared absorbances above the linear range. The variation of the prediction error as a function of the number of the factors and the k-values were considered and the minimum prediction error was evaluated for reaching to optimum. Except the natural non-negativity constraint the correlation constraint also is used on concentration vector in each iteration of ITTFA algorithm. The reliability of the method is evaluated using model data for ternary mixtures by spectral overlapping and different degrees of non-linearity. Simultaneous spectrophotometric determination of Eu3+, UO2 2+ and Th4+ with arsenazo III as chromogenic reagent is used as experimental model systems with non-linearity behavior of Eu3+and UO2 2+ components. The application to both synthetic and real data sets with different degrees of non-linearity demonstrate the ability of the proposed methodology to obtain better results than original data and ITTFA. The relative standard errors of prediction for proposed method in comparison with using the PLS calibration on original and extended data are nearly smaller.
Keywords: Non-linearity; Data extension; ITTFA; Eu3+; UO2 2+; Th4+;

Erratum to “Determination of fluoxetine and norfluoxetine in rat brain microdialysis samples following intraperitoneal fluoxetine administration” [Anal. Chim. Acta 522 (2004) 99–104] by Takeshi Fukushima; Emi Naka-aki; Xingjie Guo; Famei Li; Tineke Vankeirsbilck; Willy R.G. Baeyens; Kazuhiro Imai; Toshimasa Toyo’oka (163).