Tolfenamic Acids Research Articles

Conformational State of Fenamates at the Membrane Interface: A MAS NOESY Study.

The present work analyzes the 1H NOESY MAS NMR spectra of three fenamates (mefenamic, tolfenamic, and flufenamic acids) localized in the lipid-water interface of phosphatidyloleoylphosphatidylcholine (POPC) membranes. The observed cross-peaks in the two-dimensional NMR spectra characterized intramolecular proximities between the hydrogen atoms of the fenamates as well as intermolecular interactions between the fenamates and POPC molecules. The peak amplitude normalization for an improved cross-relaxation (PANIC) approach, the isolated spin-pair approximation (ISPA) model, and the two-position exchange model were used to calculate the interproton distances indicative of specific conformations of the fenamates. The results showed that the proportions of the A+C and B+D conformer groups of mefenamic and tolfenamic acids in the presence of POPC were comparable within the experimental error and amounted to 47.8%/52.2% and 47.7%/52.3%, respectively. In contrast, these proportions for the flufenamic acid conformers differed and amounted to 56.6%/43.4%. This allowed us to conclude that when they bind to the POPC model lipid membrane, fenamate molecules change their conformational equilibria.

Analysis of Weak Interactions in Crystals of Fenamic Acids–Ethacridine Complexes

While searching for new drugs based on combinations of fenamic acid and its derivatives with other pharmaceutically active substances, the syntheses of complexes of fenamic, flufenamic, mefenamic, and tolfenamic acids with ethacridine lactate were performed, and obtained complexes were characterized by X-ray diffraction. The inter- and intramolecular interactions were determined. An analysis of hydrogen bonds and other weak interactions was performed using Quantum Theory of Atoms in Molecules and noncovalent interaction approaches. The main force building the hydrogen-bond system are proton-accepting properties of the carboxylate group. Also, the presence of molecules of solvents in crystals results in the formation of interaction networks.

Antimicrobial and antifungal activities of bifunctional cooper(ii) complexes with non-steroidal anti-inflammatory drugs, flufenamic, mefenamic and tolfenamic acids and 1,10-phenanthroline

AbstractCooper(ii) complexes represent a promising group of compounds with antimicrobial and antifungal properties. In the present work, a series of Cu(ii) complexes containing the non-steroidal anti-inflammatory drugs, tolfenamic acid, mefenamic acid and flufenamic acid as their redox-cycling functionalities, and 1,10-phenanthroline as an intercalating component, has been studied. The antibacterial activities of all three complexes, [Cu(tolf-O,O′)2(phen)] (1), [Cu(mef-O,O′)2(phen)] (2) and [Cu(fluf-O,O′)2(phen)] (3), were tested against the prokaryotic model organisms Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) and their antifungal activities were evaluated towards the yeast, Saccharomyces cerevisiae (S. cerevisiae). The antibacterial activity of both strains has been compared with the antibiotic Neomycin. The calculated IC50 values revealed slight differences in the antibacterial activities of the complexes in the order 1 ∼ 3 > 2. The most profound growth inhibition of E. coli was observed, at its highest concentration, for the complex 1, which contains chlorine atoms in the ligand environment. The trend obtained from IC50 values is generally in agreement with the determined MIC values. Similarly, the complex 1 showed the greatest growth inhibition of the yeast S. cerevisiae and the overall antifungal activities of the Cu(ii) complexes were found to follow the order 1 > 3 ≫ 2. However, for complex 2, even at the highest concentration tested (150 μM), a 50% decrease in yeast growth was not achieved. It appears that the most potent antimicrobial and antifungal Cu(ii) complexes are those containing halogenated NSAIDs. The mechanisms by which Cu(ii) complexes cause antibacterial and antifungal activities can be understood on the basis of redox-cycling reactions between cupric and cuprous species which lead to the formation of free radicals. The higher efficacy of the Cu(ii) complexes against bacterial cells may be due to an absence of membrane-protected nuclear DNA, meaning that on entering a cell, they can interact directly with its DNA. Contrastingly, for the complexes to interact with the DNA in yeast cells, they must first penetrate through the nuclear membrane.

Bimetallic Europium and Terbium Complexes Containing Substituted Terpyridines and the NSAID Drug Tolfenamic Acid: Structural Differences, Luminescence Properties, and Theranostic Applications

A series of bioactive homobimetallic Eu(III) and Tb(III) complexes containing terpyridine derivatives (XTPY) and tolfenamic acid, as a nonsteroidal anti‐inflammatory drug (NSAID), were synthesized as cellular imaging and photocytotoxic agents. The Ln2‐dimers were interestingly showing varying solid‐state structures during the process of crystallization originated from (i) multiple coordination modes of the carboxylate group in tolfenamic acids, (ii) exchange reactions with coordinating triflate (CF3SO3–) counter anions, (iii) in situ generated hydroxyls (OH–) from basic solution and, more importantly, (iv) the non‐directionality and ionic bonding nature of lanthanide complexes. The proposed species identified in solution from the isolated complexes were broadly represented as [Ln2(XTPY)2(TFA)6] (1–6): [Eu2(TOTPY)2(TFA)6] (1), [Tb2(TOTPY)2(TFA)6] (2), [Eu2(FTPY)2(TFA)6] (3), [Tb2(FTPY)2(TFA)6] (4), [Eu2(TTPY)2(TFA)6] (5), and [Tb2(TTPY)2(TFA)6] (6), where TOTPY = 4'‐(p‐tolyl)‐2,2':6',2''‐terpyridine, FTPY = 4'‐(2‐Furanyl)‐2,2':6',2''‐terpyridine, TTPY = 4'‐(2‐Thienyl)‐2,2':6',2''‐terpyridine and TFA = tolfenamic acid. Various physicochemical and spectroscopic methods were applied to understand their structures, speciation, and optical properties. The crystal structure analysis of the complexes shows that complexes 1 and 2 are isostructural as [Ln2(TOTPY)2(TFA)4(CF3SO3)2], while 3 and 4 are isostructural as [Ln2(FTPY)2(TFA)4(µ2‐OH)](CF3SO3), and complex 5 crystallizes as [Eu2(STPY)2(TFA)6(H2O)2]. Their solid‐state structures display eight‐coordinated {LnN3O5} distorted square antiprismatic or nine‐coordinated {LnN3O6} distorted tricapped trigonal prismatic (TTP) geometry around each Ln(III) ion. The interactions of dimeric complexes with biological targets (DNA, BSA) were tested. The complexes exhibit good binding affinity to DNA (Kb ca. 105 m–1) and BSA (KBSA ca. 106 m–1) via possible partial intercalation or groove binding with XTPY/TFA ligands. Eu(III) and Tb(III) luminescence in these complexes are effectively sensitized through the reinforced dual sensitizing ligands (XTPY and TFA). The confocal fluorescence imaging studies of these probes in the HeLa and MCF‐7 cell lines were tested using the intrinsic red and green emission originates from Eu(ΙΙΙ) and Tb(ΙΙΙ). Fluorescence imaging studies displayed their sufficient uptake and distribution in the cytosol and nuclei. The complexes showed significant photocytotoxicity under 365 nm UV‐A light.

Novel copper(ii) complexes with fenamates and isonicotinamide: structure and properties, and interactions with DNA and serum albumin

Reactions of non-steroidal anti-inflammatory drugs tolfenamic, meclofenamic, mefenamic, clonixic and niflumic acids with isonicotinamide and copper(ii) acetate resulted in the formation of five novel mixed-ligand Cu(ii) coordination compounds.

Osmium tetroxide catalyzed oxidation of mefenamic and tolfenamic acids with alkaline chloramine-B: Delineation of kinetic, mechanistic and catalytic chemistry

Abstract The kinetic oxidation of mefenamic acid (MEF) and tolfenamic acid (TOL), which therapeutically belongs to the class of non-steroidal anti-inflammatory drugs (NSAIDs) finds extensive applications in pharmaceuticals. The kinetics of oxidation of MEF and TOL by chloramine-B (CAB) in the presence of osmium tetroxide (OsO4) catalyst has been investigated at 313 K in alkaline medium. The oxidation behavior of these two drugs is similar with a first-order dependence of rate on both [CAB]0 and [OsO4] and a negative-fractional-order dependence on [NaOH]. The order with respect to [Drug]0 is zero. The dielectric effect is negative. Activation parameters have been evaluated for the composite reaction as well as with respect to catalyst. The oxidation products were characterized by GC–MS analysis. The observed experimental results have been justified by a plausible reaction mechanism. The relevant kinetic rate laws have been deduced.

Redox-cycling and intercalating properties of novel mixed copper(II) complexes with non-steroidal anti-inflammatory drugs tolfenamic, mefenamic and flufenamic acids and phenanthroline functionality: Structure, SOD-mimetic activity, interaction with albumin, DNA damage study and anticancer activity

Copper(II) complexes containing non-steroidal anti-inflammatory drugs (NSAIDs) have been the subject of many research papers and reviews. Here we report the synthesis, spectroscopic study and biological activity of novel mixed copper(II) complexes with NSAIDs: tolfenamic (tolf), mefenamic (mef) and flufenamic (fluf) acids and phenanthroline (phen): [Cu(tolf-O,O′)2(phen)] (1), [Cu(mef-O,O′)2(phen)] (2), [Cu(fluf-O,O′)2(phen)] (3). Complexes were characterized by X-ray analysis and EPR spectroscopy. Complexes 1–3 are monomeric, six-coordinate and crystallize in a monoclinic space group. Interaction of Cu(II) complexes with DNA was studied by means of absorption titrations, viscosity measurements and gel electrophoresis. The relative ability of the complexes to cleave DNA even in the absence of hydrogen peroxide is in the order 3 > 2 > 1. Application of the reactive oxygen species (ROS) scavengers, L-histidine, DMSO and SOD confirmed that singlet oxygen, hydroxyl radicals (Fenton reaction) and superoxide radical were formed, respectively. Thus, in addition to mechanism of intercalation, redox-cycling mechanism which in turn lead to the formation of ROS contribute to DNA damage. Cu(II) complexes exhibit excellent SOD-mimetic activity in the order 3~1 > 2. The fluorescence spectroscopy revealed that albumin may act as a targeted drug delivery vehicle for Cu(II) complexes (K~106). The anticancer activities of complexes 1–3 were investigated using an MTS assay (reduction of the tetrazolium compound) against three cancer cell lines (HT-29 human colon adenocarcinoma, HeLa and T-47D breast cancer cells) and mesenchymal stromal cells (MSC). The most promising compound, from the viewpoint of its NSAID biological activity is 3, due to the presence of the three fluorine atoms participating in the formation of weak hydrogen-bonds at the DNA surface.

Solid-state property modification and dissolution rate enhancement of tolfenamic acid by supercritical antisolvent process

In this study, the supercritical antisolvent (SAS) process is applied to crystallization of an active pharmaceutical ingredient, tolfenamic acid, using carbon dioxide as the antisolvent. Six operating parameters in the SAS process including solvent system, operating temperature, operating pressure, solution concentration, solution flow rate and nozzle diameter are studied. The effects of operating parameters on solid-state properties of the processed tolfenamic acid including crystal habit, mean particle size and polymorphic form are compared and discussed. The crystal habit of original tolfenamic acid crystals is irregular shape with Form I polymorph. The mean particle size of original powders is about 30μm. After recrystallization using SAS process, two polymorphic forms of tolfenamic acid with different crystal habits and mean particle sizes are obtained. Form I tolfenamic acid shows a needle-like crystal habit with mean particle size of about 20μm; while Form II tolfenamic acid shows a rod-like crystal habit with mean particle size of around 10μm. In addition, the dissolution profiles of original and recrystallized tolfenamic acids are also studied and compared. Experimental results show that the recrystallized Form II tolfenamic acid crystals has an enhanced dissolution rate compared with the original sample, demonstrating that the SAS technology is an efficient process for controlling and modifying the solid-state properties of tolfenamic acid and also produces microparticles with enhanced dissolution behavior.

Fenamate Cocrystals with 4,4′-Bipyridine: Structural and Thermodynamic Aspects

Cocrystallization of nonsteroidal anti-inflammatory drug fenamates (N-phenylanthranilic acid (N-PA), niflumic acid0 (NFA), flufenamic acid (FFA), tolfenamic (TFA) and mefenamic acids (MFA)) with 4,4′-bipyridine (BP) has resulted in the formation of cocrystals with a 2:1 molar ratio. Crystal Packing Similarity analysis has revealed that the packing arrangement of the [N-PA+BP], [TFA+BP], and [MFA+BP] cocrystals consists of discrete fragments of the crystal structures of initial APIs connected to each other by BP molecules. In the case of [FFA+BP], the cocrystal contains a previously unseen packing arrangement of FFA molecules which may be a fragment of a new polymorphic FFA form. Differential scanning calorimetry studies show a good correlation between the cocrystal melting temperature and the melting points of the corresponding pure APIs. The enthalpies of cocrystal formation are small, which indicates that the packing energy gain only originates from weak van der Waals interactions between the API and BP...

1H NMR spectroscopic characterization of inclusion complexes of tolfenamic and flufenamic acids with β-cyclodextrin

The complexation between the anionic forms of tolfenamic acid and flufenamic acid with β-cyclodextrin was investigated in solution by 1D and 2D proton NMR spectroscopy. The stoichiometry of the complexes was determined by the method of continuous variation using the chemical induced shifts of both the host and guest protons. An analysis of the spectroscopic data revealed that simultaneous inclusion of both rings of tolfenamic and flufenamic acids occur, giving rise each to two isomeric 1:1 complexes. The view of a bimodal binding between these two drugs and β-cyclodextrin was also supported by ROESY experiments. Using a rough approximation, we have estimated the association constants order of magnitude of the 1:1 complexes.

Is the Fenamate Group a Polymorphophore? Contrasting the Crystal Energy Landscapes of Fenamic and Tolfenamic Acids

The concept of a polymorphophore was investigated by contrasting the crystal energy landscapes of monomorphic fenamic acid (2-(phenylamino)-benzoic acid, FA) and one of its highly polymorphic derivatives, tolfenamic acid (2-[(3-chloro-2-methylphenyl)amino]-benzoic acid, TA). The crystal energy landscapes of both molecules show that the benzoic acid R22(8) dimer motif is found in all low energy crystal structures, but conformational flexibility of the phenyl rings leads to a wide range of crystal structures with different packings of this dimer. Many of the observed fenamate crystal structures can overlay a significant proportion of the coordination environment with other observed or calculated structures, but the substituents of the phenyl group affect the ordering of the related low energy crystal structures. The crystal energy landscape of tolfenamic acid has several crystal structures, including the observed polymorphs, tightly clustered around the global minimum, whereas the corresponding cluster contains only the observed and a closely related structure for fenamic acid. Hence, the fenamate fragment is potentially permissive of a large number of structures because of the conformational flexibility, but the substituents determine whether a specific fenamate will be polymorphic. Thus, a polymorphophore promotes but does not guarantee polymorphism.

Simultaneous Determination of Mefenamic and Tolfenamic Acids in Real Samples by Terbium-Sensitized Luminescence

A simple luminescent methodology for the simultaneous determination of mefenamic and tolfenamic acids in pharmaceutical preparations and human urine is proposed. Since the native fluorescence of both analytes is not intense, the method takes advantage of the lanthanide-sensitized luminescence, which provides a higher sensitivity. Due to the strong overlapping between the luminescence spectra of both terbium complexes, the use of luminescence decay curves to resolve mixtures of the analytes is proposed, since these curves are more selective. A factorial design with three levels per factor coupled to a central composite design was selected to obtain a calibration matrix of thirteen standards plus eight blank samples that was processed using a partial least-squares (PLS) analysis. In order to assess the goodness of the proposed method, a prediction set of synthetic samples was analyzed, obtaining recovery percentages between 90 and 104 %. Limits of detection, calculated by means of a new criterion, were 14.85 μg L(-1) and 15.89 μg L(-1) for tolfenamic and mefenamic acids, respectively. The method was tested in a pharmaceutical preparation containing mefenamic acid, obtaining recovery percentages close to 100 %. Finally, the simultaneous determination of both fenamates in human urine samples was successfully carried out by means of a correction of the above-explained model. No extraction method neither prior separation of the analytes were needed.

The Involvement of Tolfenamic Acid, Mefenamic Acid and Nimesulide in Free Radical Processes

The anti-inflammatory drugs tolfenamic acid, mefenamic acid and nimesulide were investigated for their in-vitro effect on lipid peroxidation, hydroxyl and superoxide anion radicals and for their interaction with 1,1-diphenyl-2-picrylhydrazyl-stable free radical (DPPH). Lipid peroxidation of rat hepatic microsomal membranes was induced by Fe2+/ascorbic acid and assessed spectroscopically as the 2-thiobarbituric acid reactive material. The competition of these agents with dimethylsulphoxide for hydroxyl radicals generated from Fe3+/ascorbate was determined and the second-order rate constants for this reaction were calculated. Their superoxide anion radical quenching ability was assessed using the xanthine/xanthine oxidase system. It was found that tolfenamic and mefenamic acids inhibited significantly lipid peroxidation, while nimesulide had no effect. All three compounds were potent hydroxyl-radical scavengers, but they could not interact with DPPH. Tolfenamic and mefenamic acids demonstrated considerable, and nimesulide moderate superoxide anion radical-quenching activity. The results correlated with the structure and lipophilicity of the examined compounds. It is suggested that the antioxidant properties of these agents may contribute to their anti-inflammatory action.

The thermodynamic characteristics of sublimation of fenamate molecular crystals

The temperature dependences of vapor pressure were determined by the transpiration method and the thermodynamic functions of sublimation were calculated for six molecular crystals from the group of nonsteroidal antiinflammatory drugs, for niflumic, flufenamic, tolfenamic, mefenamic, and N-phenylanthranylic acids and diphenylamine. The influence of substituents on the enthalpies of sublimation of compounds of this class was studied. A correlation was observed between the enthalpies of sublimation under standard conditions and the temperatures of fusion.

Effects of fenamate on inhibitory postsynaptic currents in Purkinje’s cells

The effects of nonsteroid antiinflammatory drugs of the fenamate group (mefenamic and tolfenamic acids) on spontaneous miniature inhibitory postsynaptic currents in Purkinje's cells were studied in mouse cerebellar slices by the whole cell patch-clamp method. Both drugs in concentrations of 3-30 microM significantly prolonged miniature inhibitory postsynaptic currents and reduced their amplitude.

Infrared studies of the polymorphic states of the fenamates

Infrared spectroscopy has been used to characterize the polymorphic purity as well as to study the thermal conversion of three of the more common fenamates between their different crystalline forms via measuring changes in the NH stretch region that occur between 3300 and 3350 wavenumbers. Shifts in band frequency for mefenamic acid result from differences in internal hydrogen bonding between the NH group and either the carbonyl or hydroxyl groups of the acid moiety. Due to out-of-plane rotations about the central N C ring2 bond additional polymorphic states have been suggested for flufenamic and tolfenamic acids. Rates of conversion are given for flufenamic, mefenamic, and tolfenamic acids at temperatures between 85 and 160 °C depending on the polymorphic transition for a particular analyte. Subsequently, these rates are used to calculate the activation energy for the observed polymorphic transition. Values of 71.6, 49.0, and 50.8 kcal/mol are obtained respectively for (1) the polymorph I to II transition of mefenamic acid, (2) the polymorph I to II transition of tolfenamic acid, and (3) the polymorph III to I transition of flufenamic acid.

Effects of K + channel inhibitors on relaxation induced by flufenamic and tolfenamic acids in guinea-pig trachea

The effects of different K + channel inhibitors on flufenamic- and tolfenamic-acids-induced relaxation were studied in prostaglandin F 2α (1 μM) precontracted guinea-pig trachea. Flufenamic and tolfenamic acids (each 0.1–33 μM) and lemakalim (0.01–33 μM), but not indomethacin (0.1–33 μM), caused relaxation. Iberiotoxin (33 and 100 nM) inhibited flufenamic- and tolfenamic-acids-, but not lemakalim-, induced relaxation. Iberiotoxin (100 nM) inhibited nifedipine (10 nM–10 μM)-induced relaxation. Nifedipine (0.1 μM) inhibited the blockade of fenamate-induced relaxation by iberiotoxin. Apamin (0.1 and 1 μM) did not affect flufenamic- and tolfenamic-acids- and lemakalim-induced relaxation. Glibenclamide (10 and 33 μM) inhibited lemakalim-, but not flufenamic- and tolfenamic-acids-, induced relaxation. 4-Aminopyridine (0.5 and 2 mM) inhibited flufenamic- and tolfenamic- acids- and lemakalim-induced relaxation. Flufenamic- and tolfenamic-acids-induced relaxation is likely to be activation of Ca 2+-activated K + channels and differs from that of lemakalim.

Flufenamic and tolfenamic acids and lemakalim relax guinea-pig isolated trachea by different mechanisms

The effects of K + channel inhibitors on the relaxations induced by flufenamic and tolfenamic acids and lemakalim were examined in guinea-pig isolated trachea precontracted with prostaglandin F 2α (PGF 2α, 1 μM). Flufenamic and tolfenamic acids (0.1–33 μM) and lemakalim (0.01–33 μM) relaxed guinea-pig trachea in a concentration-dependent manner. Tetraethylammonium (0.5–2 mM), a nonspecific inhibitor of K + channels, inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation Charybdotoxin (ChTX, 33–100 nM), an inhibitor of the large Ca 2+-activated K + channels (BK Ca), also inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation. Glipizide (3.3–33 μM), an inhibitor of the ATP-sensitive K + channels (K ATP) inhibited lemakalim-induced relaxation without affecting those induced by flufenamic and tolfenamic acids. Our results indicate that the relaxations of guinea-pig isolated trachea by flufenamic and tolfenamic acids are due to activation of BK Ca. The relaxant mechanism of flufenamic and tolfenamic acids thus differs from that of lemakalim, an activator of K ATP

Fenamates inhibit contraction of guinea-pig isolated bronchus in vitro independent of prostanoid synthesis inhibition

The inhibitory and relaxant effects of flufenamic and tolfenamic acids on guinea-pig isolated bronchus were compared with those of verapamil and indomethacin. Flufenamic and tolfenamic acids (each drug, 20 μM) and verapamil (1 μM) inhibited bronchial contraction induced by Ca 2+, KC1 or PGF 2 α whereas indomethacin (20 μM) had no inhibitory effect. Only verapamil, but not flufenamic and tolfenamic acids and indomethacin, inhibited methacholine-induced contraction. Flufenamic and tolfenamic acids and verapamil (each drug, 0.1–33 μM) relaxed the bronchus precontracted by KC1 or PGF 2 α . In contrast, indomethacin (0.1–33 μM) did not relax KCl- or PGF 2 α -precontracted bronchus. Verapamil, but not flufenamic and tolfenamic acids and indomethacin, relaxed methacholine precontracted bronchus. In conclusion, fenamates inhibit Ca 2+-, KCl- and PGF 2 α -induced contractions in guinea-pig isolated bronchus in a manner involving inhibition of Ca 2+ influx but not inhibition of prostanoid synthesis.

Spectrofluorimetric determination of anthranilic acid derivatives based on terbium sensitized fluorescence.

Terbium sensitized fluorescence was used to develop a sensitive and simple spectrofluorimetric method for the determination of the anthranilic acid derivatives furosemide and mefenamic and tolfenamic acids. The method makes use of radiative energy transfer from anthranilates to terbium ions in alkaline methanolic solutions. Optimum conditions for the formation of the anthranilate-Tb3+ complexes were investigated. Under optimized conditions, the detection limits are 6 x 10(-9), 1.4 x 10(-8) and 9.0 x 10(-9) mol l-1 for furosemide, mefenamic acids and tolfenamic acid, respectively. The range of application is 2.5 x 10(-8)-5.0 x 10(-5) mol l-1 for all three drugs. The method was successfully applied to the determination of furosemide and mefenamic and tolfenamic acids in serum after extraction of the samples with ethyl acetate, evaporation of the organic layer under a stream of nitrogen at 40 degrees C and reconstitution of the residue with alkaline methanolic terbium solution prior to instrumental measurement. The mean recoveries from serum samples spiked with furosemide (5.0 x 10(-7), 2.0 x 10(-6) and 8.0 x 10(-6) mol l-1), mefenamic acid (3.0 x 10(-6), 9.0 x 10(-6) and 3.0 x 10(-5) mol l-1) and tolfenamic acid (3.1 x 10(-6), 12.5 x 10(-6) and 2.5 x 10(-5) mol l-1) were 96 +/- 8, 101 +/- 5 and 98 +/- 7%, respectively. The within-run precision (RSD) for the method for two serum samples of each drug varied from 2 to 8% and the day-to-day precision for two concentration levels varied from 2 to 13%.