A single, selective and simple validated method for simultaneous estimation of amiloride and hydrochlorothiazide in human plasma by liquid chromatography-tandem mass spectrometry.

A single, simple and selective method for simultaneous estimation of amiloride and hydrochlorothiazide in human plasma was validated using triamterine and hydrochlorothiazide (13)C,d2 as internal standard. The compounds were separated on a reverse-phase column with an isocratic mobile phase consisting of 2 mm ammonium acetate pH 3.0 and acetonitrile (30:70, v/v) and detected by tandem mass spectrometry with positive/negative ion mode. The analytes and internal standards were extracted from plasma using simple solid phase extraction. The ion transitions recorded in multiple reaction monitoring mode were m/z 230.1 → 116.0 for amiloride, m/z 254.1 → 237.1 for internal standard, triamterine in positive mode and m/z 296.1 → 204.9 for hydrochlorothiazide, m/z 299.2 → 205.8 for internal standard, hydrochlorothiazide (13)C,d2 in negative ion mode. Linearity in plasma was observed over the concentration range 0.1-10 ng/mL for amiloride and 5.0-500.0 ng/mL for hydrochlorothiazide. The mean recovery was 41.1 and 81.5% for amiloride and hydrochlorothiazide respectively. The coefficient of variation of the assay was less than 11.2 and 5.2% for amiloride and hydrochlorothiazide, respectively, and the accuracy was 89.0-98.1 and 96.6-102.9% for amiloride and hydrochlorothiazide, respectively. The validated method can be applied to the pharmacokinetic study of amiloride and hydrochlorothiazide.

Assembly of a UV-LED induced fluorescence system for rapid determination of amiloride in pharmaceutical tablet and human serum.

A simple and sensitive fluorescence method has been developed for the determination of amiloride (AMI) in pharmaceutical tablet and human serum with a portable, cost-effective, and easy-to-operate fluorescence system. The fluorescence system was assembled with some optical and electronic devices mainly including a 370 nm light-emitting-diode (LED) as light source, a fiber spectrometer and a Raspberry Pi computer. With the system, AMI produced a strong fluorescence emission at 413 nm, and the emitted intensity can maintain good stability with in a wide pH range from 2 to 8. The proposed method was successfully applied to the determinations of AMI in pharmaceutical tablet and human serum. Their detection limits were 1.67 ng mL-1 and 1.43 ng mL-1 respectively, and the recovery was in the range of 94.06-114.0%. Those obtained results proved that the proposed methods combined with the developed fluorescence system could be employed for the routine analysis of amiloride in pharmaceutical tablet and human serum, especially for the fast analysis of them under field conditions.

Amiloride fluxes across erythrocyte membranes.

Amiloride is known to inhibit both the influx of Na+ and the activation of mitogenesis in many cultured cell lines. This paper describes experiments in which the permeability coefficient of amiloride was determined from measurements of tracer fluxes across human erythrocytes and resealed ghosts. From an analysis of these fluxes, a permeability coefficient of 10(-7) cm/s for the uncharged form of amiloride was deduced. Based upon this measured permeability value, we present calculations of intracellular accumulation times of amiloride in cells of differing surface-to-volume ratio.

Blockade of ENaCs by amiloride induces c-Fos activation of the area postrema.

Epithelial sodium channels (ENaCs) are strongly expressed in the circumventricular organs (CVOs), and these structures may play an important role in sensing plasma sodium levels. Here, the potent ENaC blocker amiloride was injected intraperitoneally in rats and 2h later, the c-Fos activation pattern in the CVOs was studied. Amiloride elicited dose-related activation in the area postrema (AP) but only ~10% of the rats showed c-Fos activity in the organum vasculosum of the lamina terminalis (OVLT) and subfornical organ (SFO). Tyrosine hydroxylase-immunoreactive (catecholamine) AP neurons were activated, but tryptophan hydroxylase-immunoreactive (serotonin) neurons were unaffected. The AP projects to FoxP2-expressing neurons in the dorsolateral pons which include the pre-locus coeruleus nucleus and external lateral part of the parabrachial nucleus; both cell groups were c-Fos activated following systemic injections of amiloride. In contrast, another AP projection target--the aldosterone-sensitive neurons of the nucleus tractus solitarius which express the enzyme 11-ß-hydroxysteriod dehydrogenase type 2 (HSD2) were not activated. As shown here, plasma concentrations of amiloride used in these experiments were near or below the IC50 level for ENaCs. Amiloride did not induce changes in blood pressure, heart rate, or regional vascular resistance, so sensory feedback from the cardiovascular system was probably not a causal factor for the c-Fos activity seen in the CVOs. In summary, amiloride may have a dual effect on sodium homeostasis causing a loss of sodium via the kidney and inhibiting sodium appetite by activating the central satiety pathway arising from the AP.

Kinetics and bioavailability of two formulations of amiloride in man.

1. Two formulations of [(14)C]-amiloride were compared in six oedema-free subjects in single-dose (20 mg) studies separated by a two-week interval.2. Calculation of the elimination rate constant (K(e)), half-life (T((1/2))) and apparent volume of distribution (V(d)) from serum and urinary data showed no significant difference between the two formulations. The V(d) values (350 to 380 litres) were greater than total body fluid volume suggesting extravascular sequestration of amiloride.3. Serum and urinary amiloride levels were similar with both formulations. Pharmacokinetic parameters were similar to those of an earlier report based on one formulation.4. The calculated amiloride concentration in the renal distal tubule (3 muM to 20 muM) was similar to, but higher than, reported in vitro concentrations of amiloride which reduced sodium transport in isolated membranes.

Pharmacokinetics of an oral frusemide/amiloride combination tablet.

A randomized four-way crossover study was carried out in 12 healthy volunteers to investigate the pharmacokinetics of a new oral combination of frusemide (40 mg) and amiloride (5 mg), formulated as a single tablet. The experimental design of this bioequivalence study used commercially-available 40 mg frusemide tablets and 5 mg amiloride tablets as reference drugs, administered either separately or concomitantly. From a statistical analysis of plasma levels of frusemide and amiloride, no significant differences between the reference drugs alone and the combination tablet were seen in peak plasma levels, mean times to peak or mean areas under the plasma concentration-time curves (AUCs). The ratio of AUCs of the combination tablet to the reference drugs approached a limiting value many hours prior to complete elimination of the drug and hence reliable bioavailability comparisons were possible with blood sampling up to 24 hours post-dose.

Determination of some cardiovascular drugs in serum and urine by capillary isotachophoresis.

The separation and determination of amiloride, metoprolol, deacetylmetipranolol, labetalol and furosemide in human serum and urine by capillary isotachophoresis were investigated. Amiloride and beta-blockers were separated by cationic isotachophoresis in the electrolyte system sodium morpholinoethanesulfonate buffer (pH 5.5) (cL = 10 mM)-glutamic acid. Furosemide was separated using the anionic electrolyte system histidine hydrochloride buffer (pH 6.2) (cL = 10 mM)-morpholinopropanesulfonic acid. Endogenous and the possible exogenous compounds were almost totally removed from serum and urine by solid-phase extraction using a Separon SGX C18 cartridge. The recovery of compounds varied from 98.2 to 103.2%. The linearity range for the compounds was 50-1000 ng/ml. The relative standard deviations varied from 0.1 to 5.6%. The overall limits of determination ranged from 32 to 46 ng/ml of urine and from 39 to 46 ng/ml of serum, depending of the type of drugs.

Behavior and quantification studies of amiloride drug using cyclic and square-wave adsorptive stripping voltammetry at a mercury electrode.

The cyclic voltammograms of amiloride at the hanging mercury drop electrode showed a single well-defined four-electron irreversible cathodic peak in Britton-Robinson (B-R) buffer of pH 2. At higher pH values (pH > or =3), two irreversible cathodic peaks corresponding to the transfer of four (first peak) and two (second peak) electrons, were obtained The peak potentials were shifted to more negative values on the increase of pH of the medium, implying the involvement of protons in the electrode reaction and that the proton-transfer reaction precedes the proper electrode process. The 4-electron single peak (pH 2) or the first peak (pH > or = 3) may be attributed to the cleavage of the -CH=NH double bond of the N-imidino amide group with the release of NH(3) molecule. While the second peak may be due to the saturation of the C?O double bond of the carboxamide moiety. Based on the interfacial adsorptive character of the drug onto the mercury electrode surface, a simple, sensitive and low cost square-wave adsorptive cathodic stripping (SWAdCS) voltammetric procedure was optimized for analysis of the drug. The optimal operational conditions of the proposed procedure were: accumulation potential E(acc)= -0.7 V, accumulation time t(acc)= 60-65s, scan increment= 10 mV, pulse-amplitude = 50-60 mV, frequency =120 Hz using a B-R buffer of pH 8 as a supporting electrolyte. The linear concentration range was found to be 2 x 10(-9) to 2 x 10(-7) M amiloride with limits of detection (LOD) and quantitation (LOQ) of 1.9 x 10(-10) and 6.3 x 10(-10) M, respectively. The procedure was successfully applied for determination of amiloride in pharmaceutical formulation and spiked in human serum. The LOD and LOQ of amiloride spiked in human serum were 5.7 x 10(-10) and 1.9 x 10(-9) M amiloride, respectively. The procedure did not require sample pretreatment or any time-consuming extraction or evaporation steps, other than deproteinization and then centrifugal separation of protein from serum sample prior to analysis of the drug.

Amiloride pharmacokinetics in rat.

The kinetics of amiloride was investigated in plasma, urine, faeces and tissues of rats after oral (10 mg/kg) and i.v. (10 mg/kg bolus and 35 microg/h for 4-days infusion) administration. Initially the experimental data were analyzed by a multiexponential model, then a compartmental model was developed to describe the drug kinetics in plasma, urine, faeces and tissues after the i.v. bolus and the oral administration simultaneously. Aim of the model was also to predict the drug kinetics in plasma and tissues of rats after continuous i.v. infusion. The results of the prediction and the discrepancies between prediction and observed data allowed a deeper insight into the pharmacokinetics of amiloride.

Simultaneous determination of amiloride and hydrochlorothiazide in human plasma by liquid chromatography/tandem mass spectrometry with positive/negative ion-switching electrospray ionisation.

A new method for simultaneous determination of amiloride and hydrochlorothiazide by liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) operated in positive and negative ionization switching mode was developed and validated. Protein precipitation with acetonitrile was selected for sample preparation. The analytes were separated on a Phenomenex Curosil-PFP (250x4.6 mm, 5 microm) column by a gradient elution with a mobile phase consisting of 0.15% formic acid solution containing 0.23% ammonium acetate and methanol pumped at a flow rate of 1.0 mL.min(-1). Rizatriptan was used as the internal standard (IS) for quantification. The determination was carried out on a Waters Quattro-micro triple-quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode using the following transitions monitored simultaneously: positive m/z 230-->171 for amiloride, m/z 270-->158 for rizatriptan, and negative m/z 296-->205 for hydrochlorothiazide. The lower limits of quantification (LLOQs) were 0.1 and 1.0 ng.mL(-1) for amiloride and hydrochlorothiazide, respectively, which were lower than other published methods by using ultraviolet (UV), fluorimetric or mass spectrometric detection. The intra- and inter-day precision and accuracy were studied at three different concentration levels and were always better than 15% (n=5). This simple and robust LC/MS/MS method was successfully applied to the pharmacokinetic study of compound amiloride and hydrochlorothiazide tablets in healthy male Chinese volunteers.

Determination of active 5-(N,N-hexamethylene)amiloride in pig plasma.

A simple, cheap and specific quantitative method for the determination of the selective Na+/H+ exchange inhibitor, 5-(N,N-hexamethylene)amiloride, in plasma and aqueous solutions has been developed. The method involves extraction with ethyl acetate, thin-layer chromatography and spectrofluorodensitometry. The compound was separated from several unidentified metabolites in plasma. The detection limit was 6 x 10(-7) M. The calculated metabolic extraction by the liver was 29%, and the plasma half-life was 12.8 min. The free, active concentration of 5-(N,N-hexamethylene)amiloride was 19.4% of the total concentration, as determined by equilibrium dialysis.

Sensitive high-performance liquid chromatographic assay for determination of amiloride in biologic fluids using an ion-pair extraction method.

The paucity of data on disposition of the potassium-retaining diuretic amiloride is mainly owing to the lack of sensitive and specific analytic methods for measuring the drug in biologic fluids. Poor lipophilicity and extremely low plasma concentrations impose severe constraints. A method is described for analysing plasma and urine concentrations of amiloride using an ion-pair extraction method and high-performance liquid chromatography (HPLC) with fluorescence detection. Amiloride and internal standard triamterene form an ion-pair with bromothymol blue at pH 7.6. The ion-pairs are extracted into diethyl ether: dichloromethane (2:1), and back-extraction into 0.1% tetrabutylammonium hydroxide liberates the drugs from their ion pairs. Analyses are performed using HPLC with a reverse-phase C18 column and a mobile phase of 11% acetonitrile and 0.5% triethylamine adjusted to pH 3. Amiloride is quantified using fluorescence detection (366-nm excitation, 418-nm emission cutoff). Retention times are 2.2 and 6.0 min for amiloride and triamterene, respectively. The sensitivity limit is 0.2 ng/ml, and recovery is 82% for amiloride and 71% for triamterene. Calibration curves are linear (range 0.25-25 ng/ml for plasma and 0.05-2.0 micrograms/ml for urine). Inter- and intraday assay variability is less than 8% and precision is within 5%. The assay is of sufficient sensitivity and specificity for the study of the pharmacokinetics of amiloride in humans.

Intracellular accumulation of potent amiloride analogues by human neutrophils.

The mechanism of uptake of a series of amiloride derivatives by human neutrophils was investigated using [14C]amiloride and the 14C-labeled 5-(1-hexahydroazepinyl)-6-bromo analogue (BrMM) which is approximately 500-fold more potent than the parent compound at inhibiting Na+/H+ exchange. At an external concentration of 2 microM, the influx of BrMM at 37 degrees C was rapid, reaching a steady state by approximately 20 min. The rate of BrMM uptake (approximately 25 mumol/liter.min) was approximately 90-fold faster than for the same concentration of amiloride, a finding which correlates with differences in lipid partitioning of the two compounds. Uptake was unrelated to specific binding to Na+/H+ exchange transport sites: influx of either drug was nonsaturable whereas amiloride- and BrMM-mediated inhibition of Na+/H+ countertransport obeyed Michaelis-Menten kinetics with apparent Ki values of approximately 75 and approximately 0.2 microM. Entry occurred exclusively via the neutral (uncharged) forms (pK'a 8.40-8.55). Influx was markedly pH-dependent: it was enhanced by extracellular alkalinization and reduced by acidification. Influx was, however, insensitive to large changes in membrane voltage, thereby implying the protonated (charged) species to be impermeant. About 75% of the total intracellular pool of amiloride, but only approximately 25% of BrMM, is contained within the lysosomes, an expected consequence of the partitioning and subsequent trapping of a weak base within this strongly acidic subcellular compartment. With BrMM, there was a relative approximately 60-fold enrichment in the internal/external water concentration ratio of the drug; the value for amiloride was much less, approximately 4. This disparity is consistent with substantial binding of BrMM to internal constituents, presumably to proteins and/or nucleic acids. Thus, it is important to recognize that potentially large intracellular accumulations of potent analogues can occur that are not directly involved in inhibition of Na+/H+ exchange. These findings sound a cautionary note in the interpretation of results using these drugs in all cells, especially those of small size with high surface-to-volume ratios.

Kinetics of amiloride in rat following oral and intravenous administration.

The disposition profile of amiloride, a potassium sparing agent, was studied in rats by using an HPLC method coupled to spectrofluorometric detection. Amiloride was administered orally and intravenously at the dose of 10 mg/Kg. The most relevant pharmacokinetic parameters are described for both administration routes.

High-performance liquid chromatographic method for quantitating plasma levels of amiloride and its analogues.

An assay for amiloride was devised for efficient use with the wide variety of analogues available. Amiloride was extracted from 1-ml plasma samples by elution from a C8 preparative column with 6% acetonitrile-45% methanol-5.4% acetic acid, adjusted to pH 4.0 with trimethylamine. Samples were lyophilized, resuspended in 50% methanol, filtered through 0.22-microns Spin-X cartridges, applied to a reversed-phase C18 column, and eluted in a 0-50% acetonitrile gradient in 0.4% acetic acid, pH 4.5 (1.2 ml/min). Detection by ultraviolet absorbance at 360 nm was linear from 1 to 1000 ng. Versatility of the method was demonstrated with the analogues benzamil, 6-hydro-, 6-iodo-, 5-hexamethylene-, and 5-chlorobenzyl-2',4'-dimethylbenzyl-amiloride.

Simultaneous determination of furosemide and amiloride in plasma using high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method using fluorescence detection for the simultaneous determination of furosemide and amiloride is described. The chromatographic system is based on reversed-phase ion-pair chromatography with sodium dodecylsulphate as ion-pairing agent. The same counter-ion is used for the ion-pair liquid-liquid extraction to ethyl acetate. The minimum detectable concentration amounts to 0.3 ng of furosemide and 0.03 ng of amiloride per ml of plasma. The applicability of the method is demonstrated by the analysis of plasma samples taken from volunteers receiving both drugs.

High-performance liquid chromatographic analysis of amiloride in plasma and urine.

A high-performance liquid chromatographic method has been developed for amiloride in rabbit plasma and urine which uses a reversed-phase C18 column, a mobile phase (flow-rate 2 ml/min) consisting of 32% acetonitrile in 0.15 M perchloric acid, pH 2.2, and spectrofluorometric detection via excitation at 286 nm. A simple extraction step with ethyl acetate eliminates interfering peaks. Short retention times of about 2.3 and 3.8 min are observed for amiloride and the internal standard, triamterene, respectively. The method can measure 4 ng/ml amiloride in plasma. This assay has been used to explore the pharmacokinetics of amiloride in rabbits. The plasma disposition profile is biexponential after a 50-mg intravenous bolus dose and there is no evidence for saturable elimination at zero-order infusion rates of 1.8, 3.6 and 7.2 mg/h.

High-performance liquid chromatographic assay for amiloride in plasma and urine.

A sensitive and simplified high-performance liquid chromatographic procedure has been developed for quantification of amiloride in rabbit plasma, as well as human plasma and urine. Following protein precipitation with perchloric acid, the supernatant was directly injected into a C18 Nucleosil column. The mobile phase consisted of methanol-water (45:55) containing 0.1 M perchloric acid, and the compound was quantitated using a fluorescence detector at excitation and emission wavelengths of 286 and 418 nm, respectively. The average recovery was 97.6%. The calibration curve was linear over the range 2.0-20.0 ng/ml. The limit of detection was 0.5 ng/ml.

Renal tubular secretion of amiloride and its inhibition by cimetidine in humans and in an animal model.

The histamine H2 antagonist cimetidine has been shown to reduce the renal tubular secretion of other organic cations through competition for the specific transport system with organic cations in the renal proximal tubule. The potential interaction between cimetidine and the potassium-sparing diuretic amiloride was investigated in humans and in the isolated perfused rat kidney. A chronic dosing study was conducted in eight healthy subjects who received, in random order, amiloride (5 mg daily), cimetidine (400 mg twice daily), both drugs together, and a control phase in which no drug was present. Cimetidine reduced the renal clearance of amiloride by a mean of 17%, from 358 +/- 134 to 299 +/- 118 ml/min (p less than 0.05), and the urinary excretion of amiloride from 65 +/- 11 to 53 +/- 13% of the dose (p less than 0.05). Amiloride reduced the excretion of cimetidine from 43 +/- 7 to 32 +/- 9% of the dose (p less than 0.05) and the area under the plasma concentration-time curve for cimetidine by a mean of 14% (p less than 0.05) but had no effect on the renal clearance of cimetidine. In the perfused rat kidney, cimetidine reduced the amiloride unbound renal clearance to glomerular filtration rate ratio from 5-7:1 to 1-2:1 (p less than 0.05). These studies demonstrate that cimetidine inhibits the renal tubular secretion of amiloride in humans and in rats to a similar extent. In addition, in humans the gastrointestinal absorption of both amiloride and cimetidine appear to be reduced by each other, by an as yet unknown mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)