KATP channels do not mediate vasodilation by 3-morpholinosydnonimine in goat coronary artery

The present study investigated the role of ATP-sensitive potassium (K_ATP) channels in mediating relaxation to the nitric oxide (NO) donor, 3-morpholinosydnonimine (SIN-1) in goat coronary arteries. SIN-1 (10⁻⁸–10⁻⁵ M) caused concentration-dependent relaxations of the coronary artery ring segments contracted with K⁺ (30 mM) with an EC₅₀ of 6.61×10⁻⁷ M. Methylene blue (3×10⁻⁶ M) caused a rightward shift in the concentration–response curve of SIN-1 (10⁻⁸–3×10⁻⁵ M) with a corresponding increase in the EC₅₀ (3.62×10⁻⁶ M) of the nitrovasodilator. While the K_ATP channel blocker, glibenclamide (1 and 3×10⁻⁶ M) caused dose-dependent inhibition of vasorelaxations produced by pinacidil (10⁻⁸–10⁻⁴ M), it had no effect on the vasodilations elicited by SIN-1 (10⁻⁸–10⁻⁵ M) in the coronary arterial smooth muscle. Increasing the extracellular K⁺ concentration from 30 mM to 80 mM to reduce the K⁺ gradient across the cell membrane, inhibited the relaxations elicited by pinacidil (10⁻⁸–10⁻⁴ M). On the other hand, SIN-1 (10⁻⁸–10⁻⁵ M)-induced relaxations were potentiated in high K⁺ (80 mM) compared to those observed at K⁺ (30 mM). These results suggest that goat coronary artery vasodilations caused by the NO donor, SIN-1, do not involve K_ATP channels.     

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.     

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.     

The effect of guanidine on the accumulation of amikacin in guinea pig renal cortical slices

The role of a recently identified organic ion transport system in the accumulation of the aminoglycoside (AG), amikacin (AK) in the kidney was investigated in the present study. Because this transport system has been characterized as being a carrier for the organic cation, guanidine, the effect of guanidine on the uptake of AK into renal slices from guinea pig was examined. Renal slices incubated in medium containing AK concentrated the drug against a concentration gradient (i.e. slice: mediratio (S/M) > 1.0). This uptake was significantly reduced when an equimolar concentration (1 × 10⁻⁵ M) of another AG, gentamicin was added to the incubation medium. In contrast, AK uptake was relatively insensitive to the presence of the cation, tetraethylammonium (TEA) in the medium. Guanidine was also ineffective at inhibiting AK uptake into slices and reduced AK uptake by only 22% at guanidine concentrations of 1 × 10⁻² M. In comparison, TEA was slightly more sensitive to the presence of guanidine in the incubation media since TEA uptake was reduced by 22% at guanidine concentrations of 1 × 10⁻³ M and reduced by approximately 70% at guanidine concentrations of 1 × 10⁻² M. Thus, the results of the present study suggest that the guanidine transport system does not play a role in the renal accumulation of AK since the presence of guanidine in the incubation medium had little effect on the accumulation of AK into renal cortical slices.     

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.     

In vitro studies with ICI 169,369, a chemically novel 5-HT antagonist

ICI 169,369 is a chemically novel 5-HT antagonist that has higher affinity for the 5-HT₂ binding sites in rat cortex than it has for 5-HT₁ sites (K_i 1.79 × 10⁻⁸ and 1.58 × 10⁻⁶ M, respectively). In isolated tissue preparations ICI 169,369 was shown to be a competitive antagonist of 5-HT on the rabbit aorta, pig coronary artery and rat caudal artery. In the latter preparation it had a similar pA₂ value to ketanserin (pA₂ 8.18 ± 0.5 and 8.42 ± 0.06, respectively). Unlike ketanserin, which was inactive, ICI 169,369 was a non-surmountable antagonist at the rat stomach fundus 5-HT ‘D’ receptor, recently reclassified as 5-HT_IC. It was inactive ( > 10⁻⁶ M) at the 5-HT₃ receptors found in the isolated perfused rabbit heart and the myenteric plexus of the guinea-pig ileum. At receptors other than those for 5-HT (₁, ₂, β₁, β₂, H₁, H₂ and muscarinic), ICI 169,369 was inactive at concentrations of either 10⁻⁶ or 10⁻⁵ M. Thus the profile of ICI 169,369 should make it useful in the analysis of the role of 5-HT in physiological and pathological states.     

Adenosine alters the vascular effects of a diacylglycerol analogue and polymyxin B

Endothelium-denuded rat aorta rings were used to study the possible relationship between protein kinase C and the mechanism of adenosine-induced smooth muscle relaxation. Adenosine (5 × 10⁻⁴M) partially relaxed the aortic rings contracted by either a depolarising amount of KCl (4 × 10⁻² M) or activation of protein kinase C with l-oleoyl-2- acetyl-sn-glycerol (10⁻⁶ M). The same amount of adenosine blocked the further relaxation obtained in the presence of polymyxin B (5 × 10⁻⁵ M), a protein kinase C blocking agent. These results suggest a possible interaction in vascular smooth muscle between adenosine and protein kinase C.     

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.     

Effect of temperature on limitation by MK-801 of firing of action potentials by spinal cord neurons in cell culture

The anticonvulsant, MK-801, limited sustained high frequency repetitive firing of sodium-dependent action potentials by mouse spinal cord neurons in monolayer dissociated cell culture. Limitation was voltage- and temperature-dependent and was accompanied by decreasing rate of rise of action potentials until firing ceased during the 400 ms depolarizations. The IC₅₀ for limitation was 2 × 10⁻⁷ M at 37°C, 6.4 × 10⁻⁷ M at 35°C, and 4 × 10⁻⁵ M at 23°C. The relationship between the percentage of neurons capable of sustained repetitive firing and MK-801 concentration at 33°C was biphasic. The first phase (about 50%) of limitation had IC_50a = 1.5 × 10⁻⁷ M, and the second had IC_50b = 2 × 10⁻⁴ M; the midpoint of the connecting plateau was 10⁻⁵ M. At temperatures below 37°C, the current needed to achieve maximal firing increased. The maximal rate of rise, maximal firing frequency and sensitivity to MK-801 of action potentials elicited by 1 ms stimuli decreased at temperatures below 37°C. Passive membrane properties were unchanged. Slow firing and a temperature-sensitive conformational change in voltage-activated sodium channels could account for the higher concentrations of MK-801 required to block sodium-dependent action potentials at temperatures below 37°C.     

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.     

Thiols in Scenedesmus vacuolatus upon exposure to metals and metalloids

Phytochelatins are intracellular metal ligands produced by algae when exposed to elevated metal concentrations. In freshwater ecosystems, algae are exposed to a wide range of metals and metalloids. The aim of this study was thus to investigate phytochelatin induction in freshwater algae upon metal and metalloid exposure. To that purpose, the unicellular green alga Scenedesmus vacuolatus, was exposed to Cu, Zn, Ni, Pb and Ag, as well as to As(III), As(V), Sb(III) and Sb(V), and examined for its thiol content (gamma-glutamylcysteine, glutathione and phytochelatins). Glutathione content was found to decrease upon the exposure to Zn and to increase upon the exposure to Pb and Ag. Phytochelatins were only induced by Cu (at [Cu²⁺] = 8 × 10⁻¹¹ M) and Pb (at [Pb²⁺] = 8 × 10⁻¹¹ to 8 × 10⁻¹⁰ M), where [Cu²⁺] and [Pb²⁺] are computed free metal ion concentrations. Glutathione content also decreased upon the exposure to Sb(V) whereas an increase was observed as a result as the exposure to As(III) and As(V). The metalloids As(III), As(V) and Sb(III) in the concentration range from 8 × 10⁻⁶ to 2 × 10⁻⁴ M (total concentrations of oxyanions) were inducing phytochelatins. Glutathione and phytochelatin content in S. vacuolatus do thus sensitively respond to exposure to a number of metals and metalloids.     

Cardiac inotropic vs. chronotropic selectivity of isradipine, nifedipine and clevidipine, a new ultrashort-acting dihydropyridine

Cardiac effects of clevidipine, a new ultrashort-acting dihydropyridine Ca²⁺ channel antagonist were investigated in Langendorff-perfused rat hearts and compared to those of nifedipine and isradipine. The aim was to determine and compare the negative inotropic vs. chronotropic potency of these drugs. The hearts were perfused with oxygenated Krebs–Henseleit buffer at a perfusion pressure of 90 cm H₂O. After stabilization, one concentration of each drug was administered for 45 min followed by a higher concentration for an additional 45 min. The concentrations of each drug in this study were 10⁻⁹, 3×10⁻⁹, 10⁻⁸, 10⁻⁷, 10^−6.5 and 10⁻⁶ M for clevidipine and nifedipine, and 10⁻¹⁰, 3×10⁻¹⁰, 10⁻⁹, 10⁻⁸, 10^−7.5 and 10⁻⁷ M for isradipine. Each concentration of each drug was tested in six hearts. Coronary flow, left ventricular dP/dt max, left ventricular systolic pressure and heart rate were recorded when the hearts were beating spontaneously and during pacing at a constant rate for 1 min. Spontaneous heart rate and atrio-ventricular conduction were not affected by clevidipine at any of the concentrations studied, while nifedipine and isradipine caused a concentration-dependent decrease. These two drugs caused atrio-ventricular block at high concentrations. All three compounds reduced cardiac contractility in a concentration-dependent manner. When isradipine was administered, at a given concencentration, heart rate and contractility decreased proportionately. When clevidipine or nifedipine was given, at a given concentration, the proportionate reduction in left ventricular dP/dt max was greater than that in heart rate, resulting in a high inotropic vs. chronotropic selectivity. It is concluded that in contrast to nifedipine and isradipine, clevidipine does not impair atrio-ventricular conduction. Like nifedipine, clevidipine is selective for inotropic vs. chronotropic cardiac effects.     

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.     

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.     

Opposite effects of Bay k 8644 and nicardipine on the inhibitory effect of Ca on rat myometrium

The effect of Ca²⁺ on the oxytocin-induced, sustained contraction of rat uterine muscle in Ca-free medium after prolonged incubation with 3 mM EGTA (Ca-free contraction) was investigated. A micromolar concentration of Ca²⁺ caused phasic contraction followed by relaxation while a submicromolar concentration caused relaxation only. Cumulative addition of Ca²⁺ (10⁻⁸-3×10⁻⁶ M) caused dose-dependent relaxation (Ca reversal). This relaxation was inhibited by nicardipine and enhanced by Bay k 8644, and the effects of these two drugs were potentiated in 45.6 mM K⁺ medium. It is concluded that the inhibitory effect of Ca²⁺ on Ca-free contraction is caused by the influx of a minute amount of Ca²⁺. Thus, Ca²⁺ has dual actions in the cell: activation at concentrations higher than 10⁻⁶ M, and inhibition alone at concentrations lower than 10⁻⁷ M.     

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.     

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.     

Action of the Na ionophore monensin on vascular smooth muscle of guinea-pig aorta

The effects of the Na⁺ ionophore monensin on contractile responses were investigated in guinea-pig aorta in normal and high K⁺ solutions. In normal K⁺ (5.4 mM) solution, monensin (2 × 10⁻⁵ M) produced a rapid increase in tension followed by slow relaxation. This contraction was markedly inhibited by phentolamine (10⁻⁵ M) or prazosin (10⁻⁶ M) and was accompanied by an increase in tritium efflux from tissue preloaded with [³H]norepinephrine. In the presence of phentolamine, monensin (1–2 × 10⁻⁵ M) or ouabain (1−2 × 10⁻⁵ M) caused only a small and slowly developing contraction. Simultaneous application of these agents caused a more rapid and greater contraction. Either monensin or ouabain gradually increased cellular Na⁺ and decreased cellular K⁺ content. When monensin was applied simultaneously with ouabain, there was a rapid increase in cellular Na⁺ and loss of cellular K⁺. In high K⁺ (65.4 mM) solution, monensin (10⁻⁶ M) slightly reduced the increased tension level but when external glucose was omitted monensin markedly inhibited the contraction. A significant decrease in tissue ATP content was observed only when monensin was applied in glucose-free solution. Similarly, hypoxia (N₂ bubbling) markedly inhibited the high K⁺ contraction and decreased the tissue ATP content only in the absence of glucose. These results suggest that monensin produces a neurogenic contraction due to the release of endogenous catecholamines and also produces a myogenic contraction by a decrease in transmembrane Na⁺ and K⁺ gradients when the Na⁺ and -K⁺ pump is inhibited by ouabain, and that monensin inhibits aerobic energy metabolism of vascular smooth muscle.     

The influence of concentration on the release of drugs from gels and matrices containing Methocel

The release of propranolol hydrochloride from hydroxypropylmethylcellulose and methylcellulose matrices has been examined at constant cellulose ether contents. As the drug content decreased, the release rate of propranolol became disproportionately higher. HPMC K4M, HPMC F4M and HPMC E4M all performed similarly. However, with methylcellulose matrices, a burst release at low drug levels was apparently due to a failure of the matrix to maintain integrity. Explanations were sought on the basis of diffusional studies. Apparent diffusion coefficients were in the order of 3.1–3.8 × 10⁻⁶ cm² s⁻¹ for propranolol hydrochloride. Each of the four grades performed similarly. Using similar diffusional studies, but HPMC K15M as the polymer, an apparent diffusion coefficient of 3.6 × 10⁻⁶ cm² s⁻¹ was derived, indicating that the coefficient was independent of molecular weight. The coefficient was dependent on HPMC content decreasing from approx. 5.5 × 10⁻⁶ to 3 × 10⁻⁶ cm² s⁻¹ as the HPMC content was increased from 5 to 15% w/w. The diffusion coefficients of tetracycline hydrochloride were lower and conversion to the free base is postulated as the explanation of previously described anomolous release for this drug from matrix tablets. The tortuosity of the gels was independent of the included drug.     

Electrochemical behavior and determination of amiloride drug in bulk form and pharmaceutical formulation at mercury electrodes

The polarographic behavior of amiloride hydrochloride has been studied in Britton–Robinson buffers of pH 1.9–11. In acidic medium at pH≤2, the dc-polarograms exhibited a single 4-electron cathodic irreversible wave, while at pH values >2, a second two-electron irreversible cathodic wave appeared at a more negative potential. The single or first wave may be attributed to the cleavage of the double bond of the ---CH=NH of the imidino amide group with the release of NH₃. While the second wave may be due to the saturation of the C=O of the carboxamide moiety. A polarographic procedure of suffocate sensitivity for the determination of bulk amiloride drug in Britton–Robinson buffer at pH 2 is described. The calibration graph was obtained over the concentration range 2.5×10⁻⁵ to 2.5×10⁻⁴ M amiloride. The limits of detection (LOD) and quantitation (LOQ) of the procedure were 1×10⁻⁵ and 3.3×10⁻⁴ M bulk amiloride, respectively. Moreover, a differential-pulse adsorptive cathodic stripping voltammetric procedure has been described to assay of the drug at lower concentration levels. The optimal conditions were: E_acc=−0.9 V, t_acc=30 s, scan rate=20 mV, pulse-height=90 mV and Britton–Robinson buffer of pH 8. The calibration graph was obtained over the concentration range 2×10⁻⁸ to 1×10⁻⁶ M for bulk amiloride. Both procedures were successfully applied to the determination of amiloride in tablets without the necessity for sample pretreatment or any time-consuming extraction or evaporation steps prior to the drug analysis.