Analytica Chimica Acta (v.636, #1)

Bismuth and Sb were evaluated as internal standards (IS) to minimize matrix effects on the direct and simultaneous determination of As, Cu, and Pb in cachaça by graphite furnace atomic absorption spectrometry using W-coated platform plus Pd–Mg(NO3)2 as modifier. For 20 μL injected sample, calibration within the 0.5–10 μg L−1 As, 100–1000 μg L−1 Cu and 0.5–30 μg L−1 Pb intervals were established using the ratios As absorbance to Sb absorbance, Cu absorbance to Bi absorbance and Pb absorbance to Bi absorbance versus analytes concentration, respectively. Typical linear correlations of 0.998, 0.999 and 0.999 were, respectively, obtained. The proposed method was applied for direct determination of As, Cu and Pb in 10 commercial cachaça samples and results were in agreement with those obtained by inductively coupled plasma mass spectrometry at 95% confidence level. The found characteristic masses were 30 pg As, 274 pg Cu and 39 pg Pb. The useful lifetime of the graphite tube was around 760 firings. Recoveries of As, Cu and Pb added to cachaça samples varied, respectively, from 98% to 109%, 97% to 108% and 98% to 104% with internal standards and from 48% to 54%, 53% to 92% and 62% to 97% without internal standards. The limits of detection were 0.13 μg L−1 As, 22 μg L−1 Cu and 0.05 μg L−1 Pb. The relative standard deviations (n  = 12) for a spiked sample containing 20 μg L−1 As, Pb and 500 μg L−1 Cu were 1.6%, 1.0%, and 1.8% with IS and 4.3%, 5.2%, and 5.5% without IS.
Keywords: Arsenic; Copper; Lead; Cachaça; Internal standardization; Graphite furnace atomic absorption spectrometry;

The objective of this work was to study the effects of the following Ligands: Chelex-100, Dowex MAC-3 and Dowex 50WX-8 using Competing Ligand Exchange Method. This objective was achieved by investigating complex dissociation kinetics of trace metals: Co(II), Ni(II), Cu(II), Zn(II), Cd(II), Mn(II) and Pb(II) of a well-characterized Laurentian Fulvic Acid (LFA) in model solutions and in a natural waters of Lake Heva (Québec, Canada). The effects of variation in the competing ligands (including their quantities) on the complex dissociation kinetics were quantitatively characterized by their first-order dissociation rate coefficients. The kinetic lability of the metal complexes varied with the metal-to-LFA ratio, as expected from the theory of metal complexes of the chemically and physically heterogeneous complexants, LFA. The general trend in the metal-binding by the above competing ligands was: Dowex 50WX-8 > Chelex-100 > Dowex MAC-3. However, no difference was found between the Dowex 50WX-8 and Chelex-100 for Cd(II), Zn(II), and Co(II). The results revealed the importance of the quantity of Chelex-100 as a competing ligand in the metal(II)–LFA complexation, on the dissociation kinetics of these complexes in model solutions. By developing Competing Ligand Exchange Method as an analytical technique, for studying the relative affinities of the above competing ligands for metals complexation in natural waters this work has made a substantial contribution to analytical chemistry.
Keywords: Speciation; Inductively coupled plasma mass spectroscopy; Trace metals; Chelex-100; Dowex MAC-3; Dowex 50WX8; Natural waters;

In this study, polypyrrole (PPy) films were electrochemically deposited on gold substrates roughened by an electrochemical triangular-wave oxidation–reduction cycles (ORC) in an aqueous solution containing 0.1N KCl. Then the substrates were heated from 25 to 50 °C and the corresponding SERS performances of PPy were observed in situ. The results indicate that the SERS enhancement capabilities of substrates are gradually raised from 25 °C to a maximum at 40 °C and monotonically decreased from 40 to 50 °C. These SERS enhancement capabilities ascribed to the charge transfers from PPy to Au, which are responsible for the chemical effects of SERS mechanisms, are successfully observed via SERS and high resolution X-ray photoelectron spectroscopy (HRXPS) analyses. The variation in content of the oxidized PPy peak of the double peaks in the range of 1000–1150 cm−1 in SERS spectrum obtained on an Au substrate at different temperatures is consistent with its corresponding variation in the SERS intensity of PPy. The variation in content of the oxidized nitrogen of PPy deposited on an Au substrate at different temperatures revealed from an HRXPS analysis also confirms this consistence.
Keywords: Surface-enhanced Raman scattering; Temperature; Polypyrrole; Chemical effect;

A composite film (MWCNTs–PNF) which contains multi-walled carbon nanotubes (MWCNTs) along with the incorporation of poly(new fuchsin) (PNF) has been synthesized on glassy carbon electrode (GCE), gold (Au) and indium tin oxide (ITO) by potentiostatic methods. The presence of MWCNTs in the composite film enhances surface coverage concentration (Γ) of PNF to ≈176.5%, and increases the electron transfer rate constant (k s) to ≈346%. The composite film also exhibits promising enhanced electrocatalytic activity towards the mixture of biochemical compounds such as adenine (AD), guanine (GU) and thymine (THY). The surface morphology of the composite film deposited on ITO has been studied using scanning electron microscopy and atomic force microscopy. These two techniques reveal that the PNF incorporated on MWCNTs. Electrochemical quartz crystal microbalance study reveals the enhancement in the functional properties of MWCNTs and PNF. The electrocatalytic responses of analytes at MWCNTs and MWCNTs–PNF films were measured using both cyclic voltammetry (CV) and differential pulse voltammetry (DPV). From electrocatalysis studies, well separated voltammetric peaks have been obtained at the composite film for AD, GU and THY, with the peak separation of 320.3 and 132.7 mV between GU–AD and AD–THY respectively. The sensitivity of the composite film towards AD, GU and THY in DPV technique is 218.18, 12.62 and 78.22 mA M−1  cm−2 respectively, which are higher than MWCNTs film. Further, electroanalytical studies of AD, GU and THY present in single-strand deoxyribonucleic acid (ssDNA) have been carried out using semi-derivative CV and DPV.
Keywords: Multiwall carbon nanotubes; Composite film; Modified electrodes; Electrocatalysis; Deoxyribonucleic acid (DNA); Adenine; Guanine; Thymine;

A new dispersive liquid–liquid microextraction based on solidification of floating organic droplet method (DLLME-SFO) was developed for the determination of five kinds of polycyclic aromatic hydrocarbons (PAHs) in environmental water samples. In this method, no specific holder, such as the needle tip of microsyringe and the hollow fiber, is required for supporting the organic microdrop due to the using of organic solvent with low density and proper melting point. Furthermore, the extractant droplet can be collected easily by solidifying it in the lower temperature. 1-Dodecanol was chosen as extraction solvent in this work. A series of parameters that influence extraction were investigated systematically. Under optimal conditions, enrichment factors (EFs) for PAHs were in the range of 88–118. The limit of detections (LODs) for naphthalene, diphenyl, acenaphthene, anthracene and fluoranthene were 0.045, 0.86, 0.071, 1.1 and 0.66 ng mL−1, respectively. Good reproducibility and recovery of the method were also obtained. Compared with the traditional liquid-phase microextraction (LPME) and dispersive liquid–liquid microextraction (DLLME) methods, the proposed method obtained about 2 times higher enrichment factor than those in LPME. Moreover, the solidification of floating organic solvent facilitated the phase transfer. And most importantly, it avoided using high-density and toxic solvent in the traditional DLLME method. The proposed method was successfully applied to determinate PAHs in the environmental water samples. The simple and low-cost method provides an alternative method for the analysis of non-polar compounds in complex environmental water.
Keywords: Dispersive liquid–liquid microextraction based on solidification of floating organic droplet; Polycyclic aromatic hydrocarbons; Liquid chromatography; 1-Dodecanol;

Preparation of monodisperse immobilized Ti4+ affinity chromatography microspheres for specific enrichment of phosphopeptides by Zhiyuan Yu; Guanghui Han; Shutao Sun; Xinning Jiang; Rui Chen; Fangjun Wang; Ren’an Wu; Mingliang Ye; Hanfa Zou (34-41).
This study presented an approach to prepare monodisperse immobilized Ti4+ affinity chromatography (Ti4+-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8 μm were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13 μm were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti4+ ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti4+-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from mixture of tryptic digests of α-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5 fmol for standard tryptic digest of β-casein. The Ti4+-IMAC microspheres were compared with commercial Fe3+-IMAC adsorbent and homemade Zr4+-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe3+-IMAC, Zr4+-IMAC and Ti4+-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti4+-IMAC had the best performance for enrichment of phosphopeptides.
Keywords: Monodisperse; Microspheres; Phosphopeptides enrichment; Phosphoproteome;

Various dietary polyphenolics have been found to show an inhibitory effect on xanthine oxidase (XO) which mediates oxidative stress-originated diseases because of its ability to generate reactive oxygen species (ROS), including superoxide anion radical (O2 •−) and hydrogen peroxide. XO activity has usually been determined by following the rate of uric acid formation from xanthine–xanthine oxidase (X–XO) system using the classical XO activity assay (UV-method) at 295 nm. Since some polyphenolics have strong absorption from the UV to visible region, XO-inhibitory activity of polyphenolics was alternatively determined without interference by directly measuring the formation of uric acid and hydrogen peroxide using the modified CUPRAC (cupric reducing antioxidant capacity) spectrophotometric method at 450 nm. The CUPRAC absorbance of the incubation solution due to the reduction of Cu(II)–neocuproine reagent by the products of the X–XO system decreased in the presence of polyphenolics, the difference being proportional to the XO inhibition ability of the tested compound. The structure–activity relationship revealed that the flavones and flavonols with a 7-hydroxyl group such as apigenin, luteolin, kaempferol, quercetin, and myricetin inhibited XO-inhibitory activity at low concentrations (IC50 values from 1.46 to 1.90 μM), while the flavan-3-ols and naringin were less inhibitory. The findings of the developed method for quercetin and catechin in the presence of catalase were statistically alike with those of HPLC. In addition to polyphenolics, five kinds of herbs were evaluated for their XO-inhibitory activity using the developed method. The proposed spectrophotometric method was practical, low-cost, rapid, and could reliably assay uric acid and hydrogen peroxide in the presence of polyphenols (flavonoids, simple phenolic acids and hydroxycinnamic acids), and less open to interferences by UV-absorbing substances.
Keywords: Xanthine oxidase inhibition activity; Cupric reducing antioxidant capacity assay; Phenolics; Flavonoids;

Synchronous fluorescence determination of urinary 1-hydroxypyrene, β-naphthol and 9-hydroxyphenanthrene based on the sensitizing effect of β-cyclodextrin by Hong-Mei Yang; Yong-Sheng Wang; Jun-Hong Li; Gui-Rong Li; Ying Wang; Xuan Tan; Jin-Hua Xue; Xi-Lin Xiao; Rong-Hui Kang (51-57).
A novel method for the simultaneous determination of 1-hydroxypyrene (1-OHP), β-naphthol (β-NAP) and 9-hydroxyphenanthrene (9-OHPe) in human urine has been established by using synchronous fluorescence spectrometry. It was based on the fact that synchronous fluorescence spectrometry can resolve the broad-band overlapping of conventional fluorescence spectra, which arise from their similar molecular structures. Only one single scan is needed for quantitative determination of three compounds simultaneously when Δλ  = 15 nm is chosen. The signals detected at these three wavelengths, 369.6, 330.0 and 358.0 nm, vary linearly when the concentration of 1-OHP, β-NAP and 9-OHPe is in the range of 2.16 × 10−8–1.50 × 10−5  mol L−1, 1.20 × 10−7–1.10 × 10−5  mol L−1 and 1.07 × 10−7–3.50 × 10−5  mol L−1, respectively. The correlation coefficients for the standard calibration graphs were 0.994, 0.999 and 0.997 (n  = 7) for 1-OHP, β-NAP and 9-OHPe, respectively. The limits of detection (LOD) for 1-OHP, β-NAP and 9-OHPe were 6.47 × 10−9  mol L−1, 3.60 × 10−8  mol L−1 and 3.02 × 10−8  mol L−1with relative standard deviations (R.S.D.) of 4.70–6.40%, 2.80–4.20%, 3.10–4.90% (n  = 6), respectively. The method described here had been applied to determine traces of 1-OHP, β-NAP and 9-OHPe in human urine, and the obtained results were in good agreement with those obtained by the HPLC method. In addition, the interaction modes between β-cyclodextrin (β-CD) and 1-OHP, β-NAP or 9-OHPe, as well as the mechanism of the fluorescence enhancement were also discussed.
Keywords: Synchronous fluorescence; 1-Hydroxypyrene; β-Naphthol; 9-Hydroxyphenanthrene; β-Cyclodextrin; Urine;

A homogeneous aggregation immunoassay involving the use of gold nanoparticles (AuNPs) and light scattering detection is described for soy protein determination in food samples. AuNPs act as enhancers of the precipitate that appears when the antigen–antibody complex is formed. The AuNPs-antibody conjugate has been synthesized by physical adsorption of polyclonal anti-soy protein antibodies onto the surface of commercial AuNPs with a nominal diameter of 20 nm. The direct assay is based on the reaction of the conjugate with soy protein, which reaches the equilibrium in about 10 min, and the measurement of the light scattering intensity at 530 nm, which is proportional to the analyte concentration. The dynamic range of the calibration graph is 0.2–20 μg mL−1 and the detection limit value is 65 ng mL−1. The precision, expressed as relative standard deviation, has been assayed at two different concentrations, 0.2 and 1 μg mL−1, giving values ranging from 4.7 to 5.9%. The interference of other proteins has been assayed. The usefulness of this method has been shown by its application to the analysis of fruit juice and “nonmilk yoghourt” samples. The results obtained with the proposed method are similar to those obtained by using a commercial ELISA kit, but the assay time is significantly shorter and the detection limit was about 10 times lower. A recovery study has been also performed, giving values in the range of 84.0–119.3%.
Keywords: Soy proteins; Gold nanoparticles; Homogeneous immunoassay; Light scattering detection; Food samples;

Production of ultrasensitive generic monoclonal antibodies against major aflatoxins using a modified two-step screening procedure by Daohong Zhang; Peiwu Li; Qi Zhang; Wen Zhang; Yanling Huang; Xiaoxia Ding; Jun Jiang (63-69).
Monoclonal antibodies (McAbs) cross-reactive with four major aflatoxins were achieved using a modified two-step screening procedure. The first step was twice modified indirect enzyme-linked immunosorbent assay (ELISA) and resulted in positive hybridomas and hapten-specific antibodies. The modified indirect competitive ELISA (ciELISA) was the second step, in which the competition incubation time was decreased to 30 min, aflatoxin B 1, B 2, G 1 and G 2 were all used as competitors, the concentrations of four aflatoxins were gradiently decreased in each screening. 2–3 subclonings were performed after every modified fusion and resulted in eight hybridomas that secreted antibodies with good cross-reactivity and high affinity to four aflatoxins. Five McAbs were chosen for further analysis. Of the five, two antibodies had similar reaction efficiency with aflatoxin B 1, B 2 and G 1 but showed a weak cross-reaction to G 2. Another two had almost identical reaction capability with four aflatoxins. One clone 1C11 exhibited the highest sensitivity for all four aflatoxins. The concentrations of aflatoxin B 1, B 2, G 1 and G 2 at 50% inhibition for 1C11 were 1.2, 1.3, 2.2 and 18.0 pg mL−1 respectively. This is the most sensitive for all four major aflatoxins described so far. The results indicated that the modified two-step screening procedure had superiority and these antibodies could be used for simultaneous analysis of total aflatoxins.
Keywords: Monoclonal antibodies; Modified; Aflatoxins; Cross-reactivity; Sensitivity;

Copper and nickel speciation in mine effluents by combination of two independent techniques by Parthasarathi Chakraborty; Jiujiang Zhao; C.L. Chakrabarti (70-76).
To control potentially toxic metals in water resources it is necessary to know metal speciation and changes in the metal speciation that occur after aqueous effluents containing metals are discharged into freshwaters. This work explores the speciation of nickel and copper in metal-mining aqueous effluents. Diffusive gradients in thin films (DGT) technique and competing ligand exchange (CLE) method have been applied to determine the speciation of nickel and copper. The results of this investigation demonstrate that combination of two analytical techniques having complementary analytical capabilities can provide a better physicochemical picture of metal speciation than either one of the analytical technique can do alone. The combined use of these techniques revealed that copper formed labile complexes having slow diffusion coefficient along with the presence of small labile copper complexes. Nickel-dissolved organic complexes (DOC) complexes in the aqueous effluent have been found to have fast diffusion coefficient. The results are likely to have environmental significance for providing a link between the metal species in mine aqueous effluent and their bioavailability by determining the characteristics of copper and nickel complexes in metal-mine aqueous effluents. This knowledge is expected to promote a better understanding of the lability of DOC complexes of copper and nickel in mining effluents.
Keywords: Mining aqueous effluents; Diffusive gradients in thin films; DGT; Competing ligand exchange method; Adsorptive stripping voltammetry (AdSV);

An electrochemical biosensor for the detection of DNA based a peptide nucleic acid (PNA) capture probe (CP) modified indium tin oxide electrode (ITO) is described in this report. After hybridization, a threading intercalator, N,N′-bis[(3-propyl)-imidazole]-1,4,5,8-naphthalene diimide (PIND) imidazole complexed with Ru(bpy)2Cl (PIND-Ru, bpy = 2,2′-bipyridine), was introduced to the biosensor. PIND-Ru selectively intercalated to double-stranded DNA (ds-DNA) and became immobilized on the biosensor surface. Voltammetric tests showed highly stable and reversible electrochemical oxidation/reduction processes and the peak currents can directly be utilized for DNA quantification. When the tests were conducted in an amine-containing medium, Tris–HCl buffer for example, a remarkable improvement in the voltammetric response and noticeable enhancements of voltammetric and amperometric sensitivities were observed due to the electrocatalytic activity of the [Ru(bpy)2Cl] redox moieties. Electrocatalytic current was observed when as little as 3.0 attomoles of DNA was present in the sample solution.
Keywords: Deoxyribonucleic acid; Intercalator; Electrocatalysis; Ruthenium; Bipyridine; Amperometry;

An optical sensor for mercury ion (Hg2+), based on quenching the fluorescence of the sensing reagent porphyrin immobilized in plasticized poly(vinyl chloride) (PVC) membrane, has been developed. The responses to mercury ion were compared for the sensors modified with three porphyrin compounds including 5,10,15,20-tetraphenylporphyrin (TPP), tetra(p-dimethylaminophenyl)porphyrin (TDMAPP) and tetra(N-phenylpyrazole) porphyrin (TPPP). Among them, TDMAPP showed the most remarkable response to Hg2+. The drastic decrease of the TDMAPP fluorescence intensity was attributed to the formation of a complex between TDMAPP and Hg2+, which has been utilized as the fabrication basis of a Hg2+-sensitive fluorescence sensor. The analytical performance characteristics of the TDMAPP modified sensor was investigated. The response mechanism, especially involving the response difference of three porphyrin compounds, was discussed in detail. The sensor can be applied to the quantification of Hg2+ with a linear range covering from 4.0 × 10−8  mol L−1 to 4.0 × 10−6  mol L−1. The limit of detection was 8.0 × 10−9  mol L−1. The sensor exhibited excellent reproducibility, reversibility and selectivity. Also, the TDMAPP-based sensor was successfully used for the determination of Hg2+ in environmental water samples.
Keywords: Optical sensor; Porphyrin; Mercury ion; Fluorescence;

The objective of this study was to create a thin film optode for fast pH measurements that meets the requirements for imaging pH-responses from cells as well as for a future hybrid design for detection of multiple analytes simultaneously. The sensor is based on the covalent attachment of 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) to a film forming cellulose acetate material through a sulfonamide linkage. The synthesis routes of the cellulose material and regio-specific covalent attachment of the dye are described in detail. The sensor was sterilized in two different ways and showed excellent biocompatibility with Chinese hamster ovary cells. Imprints from cells and μm thin cell extensions were visualized when altering pH of the surrounding solution. The sensor was tested together with time-dependent sigmoidal calibrations giving pH determinations with an exceptional precision and accuracy during measurement within pH 6–8. The precision of the optode, calculated as pooled S.D. (n  = 8) according to IUPAC recommendations between pH 6.641 and 7.742 was 0.0029. The accuracy was significantly better than the electrode used as reference during the measurements. The response time (0–95%) was 100 s between pH 6.641 and 7.742 and the reverse response (95–0%) was 80 s. The sensor shows great potential for extra-cellular pH determination over time during cell growth and pharmacological exposure.
Keywords: pH; Imaging; Optode; Planar; Cells; Neurons;

A pre-column derivatization method for the sensitive determination of aldehydes using the tagging reagent 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxy] ethyl carbonylhydrazine (DBCEEC) followed by high-performance liquid chromatography with fluorescence detection and APCI-MS identification has been developed. The chromophore of fluoren-9-methoxy-carbonylhydrazine (Fmoc-hydrazine) reagent was replaced by 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxy] ethyl functional group, which resulted in a sensitive fluorescence tagging reagent DBCEEC. DBCEEC could easily and quickly labeled aldehydes. The maximum excitation (300 nm) and emission (400 nm) wavelengths did not essentially change for all the aldehyde derivatives. Derivatives were sufficiently stable to be efficiently analyzed by high-performance liquid chromatography. The derivatives showed an intense protonated molecular ion corresponding m/z [M + (CH2) n ]+ in positive-ion mode (M: molecular weight of DBCEEC, n: corresponding aldehyde carbon atom numbers). The collision-induced dissociation of protonated molecular ion formed fragment ions at m/z 294.6, m/z 338.6 and m/z 356.5. Studies on derivatization demonstrated excellent derivative yields in the presence of trichloroacetic acid (TCA) catalyst. Maximal yields close to 100% were observed with a 10 to 15-fold molar reagent excess. Separation of the derivatized aldehydes had been optimized on ZORBAX Eclipse XDB-C8 column with aqueous acetonitrile as mobile phase in conjunction with a binary gradient elution. Excellent linear responses were observed at the concentration range of 0.01–10 nmol mL−1 with coefficients of >0.9991. Detection limits obtained by the analysis of a derivatized standard containing 0.01 nmol mL−1 of each aldehyde, were from 0.2 to 1.78 nmol L−1 (at a signal-to-noise ratio of 3).
Keywords: Column liquid chromatography; Aldehydes; 2-[2-(7H-dibenzo[a,g] carbazol-7-yl)-ethoxy] ethyl carbonylhydrazine (DBCEEC); Mass spectrometry;

The measurement of cyclic nucleotide phosphodiesterase 4 activities via the quantification of inorganic phosphate with malachite green by Sha Zhu; Zhiyong Gan; Zhirong Li; Yin Liu; Xiaolan Yang; Ping Deng; Yanlin Xie; Mingan Yu; Hong Liao; Yunsheng Zhao; Lina Zhao; Fei Liao (105-110).
A spectrometric method was investigated to measure the activities of recombinant human cyclic nucleotide phosphodiesterase 4 (PDE4), based on the use of malachite green (MLG) to quantify phosphate released from adenosine-5′-monophosphate (AMP) by the action of calf intestinal alkaline phosphatase (CIAP). Glycerol at 2% stabilized the complex between MLG and phosphomolybdate, whose absorbance at 630 nm was proportional to phosphate concentrations with resistance to common substances in PDE4 reaction mixtures except papaverine. CIAP had the Michaelis–Menten constant (K m) of (12.0 ± 2.1) μM (n  = 3) for AMP at pH 7.4, and was resistant to EDTA below 0.20 mM. By the coupled end-point assay at 30.0 U L−1 CIAP with reaction durations within 30 min, the rates to release phosphate in PDE4 reaction mixtures containing 10.0 mM MgCl2 and 0.10 mM EDTA linearly responded to the amounts of PDE4 over wide ranges. Meanwhile, K m of PDE4 was (8.8 ± 0.2) μM (n  = 2), zinc ion inhibited PDE4 and rolipram had the inhibition constant about 10 nM. These results supported that by the coupled end-point assay, this method was promising to screen of PDE inhibitors that had no interference with the MLG assay of phosphate.
Keywords: Cyclic nucleotide phosphodiesterase; Calf intestinal alkaline phosphatase; Inorganic phosphate; Enzyme inhibitor screening; Malachite green;

Determination of oxytocin in milk of cows administered oxytocin by B.S. Prakash; Vijay Paul; Heike Kliem; Ulrich Kulozik; Heinrich H.D. Meyer (111-115).
To address people's concerns of exogenous oxytocin (OT) administration to lactating bovines, a study was undertaken to (a) establish an enzyme immunoassay (EIA) for OT determination in milk, (b) quantify OT in milk of cows administered OT, and (c) study influence of pasteurization on OT stability in milk. A sensitive EIA validated according to the criteria of European Union—Decision 2002/657/EC was developed for OT in skim milk in an analytical range of 10–250 pg mL−1 with a decision limit (CCα) of 30 pg mL−1 and detection capability (CCβ) of 41.5 pg mL−1. Milk samples collected from cows (n  = 38) administered either 25 or 50 IU OT prior to milking were investigated for the presence of OT. There was no significant difference among both groups with the mean concentrations of OT being 15.8 and 14.9 pg mL−1 for cows subjected to 25 and 50 IU OT administration, respectively. The OT levels in skim milk of control cows (n  = 30; untreated) were basal (around 10 pg mL−1). All the analyzed milk samples were below the CCα value of 30 pg mL−1. Pasteurization of OT spiked milk samples at different temperature and sample holding conditions reduced the immunological activity of OT to 43% at 110 °C. However, no further decline occurred in the immunological activity with increased pasteurization temperature and time. It was concluded that the milk OT concentrations after OT administrations were minimal and below the assay decision limit. However, OT was quite stable to pasteurization in OT spiked milk.
Keywords: Oxytocin; Enzyme immunoassay; Bovine milk; Pasteurization;