Eco-friendly solvent bar microextraction based on a natural deep eutectic solvent and multivariate optimization for simultaneous determination of spironolactone and canrenone in urine and plasma samples.

Simultaneous measurement of spironolactone and canrenone in urine and plasma provides valuable insight into renal function, and therapeutic efficacy and can be utilized to identify potential health risks and ensure patient safety throughout treatment. By adopting greener methods to analyze these compounds, significant reductions in the environmental impact of such studies can be achieved. For this purpose, a sensitive and eco-friendly solvent bar microextraction method using natural deep eutectic solvent (NDE) followed by high-performance liquid chromatography-diode array detection (HPLC-DAD) was developed to determine spironolactone and canrenone in urine and plasma samples. The extraction solvents were synthesized using NDE-based terpenoids containing menthol and camphor in various ratios. The extraction efficiency percentage (EE%) of both drugs was measured using response surface methodology (RSM) based on central composite design (CCD), and 29 extraction tests were conducted to determine the optimum conditions. Although all parameters were found to be significant, the extraction and elution times were critical for isolating the target analytes. Under optimized conditions, the linear dynamic ranges for spironolactone (SPI)/canrenone (CAN) were 11.7-104/13.1-104 µg L-1 and 21.7-104/24.6-104 µg L-1 in urine and plasma samples, respectively with R2 ≥ 0.993. The ranges of intra-/interprecision (relative standard deviation (RSD) %, n = 5) were 1.31-9.17%/ 2.4-11% with extraction recovery ≥ 88.6% for both drugs. The comparison findings with previously published methods confirmed that the developed NDE-solvent bar microextraction (SBME)-HPLC-DAD method for spironolactone and canrenone analysis displayed confident sensitivity, feasible operation, and simple analysis. Furthermore, the method's applicability and effectiveness were proven by successfully analyzing spironolactone and its metabolite canrenone in patients' urine and plasma samples.

A simple UHPLC-PDA method with a fast dilute-and-shot sample preparation for the quantification of canrenone and its prodrug spironolactone in human urine samples.

INTRODUCTION: Nowadays, the treatment of hypertension represents an important issue, particularly in developed countries. While in most cases the standard therapeutic approaches, consisting in the administration of 1 to 3 drugs, are adequate to reach adequate blood pressure levels, in some cases more drugs are needed: this condition is called "resistant hypertension". In this context, the administration of a diuretic, such as spironolactone or canrenoate salts, represents a standard practice. Since a reliable discrimination of real cases of resistant hypertension from cases of poor therapeutic adherence is currently difficult to obtain, the adoption of therapeutic drug monitoring has been suggested as a useful tool for this purpose. In this work, the authors developed and validated a simple, cheap and fast dilute-and-shot method with UHPLC-PDA analysis for the quantification of spironolactone and its metabolite canrenone in human urine samples. METHODS: Standards and quality controls were prepared in urine. Only 100 µL of sample were added with 80 µL of internal standard (6,7-dimethyl-2,3-di(2-pyridyl)quinoxaline) working solution and 820 µL of phosphate buffer 10 mM pH 3.2 (phase A):acetonitrile (phase B) 90:10 v:v solution. Chromatographic separation was performed on an Acquity® UPLC HSS T3 1.8 µm 2.1 × 150 mm column, with a binary gradient for 11 min at 40 °C. RESULTS: Accuracy, intra-day and inter-day precision, selectivity and sensitivity fitted FDA guidelines for all analytes (LLOQ and LOD were 156.25 ng/mL and 78.12 ng/mL, respectively, for both analytes) and recovery resulted high and reproducible. Method performances were tested on urine samples from hypertensive patients with good results. DISCUSSION: This simple analytical method could represent a useful tool for the management of antihypertensive therapy.

Simultaneous separation and rapid determination of spironolactone and its metabolite canrenone in different pharmaceutical formulations and urinary matrices by capillary zone electrophoresis.

The aim of this study was to develop a novel, sensitive, precise, simple, and rapid capillary zone electrophoresis method for the quality control of spironolactone in three different formulation types and a rapid simultaneous determination of the content of spironolactone and canrenone in urine samples using fluocinonide as an internal standard. After optimization of separation conditions, the electrolyte solution was the pH 5.5, 20 mM phosphate buffer containing 4.5 g/L sulfated-ß-cyclodextrin, 15 kV of electric filed across the capillary applied at 25°C. A diode array detector was used, and the detection wavelength was 260 nm. Under optimum conditions, good linearity was achieved with correlation coefficients from 0.9976 to 0.9997. Detection limits were 0.56 and 0.20 µg/mL, and the quantitation limits were 1.87 and 0.67 µg/mL, respectively. Excellent accuracy and precision were obtained. Recoveries of the analytes varied from 100.8 to 103.1%. The results indicated that baseline separation of analytes was obtained and this method was suitable for quantitative determination of spironolactone in pharmaceutical preparations and rapid simultaneous determination of the content of spironolactone and its major metabolite canrenone in urine samples.

Rapid UHPLC method for simultaneous determination of vancomycin, terbinafine, spironolactone, furosemide and their metabolites: application to human plasma and urine.

The ultra high performance liquid chromatography (UHPLC)-UV method for the simultaneous determination of furosemide, saluamine (furosemide metabolite), spironolactone, carnenone (spironolactone active metabolite), terbinafine, N-desmethylcarboxy terbinafine (terbinafine metabolite) and vancomycin in human plasma and urine is proposed. Good separation of the analytes was achieved with the gradient RP-UHPLC-UV with the mobile phase composed as acetonitrile and 0.1% formic acid. The determined substances were eluted from a Hypersil GOLD C(18)e (50 mm x 2.1 mm, 1.7 microm particles) column in 3.3 min. Good linear relationships were observed for all of the analytes (R(2) higher than 0.994). The limit of detection (LOD) values varied from 0.01 to 0.07 microg ml(-1), with vancomycin as an exception (0.11 microg ml(-1)). After protein precipitation and solid-phase extraction, samples of plasma and urine were analyzed. Thanks to the short analysis time and small quantities of urine or plasma needed, this method can be applied to routine clinical analysis.

[Detection of selected diuretics in biological fluids by chromatography mass spectrometry].

Modified methods for the detection and measurement of furosemide and spironolactone in biological fluids has been developed based on gas chromatography mass spectrometry. Optimal conditions for chromatography are described for the determination of the diuretic agents of interest. The proposed methods were verified by analysing urine samples obtained from patients with chronic cardiac insufficiency.

Effect of spironolactone on diuresis and urine sodium and potassium excretion in healthy dogs.

OBJECTIVES: To document the diuretic effect of different oral doses of spironolactone (SP) in healthy dogs. BACKGROUND: SP is currently mentioned as a diuretic agent in the dog. However, the recommended doses were empirically defined and their corresponding diuretic effect has never been documented in dogs. ANIMALS, MATERIALS AND METHODS: Eight adult Beagle dogs were used for two separate 2*2 cross-over designs. In the first cross-over, 4 dogs received SP orally for 8 days at 1 and 2mg/kg per day. In the second cross-over the 4 other dogs received SP similarly, but at 4 and 8 mg/kg per day. Dogs were weighed on the first and last day of each period. Plasma SP and canrenone (the main active metabolite of SP) were assayed by high performance liquid chromatography (HPLC). Daily water consumption, urine weight, urine specific gravity, and urine excretion of sodium and potassium were measured during the SP treatment. RESULTS: Two hours after SP administration, SP was metabolized into canrenone. A significant 14 and 22% decrease in urine potassium excretion was observed at 1 and 2mg/kg, respectively, but not at the two other dose levels. Daily water consumption, urine weight, urine specific gravity, and urine excretion of sodium were not significantly altered by the SP treatment regardless of dose. CONCLUSIONS: Repeated oral administration of SP at 1, 2, 4 or 8 mg/kg for 8 days had no effect on water and sodium diuresis in healthy dogs.

Pharmacokinetics of eplerenone after single and multiple dosing in subjects with and without renal impairment.

The influence of renal impairment on the pharmacokinetics of eplerenone following single and multiple dosing was evaluated. Subjects (n = 64) were stratified based on creatinine clearance values as follows: renal impairment (mild, moderate, severe), hemodialysis, and normal matches. Subjects received a single dose of eplerenone 100 mg on day 1 and then received 100 mg once daily on days 3 to 8. There were no statistically significant differences between any of the renal impairment groups and their matched-normal groups for area under the curve (AUC), C(max), or CL/F or CL/F/WT following either single or multiple dosing (P > or = .093). The inactive metabolite and inactive ring-opened form displayed greater AUCs in renal impairment. Hemodialysis removed approximately 10% of the eplerenone dose. Eplerenone 100 mg once daily was well tolerated in all groups. Considering that renal function had no significant effects on eplerenone CL/F and that eplerenone metabolites are inactive, no dose adjustment appears necessary in patients with renal dysfunction.

A validated SPE-LC-MS/MS assay for Eplerenone and its hydrolyzed metabolite in human urine.

An automated LC-MS/MS assay was validated to quantitate the first selective aldosterone blocker Eplerenone (I) and its hydrolyzed metabolite (II) in human urine. After the addition of the stable isotope labeled internal standards, human urine samples were extracted on a C(18) solid phase extraction (SPE) cartridge using a Zymark RapidTrace automation system. The extraction eluates were diluted with 20 mM ammonium acetate aqueous solution and directly injected onto the LC-MS/MS system. The chromatographic separation was performed on a reverse phase Zorbax XDB-C(8) HPLC column (2.1 x 50 mm, 5 microm) with a mobile phase of acetonitrile:water (40:60, v/v) containing 10 mM ammonium acetate (pH 7.4). I and II were ionized using positive and negative ionization mass spectrometry, respectively, to achieve the best sensitivity. The ionization polarity was switched during the run at approximately 2.5 min after the injection. Multiple reaction monitoring (MRM) with a tandem mass spectrometer was used to detect the analytes. The precursor to product ion transitions of m/z 415-->163 and m/z 431-->337 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 50-10000 ng/ml of urine for both of I and II. The lower limit of quantitation (LLOQ) was 50 ng/ml for I and II. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. Sample analysis time for each injection was 5 min; a throughput of 100 human urine standards and samples per run was achieved.

Use of partial least-squares regression for multicomponent determinations based on kinetic spectrofluorimetric data. Simultaneous determination of canrenone and spironolactone in urine.

A new spectrofluorimetric method for the simultaneous determination of canrenone and spironolactone in urine is proposed. The method is based on the different rates at which the two analytes react with hot sulfuric acid to form a trienone. The kinetic spectrofluorimetric data are processed by partial least-squares regression. The effects of sulfuric acid concentration and temperature on the system under study were also evaluated and the optimum values for carring out the reaction were 50% and 50 degrees C, respectively. The method was checked by analyzing urine samples that they contained both diuretics. The accuracy and the precision of the method were tested. The relative standard errors in the quantification of each analyte in all tested samples were 3.69 and 3.59%. The proposed method was validated by comparison with a high performance liquid chromatographic method for urine samples.

Absorption and disposition of a selective aldosterone receptor antagonist, eplerenone, in the dog.

PURPOSE: The present study was conducted to characterize the pharmacokinetics of eplerenone (EP), a selective aldosterone receptor antagonist, and its open lactone ring form in the dog. METHODS: Pharmacokinetic studies of EP were conducted in dogs following i.v., oral, and rectal dosing (15 mg/kg) and following intragastric, intraduodenal, intrajejunal, and intracolonic dosing (7.5 mg/kg). RESULTS: After oral administration, the systemic availability of EP was 79.2%. Systemic availabilities following administration via other routes were similar to that following oral administration. The half-life and plasma clearance of EP were 2.21 hr and 0.329 l/kg/hr, respectively. Plasma concentrations of the open lactone ring form were lower than EP concentrations regardless of the route of administration. The C-14 AUC in red blood cells was approximately 64% and 68% of the plasma AUC for i.v. and oral doses. Percentages of the dose excreted as total radioactivity in urine and feces were 54.2% and 40.6%, respectively, after i.v. administration, and 40.7% and 52.3%, respectively, after oral administration. The percentages of the dose excreted in urine and feces as EP were 13.7% and 2.5%, respectively, after i.v. administration, and 2.1% and 4.6% after oral administration, respectively. Approximately 11% and 15% of the doses were excreted as the open form following i.v. and oral doses. CONCLUSIONS: EP was rapidly and efficiently absorbed throughout the gastrointestinal tract, resulting in a good systemic availability. The drug did not preferentially accumulate in red blood cells. EP was extensively metabolized; however, first-pass metabolism after oral and rectal administration was minimal. EP and its metabolites appear to be highly excreted in the bile.

High-performance liquid chromatographic determination of spironolactone and its major metabolite canrenone in urine using ultraviolet detection and column-switching.

A rapid and simple column liquid chromatographic method involving a column-switching system for the determination of spironolactone and its main metabolite canrenone in urine is described. Purification and concentration was performed using an Hypersil ODS-C18, 30 microns (20 x 2.1 mm I.D.) pre-column. The most polar urinary compounds were removed by washing the pre-column with water, and the analytes were subsequently switched to a LiChrospher RP C18, 5 microns (125 x 4 mm I.D.) analytical column and separated by means of an acetonitrile-water mobile-phase. Under the proposed conditions, the extraction efficiency was approximately 100% over the 0.5-10.0 micrograms/ml concentration range. The limits of detection were 20 ng/ml for both compounds. The proposed method has been applied to urine samples obtained after the oral administration of spironolactone.

High-performance liquid chromatographic determination of spironolactone and its metabolites in human biological fluids after solid-phase extraction.

A simple and sensitive high-performance liquid chromatographic procedure to determine spironolactone and its three major metabolites in biological specimens is described. The assay involves sequential extraction on C18 and CN solid phases, and subsequent separation on a reversed-phase column. In plasma samples, spironolactone and its metabolites were completely separated within 8 min using an isocratic mobile phase, while in urine samples a methanol gradient was necessary to achieve a good separation within 14 min. Recoveries for all analytes were greater than 80% in plasma and 72% in urine. Linear responses were observed for all compounds in the range 6.25-400 ng/ml for plasma and 31.25-2000 ng/ml for urine. The plasma and urine methods were precise (coefficient of variation from 0.8 to 12.5%) and accurate (-12.1% to 7.4% of the nominal values) for all compounds. The assay proved to be suitable for the pharmacokinetic study of spironolactone in healthy human subjects.

Discovery of a natural spirolactone derivative in man and animal.

The presence of 6,7-dihydroxy-6,7-dihydrocanrenone (DHC) in man and in animal has been shown. Sodium loading results in a decrease of urinary DHC. On the contrary, sodium depletion increases its concentration.

Pharmacokinetics of spironolactone and potassium canrenoate in humans.

Plasma concentrations and urinary excretion of canrenone (III), canrenoic acid (IV) and canrenoic acid glucuronide (V) were determined by means of high performance liquid chromatography (HPLC) and fluorometry after oral administration of spironolactone (I) and potassium canrenoate (II) to human subjects. Comparison of both assays for III in plasma as well as in urine after administration of I showed marked differences. Plasma concentrations of III were significantly higher after administration of II than I, Cmax and AUC from II being 3--5 times larger than those from I by means of HPLC assay, while the fluorometrically determined values for III in plasma after administration of I and II did not differ as much from each other. On the other hand, in contrast to plasma, the amount of III excreted in urine after administration of I was much larger than that after II, i.e. 3--4 times greater by means of HPLC and over 10 times greater by means of fluorometry. These results strongly suggest that precursors of III are formed which have a higher renal clearance than that for III alone after oral administration of I. Considering the relative biological potency ratio of I and II, it is presumed that their pharmacological activities may relate to the urinary excretion of III. Plasma concentrations of IV were definitely higher after administration of II compared to those after I. Canrenoic acid (IV) was excreted mainly as glucuronide (V) in urine.

Application of ion-exchange and lipophilic-gel chromatography to the purification and group fractionation of steroidal spirolactones, isolated from biological fluids.

Chromatography of steroidal spirolactones on DEAE-Sephadex A-25 under selected pH conditions allowed efficient separation of these compounds from other steroids and many of the endogenous components of human urine. The spirolactones were recovered in high yield, mostly over 90%. Lipophilic-gel chromatography provided a useful method for group fractionation of mixtures of these spirolactones with high recoveries (generally over 90%), unaffected by the presence of endogenous material from normal human urine.

Identification of selected antihypertensive drugs by thin-layer chromatography.

A thin-layer chromatographic procedure is described for the qualitative identification of several antihypertensive drugs including certain thiazide diuretics spironolactone, triamterene, methyldopa and their metabolites. Utilization of new solvent developing systems and spray detecting reagents provides a method useful for the identification of these compounds in biologic fluids at low therapeutic concentrations. Sensitivity limits for these antihypertensive drugs are given, and alternate techniques to provide confirmatory analyses are also presented.

Pharmacokinetics of spironolactone in man.

Five healthy male volunteers received 500 mg Aldactone orally together 100 muCi 3H-20-21-spironolactone; one elderly patient received 1 mCi 3H-spironolactone without additional 'cold' drug. For 6 days the disposition kinetics of the drug were studied in plasma, urine and feces. The tritium concentrations in plasma reached a peak between 25-40 min after administration amounting to 2-3% of the dose/1. Up to the 12th h, they fell rapidly and showed a monoexponential decline (t 1/2: 2.57 +/- 0.27 days) between the 36th and 96th h. Later, a striking increase in the speed of elimination of radioactivity from plasma (t 1/2: 1.66 +/- 0.21 days) was observed. The biological half-life of labeled material in plasma was longer than that of fluorigenic compounds. 47-57% of the dose were excreted in urine and the remaining amount could be detected in feces (total recovery 90%). The half-life of the urinary excretion rate was distinctly shorter (t 1/2: 0.9 +/- 0.11 days) than that of total radioactivity in plasma. This, together with an observed increase of the polar fraction in urine from 35 up to 85%, which was accompanied by a decrease in plasma from 55 to 35%, suggests either tubular reabsorption or enterohepatic recirculation of lipophilic compounds. TLC-separation of the lipophilic fraction in urine revealed two previously unknown compounds of which the main congener was identified as 3-(3-oxo-7 alpha-methylsulfonyl-6 beta, 17 beta-dihydroxy-4-androsten-17 alpha-yl) propionic acid gamma-lactone, as well as canrenone and the metabolites which have already been described (Karim and Brown, 1972; Karim et al., 1975). This metabolite represents the main lipophilic degradation product in urine within the first hours, whereas the 6 beta-OH-7 alpha-methylsulfinyl-spirolactone leveled off and seemed to be and endexcretion product. For further characterisation, the polar fraction was subjected to acidic hydrolysis. The known metabolic pathways of spironolactone degradation are discussed.

Species differences in the metabolism and disposition of spironolactone.

The absorption, excretion and metabolism of [22-14C]spironolactone was compared in Charles River rats, beagle dogs and rhesus monkeys. The drug was administered at the fixed dose of 5 mg/kg po and iv. From the po/iv ratios of the areas under the plasma radioactivity-time curves, the gastrointestinal absorption of the drug was estimated to be 82% in the rat, 62% in the dog, and 103% in the monkey. The absolute bioavailability of a pharmacologically active metabolite, canrenone, was 57% in the dog and 48% in the monkey. Spironolactone was extensively metabolized in all three species and differences existed in the composition of the metabolites in their plasma, urine, and feces. The amount of radioactivity that was excreted in the urine and feces of all three species was similar after either po or iv administration of the drug. The cumulative average excretion of radioactivity in the urine as percentage of the po dose in 6 days was 4.69% in the rat (N = 5), 18.5% in the dog (N = 3), and 46.0% in the monkey (N = 3). In the feces, the corresponding excretion values were 74.2, 69.3 and 40.1%, respectively. Canrenone excretion in the urine constituted 0.65% of the po dose in the rat, 0.82% in the dog, and 5.86% in the monkey, whereas the excretion of total fluorogenic metabolites constituted 1.1, 1.9, and 12.1% respectively. Comparison of animal data with those published for humans indicated that the disposition and metabolism of spironolactone in the rhesus monkey, rather than those in the rat or the dog, was closest to that in man.