Genotoxic properties of 2-dodecylcyclobutanone, a compound formed on irradiation of food containing fat

When food containing fat is treated by ionizing radiation, a group of 2-alkylcyclobutanones is formed. These components contain the same number of carbon atoms as their precursor fatty acids and the alkyl group is located in ring position 2. Thus, from palmitic acid 2-dodecylcyclobutanone is derived. To date, there is no evidence that the cyclobutanones occur in unirradiated food. Therefore, these components cannot be considered inherent to food, and for questions pertaining to risk assessment of irradiated food it would be advisable to determine the genotoxic and toxic potentials of cyclobutanones. Measurements of DNA damage in cells exposed to 2-dodecylcyclobutanone, employing the single cell microgel electrophoresis technique, have been carried out. In vitro experiments using rat and human colon cells indicate that 2-docylcyclobutanone in the concentration range of about 0.30 – 1.25 mg/ml induces DNA strand breaks in the cells. Simultaneously, a concentration related cytotoxic effect is observed as was determined by trypan blue exclusion. To which extent these in vitro findings are of relevancy for the in vivo human exposure situation needs to be investigated in further studies. In vivo tests in rats are in progress.     

The Presence of a Cyclohexyldiamine Moiety Confers Cytotoxicity to Pentacyclic Triterpenoids

Pentacyclic triterpenoids oleanolic acid, ursolic acid, betulinic acid, and platanic acid were acetylated and converted into several amides 9–31; the cytotoxicity of which has been determined in sulforhodamine B assays employing seral human tumor cell lines and nonmalignant fibroblasts. Thereby, a betulinic acid/trans-1,4-cyclohexyldiamine amide showed excellent cytotoxicity (for example, EC₅₀ = 0.6 μM for HT29 colon adenocarcinoma cells).     

Chemically surface-modified carbon nanoparticle carrier for phenolic pollutants: Extraction and electrochemical determination of benzophenone-3 and triclosan

Chemically surface-modified (tosyl-functionalized) carbon nanoparticles (Emperor 2000 from Cabot Corp.) are employed for the extraction and electrochemical determination of phenolic impurities such as benzophenone-3 (2-hydroxy-4-methoxybenzophenone) or triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol). The hydrophilic carbon nanoparticles are readily suspended and separated by centrifugation prior to deposition onto suitable electrode surfaces and voltammetric analysis. Voltammetric peaks provide concentration information over a 10–100 μM range and an estimated limit of detection of ca. 10 μM (or 2.3 ppm) for benzophenone-3 and ca. 20 μM (or 5.8 ppm) for triclosan.

Alternatively, analyte-free carbon nanoparticles immobilized at a graphite or glassy carbon electrode surface and directly immersed in analyte solution bind benzophenone-3 and triclosan (both with an estimated Langmuirian binding constants of K ≈ 6000 mol⁻¹ dm³ at pH 9.5) and they also give characteristic voltammetric responses (anodic for triclosan and cathodic for benzophenone-3) with a linear range of ca. 1–120 μM. The estimated limit of detection is improved to ca.5 μM (or 1.2 ppm) for benzophenone-3 and ca. 10 μM (or 2.3 ppm) for triclosan. Surface functionalization is discussed as the key to further improvements in extraction and detection efficiency.     

Indirect fluorescent determination of selected nitro-aromatic and pharmaceutical compounds via UV-photolysis of 2-phenylbenzimidazole-5-sulfonate

An indirect fluorescence (FL) detection method via the reactivity of UV-photolyzed 2-phenylbenzimidazole-5-sulfonate (PBSA) has been developed for non-fluorescent aromatic compounds. At high pH with UV photolysis, PBSA in the excited state is known to be quenched by reaction with oxygen species and analyte compounds that are reactive toward these oxygen species produced during photolysis can lessen the loss of PBSA FL. After off-line photolysis of PBSA in the presence of various nitro-aromatic test compounds, the increase in PBSA FL is clearly evident. A flow injection (FI) instrument using a PBSA mobile phase propelled through a Teflon coil wrapped around a Hg lamp is optimized and modified for use for liquid chromatography (LC). For the on-line FI determination of the non-fluorescent nitro-aromatic compounds such as 4-nitroaniline, 2-nitrophenol, 3-nitrophenol, 4-nitrophenol, and -nitronaphthalene, a positive linear response for PBSA FL from about 0.5 to 15 μM and detection limits generally between 0.2 and 1 μM (4–20 pmol) are found. Linear responses and detection limits of selected pharmaceutical compounds such as the antibacterial nitrofurantoin, antihistamines chlorpheniramine and brompheniramine, and other compounds were similar. In general, detection limits using UV detection at about 214 nm were not as good in the 1–2 μM range but linearity extended up to 100 μM. The amino acid phenylalanine and small peptides containing this aromatic amino acid were also determined using this method. Application of this detection method for the liquid chromatography determination of 4-nitroaniline, 2-nitrophenol, nitrofurantoin, and salicylate is shown.     

Direct electrochemistry and electrocatalysis of hemoglobin immobilized in TiO2 nanotube films

Titanium oxide nanotubes (TiO₂-NTs) synthesized by the hydrothermal method had been prepared as the co-immobilization matrix to incorporate hemoglobin (Hb) successfully. The nanostructures of TiO₂-NTs were investigated by X-ray diffraction and high-resolution electron microscopy. The Hb immobilized in TiO₂-NTs had a similar structure to the native of Hb and retained its near-native conformations as characterized by the UV–vis and FT-IR spectroscopy. A couple of quasi-reversible redox peaks with a formal potential of −0.34 V (vs. SCE) in 0.10 M pH 7.0 phosphate buffered saline (PBS) were observed. The amperometric response of the immobilized Hb linearly to H₂O₂ concentration ranged from 4 μM to 64 μM with a detection limit of 4.637 × 10⁻⁶ M and the high stability of the immobilized Hb in TiO₂-NTs constituted a promising platform for the development of biosensors.     

Sequential injection analysis (SIA)-chemiluminescence determination of indomethacin using tris[(2,2'-bipyridyl)]ruthenium(III) as reagent and its application to semisolid pharmaceutical dosage forms

Automated sequential injection (SIA) method for chemiluminescence (CL) determination of nonsteroidal anti-inflammatory drug indomethacin (I) was devised. The CL radiation was emitted in the reaction of I (dissolved in aqueous 50% v/v ethanol) with intermediate reagent tris(2,2′-bipyridyl)ruthenium(III) (Ru(bipy)₃³⁺) in the presence of acetate. The Ru(bipy)₃³⁺ was generated on-line in the SIA system by the oxidation of 0.5 mM tris(2,2′-bipyridyl)ruthenium(II) (Ru(bipy)₃²⁺) with Ce(IV) ammonium sulphate in diluted sulphuric acid. The optimum sequence, concentrations, and aspirated volumes of reactant zones were: 15 mM Ce(IV) in 50 mM sulphuric acid 41 μL, 0.5 mM Ru(bipy)₃²⁺ 30 μL, 0.4 M Na acetate 16 μL and I sample 15 μL; the flow rates were 60 μL s⁻¹ for the aspiration into the holding coil and 100 μL s⁻¹ for detection. Calibration curve relating the intensity of CL (peak height of the transient CL signal) to concentration of I was curvilinear (second order polynomial) for 0.1–50 μM I (r = 0.9997; n = 9) with rectilinear section in the range 0.1–10 μM I (r = 0.9995; n = 5). The limit of detection (3σ) was 0.05 μM I. Repeatability of peak heights (R.S.D., n = 10) ranged between 2.4% (0.5 μM I) and 2.0% (7 μM I). Sample throughput was 180 h⁻¹. The method was applied to determination of 1 to 5% of I in semisolid dosage forms (gels and ointments). The results compared well with those of UV spectrophotometric method.     

Determination of 40 synthetic food colors in drinks and candies by high-performance liquid chromatography using a short column with photodiode array detection

Forty synthetic food colors were determined in drinks and candies by reversed-phase high-performance liquid chromatography with photodiode array detection. The following food colors were analyzed within 19 min using a short analytical column (50 mm × 4.6 mm i.d., 1.8 μm) at 50 °C with gradient elution: Ponceau 6R, Tartrazine, Fast yellow AB, Amaranth, Indigotine, Naphthol yellow S, Chrysoine, Ponceau 4R, Sunset yellow FCF, Red 10B, Orange G, Acid violet 7, Brilliant black PN, Allura red AC, Yellow 2G, Red 2G, Uranine, Fast red E, Green S, Ponceau 2R, Azorubine, Orange I, Quinoline yellow, Martius yellow, Ponceau SX, Ponceau 3R, Fast green FCF, Eosine, Brilliant blue FCF, Orange II, Orange RN, Acid blue 1, Erythrosine, Amido black 10B, Acid red 52, Patent blue V, Acid green 9, Phloxine B, Benzyl violet 4B, and Rose bengal. The recoveries of these compounds added to soft drinks and candies at 5 μg/g ranged from 76.6 to 115.0%, and relative standard deviations (R.S.D.s) were within 6.0%. The limits of detection and the limits of quantitation were 0.03 and 0.1 μg/g, respectively.     

Challenges associated with the synthesis of unusual o-carboxamido stilbenes by the Heck protocol: Intriguing substituent effects, their toxicological and chemopreventive implications

The syntheses of fourteen unusual o-carboxamido stilbenes by the Heck protocol revealed surprising complexity related to intriguing substituent effects with mechanistic implications. The unexpected cytotoxic and chemopreventive properties also seem to be substituent dependent. For example, although stilbene 15d (with a 4-methoxy substituent) showed cytotoxicity on HT29 colon cancer cells with an IC(50) of 4.9 μM, the 3,4-dimethoxy derivative (15c) is inactive. It is interesting to observe that the 3,5-dimethoxy derivative (15e) showed remarkable chemopreventive activity in WRL-68 fetal hepatocytes, surpassing the gold standard, resveratrol. The resveratrol concentration needed to be 5 times higher than that of 15e to produce comparable elevation of NQO1.     

Cytotoxicity and Apoptosis Effects of Curcumin Analogue (2E,6E)-2,6-Bis(2,3-Dimethoxybenzylidine) Cyclohexanone (DMCH) on Human Colon Cancer Cells HT29 and SW620 In Vitro

Colorectal cancer (CRC) is the third most common type of cancer worldwide and a leading cause of cancer death. According to the Malaysian National Cancer Registry Report 2012–2016, colorectal cancer was the second most common cancer in Malaysia after breast cancer. Recent treatments for colon cancer cases have caused side effects and recurrence in patients. One of the alternative ways to fight cancer is by using natural products. Curcumin is a compound of the rhizomes of Curcuma longa that possesses a broad range of pharmacological activities. Curcumin has been studied for decades but due to its low bioavailability, its usage as a therapeutic agent has been compromised. This has led to the development of a chemically synthesized curcuminoid analogue, (2E,6E)-2,6-bis(2,3-dimethoxybenzylidine) cyclohexanone (DMCH), to overcome the drawbacks. This study aims to examine the potential of DMCH for cytotoxicity, apoptosis induction, and activation of apoptosis-related proteins on the colon cancer cell lines HT29 and SW620. The cytotoxic activity of DMCH was evaluated using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) cell viability assay on both of the cell lines, HT29 and SW620. To determine the mode of cell death, an acridine orange/propidium iodide (AO/PI) assay was conducted, followed by Annexin V/FITC, cell cycle analysis, and JC-1 assay using a flow cytometer. A proteome profiler angiogenesis assay was conducted to determine the protein expression. The inhibitory concentration (IC₅₀) of DMCH in SW620 and HT29 was 7.50 ± 1.19 and 9.80 ± 0.55 µg/mL, respectively. The treated cells displayed morphological features characteristic of apoptosis. The flow cytometry analysis confirmed that DMCH induced apoptosis as shown by an increase in the sub-G0/G1 population and an increase in the early apoptosis and late apoptosis populations compared with untreated cells. A higher number of apoptotic cells were observed on treated SW620 cells as compared to HT29 cells. Human apoptosis proteome profiler analysis revealed upregulation of Bax and Bad proteins and downregulation of Livin proteins in both the HT29 and SW620 cell lines. Collectively, DMCH induced cell death via apoptosis, and the effect was more pronounced on SW620 metastatic colon cancer cells, suggesting its potential effects as an antimetastatic agent targeting colon cancer cells.     

Flow injection analysis of zinc pyrithione in hair care products on a cobalt phthalocyanine modified screen-printed carbon electrode

Zinc pyrithione (ZPT) is an antibacterial and antifungal reagent that is often utilized for the antidandruff activity in hair-care shampoos with a composition level up to 1% in the formulation. It has some adverse effects to human and animal if consumed orally. A disposable type of cobalt phthalocyanide modified screen-printed carbon electrode (CoPc/SPE) in couple with flow injection analysis (FIA) was developed for easy and selective analysis of ZPT in commercial hair-care products. Under the optimized FIA conditions, the CoPc/SPE yielded a linear calibration plot in the window of 6–576 μM with sensitivity and detection limit of 1.65 nA μM⁻¹ and 0.9 μM (i.e. 1.42 pg in 5 μl sample loop), respectively, in 0.1 M KOH solution at an applied potential of 0.3 V versus Ag/AgCl. Since the approach is simple, easy, selective, and inexpensive, it offers a potential application of daily ZPT analysis in hair-care products.     

Sensitive absorption detection for micro-column liquid chromatography by ultraviolet forward-scattering degenerate four-wave mixing

The performance of forward-scattering degenerate four-wave mixing (F-D4WM) in the mid-ultraviolet (UV) region (351 nm) as a detection technique for micro-column liquid chromatography (μLC) is studied, using nitro-substituted polycyclic aromatic hydrocarbons (NO₂-PAHs) and amino-substituted anthraquinones (AAQs) as test compounds. When using round capillaries, the concentration limits of detection (LOD) for the NO₂-PAHs were 20–70-fold better compared with absorption detection using a U-shaped detector cell. The final result is influenced by photochemical degradation during 351 nm F-D4WM detection. Furthermore, it is demonstrated that the use of recently commercially available square capillaries (i.e., capillaries with both a square cross section and a square bore) instead of round ones is quite suitable for F-D4WM detection. The square capillary (internal dimensions, 75×75 μm²; external dimensions 300×300 μm²) did not cause significant chromatographic band broadening in μLC. The angle of incidence of the laser light should be 56°, thus fulfilling the Brewster condition at the air–quartz boundary. Using this approach for the AAQs, compounds not prone to significant photodegradation under the experimental conditions, a further 4-fold improvement was achieved. As a result, the overall gain in concentration LOD for 2-amino-9,10-anthraquinone (molar absorptivity 4000 M⁻¹ cm⁻¹ at 351 nm) was from 4 to 0.05 μM, and baseline irregulations were reduced.     

Evaluation of an amperometric glucose biosensor based on a ruthenium complex mediator of low redox potential

An amperometric glucose ring-disk biosensor based on a ruthenium complex mediator of low redox potential was fabricated and evaluated. This thin-layer radial flow microsensor (10 μl) with ring-disk working electrode displayed remarkable amperometric sensitivity. For Ru₃(μ₃-O)(AcO)₆(Py)₃(ClO₄) (Ru-Py), a trinuclear oxo-acetate bridged cluster, a reversible redox curve of low redox potential and narrow potential window (redox potentials were −0.190 and −0.106 V versus Ag/AgCl wire, respectively) was observed, which is comparable to many reported mediators such as ferrocene derivatives and other ruthenium complexes. The glucose and hydrogen peroxide assays were carried out with this complex-modified electrode Ru-Py-HRP-GOx/Nafion. The sensitivity was obtained 24 nA (15.4 mA M⁻¹ cm⁻²) for 10 μM glucose and 126 nA (160 mA M⁻¹ cm⁻²) for 5 μM H₂O₂, respectively with a working potential at 0 V versus Ag/AgCl. Ascorbic acid was studied as interference to the glucose assay. The application of 0 V potential versus Ag/AgCl did not avoid the occurrence of the oxidation of ascorbic acid, however, the pre-coating of ascorbate oxidase on the disk part of the ring-disk working electrode efficiently pre-oxidized the ascorbic acid and hence eliminated its interference on the glucose response. The practical reliability was also evaluated by assaying the dialysate from the prefrontal cortex of Wistar rats.     

Determination of arsenic by pervaporation-flow injection hydride generation and permanganate spectrophotometric detection

A novel pervaporation-flow injection (PFI) system for the determination of As(III) in aqueous samples at μg l⁻¹ level is described. The analytical procedure involved stopping the acceptor stream and injecting acidified As(III) samples into a 0.3 M HCl stream which was mixed with a 0.14 M sodium borohydride in 0.025 M NaOH stream. The arsine generated was transported in the pervaporation unit across a semi-permeable membrane (1.5 mm thickness) into the static acceptor solution containing 1.0×10⁻⁴ M KMnO₄ in 0.1 M H₂SO₄ where it was oxidised. The acceptor stream was restarted after 6.5 min, and the decrease in permanganate absorbance at 528 nm was monitored to determine the initial concentration of As(III) in the samples. The method is characterised by a linear calibration range from 0.25 to 2000 μg l⁻¹, a detection limit of 0.18 μg l⁻¹ and a sampling frequency of 7 h⁻¹. Samples containing As(V) were pre-treated with KI and HCl prior to injection to reduce As(V) to As(III). The effects of common anionic and cationic interferences, and the elimination of some metallic interferences using -cysteine are discussed. The method was applied to the analysis of environmental waters and the results were in good agreement with hydride generation atomic absorption spectrometric data.     

Monoclonal antibody-based ELISA for the quantification of nitrofuran metabolite 3-amino-2-oxazolidinone in tissues using a simplified sample preparation

A sensitive and specific monoclonal ELISA for the determination of tissue bound furazolidone metabolite 3-amino-2-oxazolidinone (AOZ) is described. The procedure enables the detection of AOZ in matrix supernatant after homogenisation, protease treatment, acid hydrolysis and derivatisation of AOZ released from the tissue by o-nitrobenzaldehyde. The formed p-nitrophenyl 3-amino-2-oxazolidinone (NPAOZ) is determined by ELISA calibrated with matrix-matched standards in the concentration range of 0.05–5.0 μg I⁻¹. The assay was validated according to criteria set down by Commission Decision 2002/657/EC for the performance and validation of analytical methods for chemical residues. Detection capability, set on the basis of acceptance of no false negative results, was 0.4 μg kg⁻¹ for shrimp, poultry, beef and pork muscle. This sensitivity approaches the established confirmatory LC–MS/MS able to quantify tissue-bound AOZ at levels as low as 0.3 μg kg⁻¹. An excellent correlation of results obtained by ELISA and LC/MS–MS within the concentration range 0–32.1 μg kg⁻¹ was found in the naturally contaminated shrimp samples (r = 0.999, n = 8). A similar correlation was found for the incurred poultry samples within the concentration range of 0–10.5 μg kg⁻¹ (r = 0.99, n = 8).     

Kinetic spectrophotometric method for rapid determination of trace formaldehyde in foods

A simple and sensitive kinetic-spectrophotometric method was developed for the determination of ultra trace amount of formaldehyde in food samples. The method was based on the oxidation of rhodamine B (RhB) by potassium bromate in sulfuric acid medium (formaldehyde as catalyst). The reaction was monitored by measuring the decrease in absorbance of the dye at 515 nm after 6 min. The developed method allowed the determination of formaldehyde in the range of 10–100 μg L⁻¹ with good precision, accuracy and the detection limit was down to 2.90 μg L⁻¹. The relative standard deviations for the determination of 10 and 60 μg L⁻¹ of formaldehyde were 3.0% and 1.9% (n = 10), respectively. The method was found to be sensitive, selective and was applied to the determination of formaldehyde in foods with satisfactory results.     

Sculponins A–C, three new 6,7-seco-ent-kauranoids from Isodon sculponeatus

Three new 6,7-seco-ent-kauranoids (1–3) were isolated and structures were elucidated from Isodon sculponeatus. Diterpenoids 1–3 possessing multicyclic skeletons formed via oxygen atoms are all unprecedented among ent-kauranes. Compound 1 displayed significant cytotoxic activity against K562, A549, and HepG2 human tumor cell lines, with IC₅₀ values of 1.4, 2.3, and 2.0 μM, respectively, equal to the positive control. Plausible pathways for the biosynthesis of 1 and 2 from one related diterpenoid were also postulated.     

Supported liquid membranes for the determination of vanillin in food samples with amperometric detection

A supported liquid membrane system has been developed for the extraction of vanillin from food samples. A porous PTFE membrane is impregnated with an organic solvent, which forms a barrier between two aqueous phases. The analyte is extracted from a donor phase into the hydrophobic membrane and then back extracted into a second aqueous solution, the acceptor. The determination (100–1400 μg ml⁻¹ vanillin) was performed using a PVC-graphite composite electrode versus Ag/AgCl/3MKCl at +0.850 V placed in a wall-jet flow cell as amperometric detector. The solid sample is directly placed in the membrane unit without any treatment, and the analyte was extracted from the sample, passes through the membrane and conduced to the flow cell by the acceptor stream. The limit of detection (3σ) was 44 μg ml⁻¹. The method was applied to the determination of vanillin (9–606 μg g⁻¹) in food samples.     

Time measurement-visual analysis of nickel(II) using autocatalytic reaction with sodium sulfite/hydrogen peroxide system and its application to the length detection-flow analysis

Trace amounts of nickel(II) can function as a trigger (=reaction initiator) in an autocatalytic reaction with the sodium sulfite/hydrogen peroxide system. Based on this finding, sub-μg L⁻¹ levels of nickel(II) were determined by a time measurement using the autocatalytic reaction. The detection range using the above method was 10⁻⁹–10⁻⁵ M, the detection limit (3σ) was 8.1 × 10⁻¹⁰ M (0.047 μg L⁻¹), and the relative standard deviation was 2.66% at nickel(II) concentration of 10⁻⁷ M (n = 7). This method was applied to length detection-flow injection analysis. The detection range for the flow injection analysis was 2 × 10⁻⁹–2 × 10⁻³ M. The detection limit (3σ) was 1.4 × 10⁻⁹ M (0.082 μg L⁻¹), and the relative standard deviation was 1.86 at initial nickel(II) concentration of 10⁻⁶ M (n = 7).     

Trace analysis of chlorobenzenes in water samples using headspace solvent microextraction and gas chromatography/electron capture detection

In the present work, a rapid method for the extraction and determination of chlorobenzenes (CBs) such as monochlorobenzene, 1,2-dichlorobenzene, 1,3-dichlorobenzene, 1,4-dichlorobenzene, 1,2,3-trichlorobenzene and 1,2,4-trichlorobenzene in water samples using the headspace solvent microextraction (HSME) and gas chromatography/electron capture detector (ECD) has been described. A microdrop of the dodecane containing monobromobenzene (internal standard) was used as extracting solvent in this investigation. The analytes were extracted by suspending a 2.5 μl extraction drop directly from the tip of a microsyringe fixed above an extraction vial with a septum in a way that the needle passed through the septum and the needle tip appeared above the surface of the solution. After the extraction was finished, the drop was retracted back into the needle and injected directly into a GC column. Optimization of experimental conditions such as nature of the extracting solvent, microdrop and sample temperatures, stirring rate, microdrop and sample volumes, the ionic strength and extraction time were investigated. The optimized conditions were as follows: dodecane as the extracting solvent, the extraction temperature, 45 °C; the sodium chloride concentration, 2 M; the extraction time, 5.0 min; the stirring rate, 500 rpm; the drop volume, 2.5 μl; the sample volume, 7 ml; the microsyringe needle temperature, 0.0 °C. The limit of detection (LOD) ranged from 0.1 μg/l (for 1,3-dichlorobenzene) to 3.0 μg/l (for 1,4-dichlorobenzene) and linear range of 0.5–3.0 μg/l for 1,2-dichlorobenzene, 1,3-dichlorobenzene and from 5.0 to 20.0 μg/l for monochlorobenzene and from 5.0 to 30 μg/l for 1,4-dichlorobenzene. The relative standard deviations (R.S.D.) for most of CBs at the 5 μg/l level were below 10%. The optimized procedure was successfully applied to the extraction and determination of CBs in different water samples.     

Inhibitive detection of benzoic acid using a novel phenols biosensor based on polyaniline–polyacrylonitrile composite matrix

A novel sensitive and stable phenols amperometric biosensor, based on polyaniline–polyacrylonitrile composite matrix, was applied for determination of benzoic acid. The electrochemical biosensor functioning was based on the inhibition effect of benzoic acid on the biocatalytic activity of the polyphenol oxidase (PPO) to its substrate (catechol) in 0.1 M phosphate buffer solution (pH 6.5). A potential value of −50 mV versus SCE, and a constant catechol concentration of 20 μM were selective to carry out the amperometric inhibition measurement. The kinetic parameters Michaelis-Menten constant and maximum current (I_max) in the absence and in the presence of benzoic acid were also evaluated and the possible inhibition mechanism was deduced. The inhibiting action of benzoic acid on the polyphenol oxidase electrode was reversible and of the typical competitive type, with an apparent inhibition constant of 38 μM. This proposed biosensor detected levels of benzoic acid as low as 2 × 10⁻⁷ M in solution. In addition, the effects of temperature, pH value of solution on the inhibition and the interferences were investigated and discussed herein. Inhibition studies revealed that the proposed electrochemical biosensor was applicable for monitoring benzoic acid in real sample such as milk, yoghurt, sprite and cola.