Carbon film resistor electrode for amperometric determination of acetaminophen in pharmaceutical formulations

Flow injection analysis (FIA) with amperometric detection was employed for acetaminophen quantification in pharmaceutical formulations using a carbon film resistor electrode. This sensor exhibited sharp and reproducible current peaks for acetaminophen without chemical modification of its surface. A wide linear working range (8.0 × 10⁻⁷ to 5.0 × 10⁻⁴ mol L⁻¹) in phosphate buffer solution as well as high sensitivity (0.143 A mol⁻¹ L cm⁻²) and low submicromolar detection limit (1.36 × 10⁻⁷ mol L⁻¹) were achieved. The repeatability (R.S.D. for 10 successive injections of 5.0 × 10⁻⁶ and 5.0 × 10⁻⁵ mol L⁻¹ acetaminophen solutions) was 3.1 and 1.3%, respectively, without any memory effect between injections. The new procedure was applied to the analyses of commercial pharmaceutical products and the results were in good agreement with those obtained utilizing a spectrophotometric method. Consequently, this amperometric method has been shown to be very suitable for quality control analyses and other applications with similar requirements.     

Electrochemical determination of Cephalothin antibiotic by adsorptive stripping voltammetric technique

A sensitive and reliable stripping voltammetric method was developed to determine Cephalothin antibiotic drug. This method is based on the adsorptive accumulation of the drug at a hanging mercury drop electrode and then a negative sweep was initiated, which yield a well defined cathodic peak at −625 mV versus Ag/AgCl reference electrode. To achieve high sensitivity, various experimental and instrumental variables were investigated such as supporting electrolyte, pH, accumulation time and potential, drug concentration, scan rate, convection rate and working electrode area. The monitored adsorptive current was directly proportional to the concentration of Cephalothin and it shows a linear response in the range from 4×10⁻⁷ to 1.2×10⁻⁶ mol l⁻¹ (correlation coefficient=0.9995) and the detection limit (S/N=3) is 3.3×10⁻⁹ mol l⁻¹ at an accumulation time of 3 min. The developed AdSV procedure shows a good reproducibility, the relative standard deviation R.S.D.% (n=10) at a concentration level of 5×10⁻⁷ mol l⁻¹ was 0.94%. Possible interferences by other pharmaceutical drugs and surfactants have been also evaluated. The applicability of this approach was illustrated by the determination of Cephalothin in pharmaceutical preparation and biological fluids such as serum and urine.     

Enzyme-amplified lanthanide luminescence based on complexation reaction--a new technique for the determination of doxycycline

A new spectrofluorimetric method is described for the determination of doxycycline, based on modified enzyme-amplified lanthanide luminescence. Under the optimum conditions, Eu³⁺–doxycycline forms a ternary complex with lysozyme in close proximity and lysozyme can remarkably enhance the characteristic fluorescence intensity of Eu³⁺ at 612 nm in doxycycline–Eu³⁺ binary complex. The enhanced fluorescence intensity is in proportion to the concentration of doxycycline. The limit of detection is 1.28×10⁻⁸ mol l⁻¹, with a linear range from 1.7×10⁻⁷ to 1.7×10⁻⁶ mol l⁻¹. Interferences of other coexisting substances were studied. The developed method was successfully applied to the determination of doxycycline in serum, urine and real samples. The mechanism of fluorescence enhancement was also studied.     

Potentiometric sensor for methylene blue based on methylene blue–silicotungstate ion association and its pharmaceutical applications

A methylene blue (MB) poly(vinyl chloride) membrane sensor based on MB–silicotungstate (SLT) ion association as electroactive material was described. The linear response covered the range 1×10⁻³–1×10⁻⁶ mol dm⁻³ MB solution, with a slope 52.0±0.8 mV decade⁻¹ (pH range 3.0–10.0). The detection limit was 7.65×10⁻⁷ mol dm⁻³. The electrode showed stability, good reproducibility and fast response. Interferences from common inorganic cations, some organic base were negligible. These characteristics of the electrode enabled it to be used successfully for the determination of MB in injection. There was a good agreement for the results of MB content in injection between potentiometric method and USP standard procedure.     

Polarographic behavior of cephalexin and its determination in pharmaceuticals and human serum

Cephalexin gives a reduction wave in 0.03 mol/l HCl medium at ca. −1.24 V. With cephalexin concentration higher than 2.5×10⁻⁵ mol/l, another reduction wave is observed at ca. −0.90 V. These reduction waves are attributed to the reduction of ethylenic bond of a six-membered dihydrothiazine ring. When H₂O₂ is present, the reduction wave at ca. −0.90 V is catalyzed by H₂O₂ and its reduction intermediate hydroxyl radical √OH, producing a catalytic wave. However, the reduction wave at ca. −1.24 V remains nearly unchanged. A sensitive polarographic method for the determination of cephalexin is proposed based on the reduction wave of cephalexin. The second-order derivative peak current of the wave at ca. −1.24 V is rectilinear to the cephalexin concentration in the range 1.0×10⁻⁷ to 2.5×10⁻⁵ mol/l, and the detection limit is 5.0×10⁻⁸ mol/l. The proposed method is applied to the individual tablet dosage form and human serum.     

Determination of catecholamines by flow-injection analysis and high-performance liquid chromatography with chemiluminescence detection

A chemiluminescence (CL) detection of catecholamines [norepinephrine (NE), epinephrine (E), dopamine (DA) and l-dopa (LD)] is described for the flow-injection (FI) and high-performance liquid chromatographic (HPLC) determination of these compounds. The detection method is based on the inhibition effect of catecholamines (CAs) on the CL reaction of luminol with iodine in the alkaline medium. The proposed FI method allows the determination of CAs in pharmaceutical preparations for the purpose of drug quality control. The calibration curves show good linearity in the concentration range of: 1.1–20.0 μg l⁻¹ (NE), 0.5–5.0 μg l⁻¹ (E), 0.6–9.0 μg l⁻¹ (DA) and 0.6–10.0 μg l⁻¹ (LD). The limits of detection (defined as a signal-to-noise ratio of 3) are: 0.34 μg l⁻¹ (NE), 0.15 μg l⁻¹ (E) and 0.18 μg l⁻¹ (DA, LD). The HPLC procedure was successfully applied for the determination of catecholamines (NE, E, DA) in human urine after solid-phase extraction (SPE). In a simple run time CAs can be determined in 20 min. The chromatographic linear ranges are: 5.0–72.0 μg l⁻¹ (NE), 5.0–48.0 μg l⁻¹ (E) and 5.0–96.0 μg l⁻¹ (DA). The limits of detection for three urinary CAs are: 0.71 μg l⁻¹ (NE), 0.26 μg l⁻¹ (E) and 0.73 μg l⁻¹ (DA).     

Polarographic determination of diazepam with its parallel catalytic wave in the presence of persulfate

A new method for the determination of diazepam was proposed based on its polarographic catalytic wave in the presence of persulfate. In 0.20 M NaAc–HAc (pH 4.7)–2.0×10⁻² M K₂S₂O₈ supporting electrolyte, the reduction wave of diazepam with peak potential −0.89 V (versus SCE) was catalyzed, producing a parallel catalytic wave. The peak current of the catalytic wave was 15 times higher than that of the corresponding reduction wave for 4.0×10⁻⁶ M diazepam, and was rectilinear to diazepam concentration in the range of 5.6×10⁻⁸ to 8.8×10⁻⁶ and 8.8×10⁻⁶ to 2.0×10⁻⁴ M. The detection limit was 9.6×10⁻⁹ M. The mechanism of the parallel catalytic wave of diazepam was discussed.     

Flow-injection amperometric determination of dopamine in pharmaceuticals using a polyphenol oxidase biosensor obtained from soursop pulp

The amperometric determination of dopamine (Do) in pharmaceuticals formulations by flow injection analysis (FIA) is proposed. An enzymatically modified carbon paste electrode constituted by 25% (w/w) of polyphenol oxidase obtained from Annona muricata L. tissue, 30% (w/w) of graphite, 30% (w/w) of silicone and 15% (w/w) of 7,7,8,8 tetracyanoquinodimethane (TCNQ), was used as flow-through detector. The flow amperometric detection was carried out at a potential of 0.10 V (vs. Ag/AgCl) when an injected sample volume of 250 μl was inserted on a 0.3 M phosphate buffer carrier solution (pH 7.8) flowing at 2.5 ml/min. The developed biosensor showed good stability and reproducibility, enabling up to 500 determinations in 60 days, without considerable loss of enzymatic activity. The FIA system presented a linear response to Do concentrations in the interval from 2×10⁻² to 2×10⁻⁴ M, with relative standard deviations lower than 1.5%. The kinetic parameter K_M for the soluble and immobilized enzyme was 1.45×10⁻² and 1.91×10⁻² M, respectively. In the analyses of different commercially pharmaceutical formulations a relative deviation lower than about 3.4% was obtained.     

Preparation of a cimetidine ion-selective electrode and its application to pharmaceutical analysis

A novel cimetidine ion-selective electrode is prepared, characterized and used in pharmaceutical analysis. The electrode incorporates PVC-membrane with cimetidine–phospohotungstate ion pair complex. The electrode exhibits a Nernstian response for cimetidine in the concentration range 1.0×10⁻⁵–1.0×10⁻² M with a slope of 58±1 mV per decade. The limit of detection is 5.0×10⁻⁶ M. The electrode displays a good selectivity for cimetidine with respect to a number of common foreign inorganic and organic species. It can be used in the pH range 3.0–5.5. The membrane sensor was successfully applied to the determination of cimetidine in its tablets as well as its recovery from a urine sample.     

Effects of adrenomedullin on rat cerebral arterioles

The effects of adrenomedullin on isolated rat intracerebral arterioles were investigated and compared with those of calcitonin gene-related peptide (CGRP) and amylin. Adrenomedullin produced dose-dependent vasodilation (maximum dilation 27.1±2.1% at 3×10⁻⁷ M, median effective dose (EC₅₀) 1.6×10⁻⁹ M). CGRP produced similar vasodilation (19.8±4.1%) at 10⁻⁷ M with a lower EC₅₀ of 2.8×10⁻¹¹ M. Amylin did not cause vasodilation at concentrations up to 10⁻⁶ M. Adrenomedullin-induced vasodilation was significantly suppressed by CGRP-(8–37). These data suggest that adrenomedullin is a potent vasodilator for arterioles in the cerebral microcirculation that acts through CGRP receptors.     

Effects of papaverine on isolated rabbit papillary muscle

The effect of papaverine on the positive inotropic response to isoprenaline and to calcium was studied on the rabbit isolated papillary muscle; theophylline and the calcium antagonistic D600 were used for comparison. The dose—response curve for isoprenaline was shifted to the left by papaverine (3 × 10⁻⁶ to 3 × 10⁻⁵ M), in a dose-dependent manner, while that for calcium was not affected by the same concentration. In this respect papaverine was about 30 times more potent than theophylline. In the presence of papaverine isoprenaline induced arrhythmic contractions of the papillary muscle: the incidence of arrhythmic contractions correlated to the concentration of papaverine. Papaverine 10⁻⁵ to 10⁻⁴ M caused only a positive inotropic response whereas 3 × ⁻¹⁰ to 10⁻³ M induced a biphasic response, i.e., after a positive inotropic effect followed a negative one. In the presence of 3 × 10⁻⁴ M papaverine isoprenaline failed to cause a positive inotropic response but exclusively induced arrhythmic contractions. Calcium, on the other hand, readily antagonized the negative inotropic effectof papaverine (3 × 10⁻⁴ M) and caused a contracture of the papillary muscle. The results indicate that papaverine (3 × 10⁻⁶ to 10⁻⁵ M) like theophylline (10⁻⁴ to 10⁻³ M) produces its effect by phosphodiesterase inhibition and thereby specifically potentiates the response through β-adrenoceptor stimulation. In higher concentrations (3 × 10⁻⁴ to 10⁻³ M) it act as a calcium antagonistic, like D600, and furthermore may interact with calcium moving through myocardial cell membranes to cause a contracture via a mechanism which it shares with theophylline.     

Effect of different alcohols on the contractile force of the isolated guinea-pig myocardium

The effect of methanol ethanol, n-propanol, i-propanol, n-butanol, n-butanol, n-pentanol and i-pentanol on myocardial contractile force was studied using the isolated guinea-pig ventricular strip. Ethanol, 3 × 10⁻² − 3 × 10⁻³ M caused a concentration-related reduction of myocardial contractile force. Similarly geometrically increasing concentrations of the 7 other alcohols, 1 × 10⁻³ − 1 M, decreased contractile force essentially in proportion to the concentration. The magnitude of myocardial depression produced by the 8 alcohols was proportional to the length of their carbon chains. When the pD′₂ value of each alcohol studied was related to its structure, among the alkyl alcohols, methanol and n-pentanol caused, respectively, the least and the greatest depression of contractile force. When sucrose, 1 × 10⁻² − 3 × 10⁻¹ M was added to the media to produce hyperosmolarity equivalent to that induced by ethanol, sucrose did not alter the contractile force significantly. The present study suggests that the negative inotropic action of ethanol and other alcohols is not caused indirectly by the accompanying hyperosmolarity, but most likely is caused by their direct affinity for myocardial cell structures and their consequent effects on cellular function.     

Determination of salbutamol in syrups by capillary electrophoresis with contactless conductivity detection (CE-C(4)D)

This paper describes the separation and quantification of salbutamol in pharmaceutical products (salbutamol syrups) by capillary electrophoresis (CE) with contactless conductivity detection (C⁴D). The system was studied by micellar electrokinetic capillary chromatography (MEKC) and free solution capillary electrophoresis (FSCE), being the latter chosen in function of best resolution and sensitivity in comparison with the MEKC method. CE-C⁴D was applied to analysis of salbutamol in syrups utilizing 1.0 × 10⁻² mol L⁻¹ acetic acid/sodium acetate buffer (pH 4.9) as running electrolyte. Tetraethylammonium (TEA) solution was used as internal standard. The results obtained include a linear dynamic range from 7.0 × 10⁻⁵ to 3.0 × 10⁻⁴ mol L⁻¹ and good repeatability (R.S.D. = 4.7% for n = 10 for a 7.0 × 10⁻⁵ mol L⁻¹ salbutamol solution). The detection limit was calculated as 1.0 × 10⁻⁵ mol L⁻¹ and the limit of quantification was estimated as 3.3 × 10⁻⁵ mol L⁻¹. For syrups analysis the reproducibility presented deviations between 1.5% and 2.5% (three different days) obtained for measurements in triplicate.     

Study on the fluorescence system of chlortetracycline-Eu-TOPO-sodium dodecyl sulfonate and the determination of chlortetracycline

The fluorescence system of Eu-chlortetracycline-TOPO-sodium dodecyl sulfonate was studied. It was found that chlortetracycline formed a complex with Eu(III) at pH 8.0–9.0 and then emitted the characteristic fluorescence of Eu(III). TOPO and sodium dodecyl sulfonate greatly enhanced the fluorescence intensity of the system. The experiments indicated that under the optimum determining conditions a linear relationship was obtained between the fluorescence intensity and chlortetracycline concentration in the range of 2.0 × 10⁻⁸−1.0 × 10⁻⁵ M. The detection limit was 6.0 × 10⁻⁹ M. In addition, the luminescence mechanism of the complex system has been discussed.     

Sanguinarine: A positive inotropic alkaloid which inhibits cardiac Na,K-ATPase

In isolated, isometrically contracting left guinea pig atria, sanguinarine, a benzophenanthridine alkaloid from the papaveracea Sanguinaria canadensis, produced a concentration-dependent positive inotropic effect. Between 2.3 × 10⁻⁶ M and 6.5 × 10⁻⁵ M, sanguinarine increased contractility by 108% which was comparable to the maximal inotropic effect of ouabain. Within the same concentration range, sanguinarine caused inhibition of Na⁺,K⁺-ATPase isolated from guinea pig myocardium. 100% inhibition of Na⁺,K⁺-ATPase activity occurred at 1 × 10⁻⁴ M sanguinarine. The I₅₀ for enzyme inhibition and the ED₅₀ for the inotropic action of sanguinarine were the same (6–6.5 × 10⁻⁶ M) indicating that both effects may be causally related.     

Preparation of a diclofenac potentiometric sensor and its application to pharmaceutical analysis and to drug recovery from biological fluids

A novel diclofenac ion-selective electrode is prepared, characterized and used in pharmaceutical analysis. The diclofenac complex with hexadecylpyridinium bromide is obtained in situ by soaking the PVC-membranes in a 1 × 10⁻² M diclofenac solution. Among four different solvent mediators tested, dibutyl phthalate (DBP) exhibited a proper behavior including Nernstian slope of the calibration curve, fast response time and good reproducibility of the emf values. The electrode exhibits a Nernstian slope of −59 ± 1 mV decade⁻¹ for diclofenac in the concentration range 1.0 × 10⁻⁵ to 1.0 × 10⁻² M with a limit of detection of 4.0 × 10⁻⁶ M. The electrode displays a good selectivity for diclofenac with respect to a number of common inorganic and organic species. It can be used in a pH range of 6.0–9.0. The membrane sensor was successfully applied to the determination of diclofenac in its tablets as well as for its recovery from blood serum and urine samples.     

Fluorimetric determination of some antibiotics in raw material and dosage forms through ternary complex formation with terbium (Tb(3+))

A highly sensitive and specific fluorimetric method was developed for the determination of cefazolin sodium I, cefoperazone sodium II, ceftriaxone sodium III, and cefixime IV. The proposed method involves the formation of ternary complex with Tb³⁺ in the presence of Tris buffer. The quenching of the terbium fluorescence due to the complex formation was quantitative for the four studied drugs. The effect of pH, concentration of Tris buffer and terbium were studied. The formation of the complex was highly dependent on the pH. The optimum pH was found to be pH 8 for cefazolin sodium I, ceftriaxone sodium III, cefixime IV and pH 10 for cefoperazone sodium II. The optimum concentration for Tb³⁺ was found 1 ml of 10⁻⁴ M solution and for Tris buffer 1 ml of the prepared solution. Under the described conditions, the proposed method was applicable over the concentration range 8.79×10⁻⁶–7.91×10⁻⁵, 9.7×10⁻⁶–4.49×10⁻5, 6.10×10⁻⁶–2.50×10⁻⁵, and 4.92×10⁻⁶–2.95×10⁻⁵ mol with mean percentage accuracy of 99.79±0.24, 98.97±1.25, 100.05±0.79, and 100.15±0.54 for I, II, III, and IV, respectively. The proposed method was applied successfully for the determination of studied drugs in bulk powder and in pharmaceutical formulations. The results obtained by applying the described method were statistically analyzed and compared with those obtained by applying the official method. The proposed method was used as stability indicating method for the determination of the studied drugs in the presence of their degradation products.     

Solid-phase electrochemical enzyme immunoassay with attomole detection limit by flow injection analysis

A sandwich electrochemical enzyme immunoassay with flow injection analysis for the model antigen mouse IgG has been developed with alkaline phosphatase as the enzyme label. The enzyme substrate, 4-aminophenyl phosphate and its enzymatic reaction product, 4-aminophenol have been studied by cyclic and hydrodynamic voltammetry. The determination of 4-aminophenol by flow injection analysis with electrochemical detection (FIAEC) has a linear range of 5.0 × 10⁻⁸ to 1.0 × 10⁻⁵ M, a detection limit of 2.4 × 10⁻⁸ M, and a sample throughput of 72 samples/h. The detection limit is set by a background capacitance response, which depends on the ionic strength difference between the sample and the mobile phase. The sandwich immunoassay has been characterized with respect to substrate concentration for the enzymatic reaction, detection limit, dynamic range and sources of error. Mouse IgG can be determined with a detection limit of 0.81 pg ml⁻¹ by a 30-min substrate incubation time and a six orders of magnitude linear dynamic range.     

The effect of trimethyltin on acetylcholine release in the guinea-pig trachea

The purpose of the present work was to characterise the effects of trimethyltin on the release of acetylcholine from parasympathetic nerves and its effect on the postjunctional cholinergic stimulation of a smooth muscle. The guinea-pig trachea has been used as a model. Prejunctionally, trimethyltin (3.0 × 10⁻³ M) significantly enhanced in a reversible manner the high K⁺ (75 mM) evoked release of endogenous acetylcholine and [³H]acetylcholine. The evoked release of endogenous acetylcholine and [³H]acetylcholine was released from a pool of acetylcholine being independent of extraneuronal Ca²⁺ in the presence, but not in the absence of trimethyltin. The effect of trimethyltin on the release was not inhibited by low Ca²⁺ (0 mM and 1.0 × 10⁻⁴ M) or by Ca²⁺ channel blockers (verapamil, 1.0 × 10⁻⁴ M, flunarizine, 1.0 × 10⁻⁴ M, ω-conotoxin GVIA, 2.0 × 10⁻⁷ M and ω-agatoxin, 2.0 × 10⁻⁷ M). The present results also demonstrate that trimethyltin induce emptying of a non-vesicular, probably a cytoplasmic storage pool of acetylcholine, since AH5183 (2.0 × 10⁻⁵ M), an inhibitor of the translocation of acetylcholine into synaptic vesicles, and -latrotoxin (1.0 × 10⁻⁸ M), a toxin from black widow spider venom inducing vesicle depletion, had no inhibitory effects on the release of [³H]acetylcholine evoked by trimethyltin (3.0 × 10⁻³ M). The release of [³H]acetylcholine was moreover enhanced by trimethyltin when the vesicular uptake of [³H]acetylcholine was inhibited by AH5183, probably as a result of a higher cytoplasmic concentration of [³H]acetylcholine. Trimethyltin also reduced the neuronal uptake of [³H]choline and this was probably due to a depolarising effect of trimethyltin on the cholinergic nerve terminals. A similar depolarisation induced by trimethyltin was observed during patch clamping of GH₄ C₁ neuronal cells. Postjunctionally, trimethyltin had no effect by itself or on the carbachol-induced smooth muscle contraction, indicating that trimethyltin did not have a general depolarising effect on smooth muscle cells or an effect on muscarinic receptors. Furthermore, the reduced electrical field-induced contraction and the subsequent increase in the basal smooth muscle tension that was observed by addition of trimethyltin was activity-dependent, and was most probably due to emptying of a nervous non-vesicular storage pool of acetylcholine, followed by rapid hydrolysis of acetylcholine by acetyl- and pseudocholinesterases.     

Voltammetric investigation of diethylstilbestrol

In this work electrooxidation of diethylstilbestrol (DES) was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using a glassy carbon (GC) electrode. It was statistically shown that both methods could be used for the determination of DES in the concentration range of 2×10⁻⁵–6×10⁻⁴ M by CV and 1×10⁻⁵–1×10⁻³ M in methanol (MeOH) and 4×10⁻⁵–6×10⁻⁴ M in acetonitrile (ACN) by DPV and both of the methods could be applied to human serum. A mechanism was proposed about the electrooxidation of this substance.