Sensitive phosphorimetric determination of bumetanide in human urine with its inhibition effect on the formation of [Fe-morin]3+ complex.

A novel solid substrate-room temperature phosphorimetry (SS-RTP) was developed for determination of bumetanide (BMTN). It was validated by determining selectivity, linearity, accuracy, precision, and signal to noise ratio (S/N) for analysis. And all the experiments presented in this work were based on that BMTN inhibited the formation of [Fe-morin](3+) ([FeR](3+)) complex by the reaction between Fe(3+) and R, which led to severe quenching of room temperature phosphorescence (RTP) signal. The rate constant of the reaction (k) was 2.44 x 10(-4) s(-1), the activation energy (E) was 21.39 kJ mol(-1). Detection limit of this method (LD, 5.0 ag spot(-1), corresponding concentration was 1.2 x 10(-14) g mL(-1)) was evaluated and compared with other methods, indicating better sensitivity for BMTN determination using this technique. And due to the high sensitivity of the method, it has been successfully applied to determine BMTN in human urine samples. The linear range was from 0.040 pg mL(-1) to 4.0 pg mL(-1), allowing wide determined range of BMTN. Meanwhile, the mechanism of this method was also discussed.

Determination of diuretics in human urine by hollow fiber-based liquid-liquid-liquid microextraction coupled to high performance liquid chromatography.

In competition sports, a diuretic is a substance widely prohibited by the World Anti-Doping Agency (WADA). In this paper, a sensitive, rapid and convenient analytical method for the determination of acidic [furosemide (FUROS) and bumetanide (BUMET)] and basic [triamterene (TRIAM)] diuretics in human urine was developed by hollow fiber-based liquid-liquid-liquid microextraction (LLLME) coupled with HPLC-UV. The LLLME conditions, such as the organic extraction solvent, the acidity and basicity of the donor- and acceptor-phases, stirring speed, extraction time and ionic strength, were studied in detail. Under the optimum conditions, the linear ranges of furosemide, bumetanide and triamterene were 1.2-250, 5.0-250 and 5.0-500 ng mL(-1), respectively. The detection limits were 0.5 ng mL(-1) for furosemide, 1.2 ng mL(-1) for bumetanide and 2.0 ng mL(-1) for triamterene. The LLLME obtained a great improvement of the detection limits for all the analytes considered here, to the ng mL(-1) level, which almost reaches the level of the LC-MS method. This new LLLME method provided very high enrichments: 117-fold for furosemide, 175-fold for bumetanide and 68-fold for triamterene. Since the hollow fiber membrane was sealed, it could be used for extracting the diuretics directly from 'dirty' human urine samples without any clean-up procedures. With LLLME-HPLC, the corresponding recoveries ranged from 79.2 to 109% with the RSDs not exceeding 5.5% for the three diuretics in the spiked urine samples. The method was successfully applied to analyse the amounts of the three diuretics in real urine samples of volunteers after oral drug-taking. This new method proves to be sensitive and reliable and thus renders a very suitable means for the determination of trace diuretics in human urine based on the common HPLC instrument.

Detection of bumetanide in an over-the-counter dietary supplement.

Bumetanide is a loop diuretic used clinically to treat heart failure, acute renal failure, high blood pressure, and edema. However, diuretics may also be used by athletes as masking agents and to decrease weight. Taken as masking agents, diuretics increase urine production and decrease urinary concentrations of banned performance-enhancing agents, such as anabolic steroids. StarCaps is an over-the-counter dietary supplement marketed as a diet aid. The manufacturer claims that the product contains only natural cleansing agents and emphasizes that it is free from traditional appetite suppressants such as sympathomimetic amines. However, no such disclaimer is made concerning diuretic agents. A single StarCaps capsule was administered to two male and two female volunteers, and their urine specimens were collected at discrete intervals (2, 4, 8, and 12 h) post administration. The specimens were analyzed by a high-performance liquid chromatography-mass spectrometry quadrupole (HPLC-MS) method, and bumetanide was detected in all specimens (4.6 to 351.3 ng/mL). Adjusting the bumetanide concentrations for creatinine content did little to normalize the excretion profiles. Bumetanide was also detected in the StarCaps capsules at concentrations approaching therapeutic doses. HPLC-quadrupole-time-of-flight mass spectrometry was used to confirm the presence of bumetanide in the urine samples and StarCaps capsules. The results showed that unregulated dietary supplements may put consumers at risk for unwitting consumption of prescription medications, and that it is possible for athletes to inadvertently test positive for bumetanide and face disciplinary actions.

Fast quantitative determination of diuretic drugs in tablets and human urine by microchip electrophoresis with native fluorescence detection.

Microchip electrophoresis (MCE) with native fluorescence detection has been applied for the fast quantitative analysis of pharmaceutical formulations. For this purpose, methods for fast separation and sensitive detection of the unlabeled diuretic drugs, amiloride, triamterene, bendroflumethiazide (BFMTZ), and bumetanide were developed. An epifluorescence setup was used enabling the coupling of different lasers into a commercial fluorescence microscope. The detection sensitivity of different excitation light sources was compared utilizing either a HeCd laser (lambda(exc) = 325 nm), a frequency quadrupled Nd:YAG laser (lambda(exc) = 266 nm), or a mercury lamp (lambda(exc) = 330-380 nm). At optimal conditions using the HeCd laser, the drugs were separated within 15 s with LODs less than 1 mug/mL for the four compounds. A linear relationship between concentration and peak area was obtained in the concentration range of 0.05-20 microg/mL with a mean correlation coefficient of around 0.996 for all analytes. The method was successfully applied to the analysis of the respective drugs in commercial formulations and in human urine without interference from other constituents. These data show that MCE has a great potential for reliable drug analysis.

A study of the pharmacodynamic differences between immediate and extended release bumetanide formulations.

Optimized bumetanide extended (ER) and immediate release (IR) formulations were developed using fluid bed layering and coating techniques. We postulated that the ER bumetanide formulation would have more effective and sustained diuretic and saluretic effects than IR. The diuretic/saluretic effects of both formulations were measured in rabbits (n=8) for two days after dosing with 1mg/kg bumetanide. During the first day, both formulations produced 2-3 times more urine volume and sodium excretion than baseline. In the first 24h, despite less bumetanide excretion from the ER formulation (101+/-13.9microg/kg compared to 146+/-14.6microg/kg for the IR formulation; P<0.04); the ER formulation produced diuresis and natriuresis that was equivalent to that of the IR formulation. In contrast, urine production in the IR formulation group fell below that of placebo controls on day 2. During the second day, the ER formulation was noted to produce persistent bumetanide excretion; the diuretic and natriuretic effects were not statistically significant from baseline control. We speculate that the decrease in response to bumetanide observed especially for the IR formulation during the second day may be due to the activation of compensatory counter-regulatory homeostatic mechanism(s). We conclude that the ER formulation had similar diuretic/saluretic effects but better drug excretion to urine production efficiencies than the IR formulation in the healthy rabbit model. The ER formulation, while providing comparable diuretic/saluretic effect to the IR formulation, offers the advantage of avoiding the initial, rapid and robust diuretic effect experienced with the IR formulations. Taken together, the data provide sufficient basis to warrant further investigation and refinement of our ER bumetanide formulation in humans.

Quantitative determination of the loop diuretic bumetanide in urine and pharmaceuticals by high-performance liquid chromatography with amperometric detection.

A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of the diuretic bumetanide using a microBondapak C18 column. The mobile phase consists of a 50:50 acetonitrile-water mixture containing 5mM KH2PO4-K2HPO4 (pH 4.0). The compound is monitored at +1350 mV with an amperometric detector equipped with a glassy carbon working electrode. A liquid-liquid or solid-liquid extraction is done prior to chromatographic analysis in order to avoid the interferences found in the urine matrix. The percentages of recovery obtained are 71%+/-1% for liquid-liquid extraction and 84.2%+/-0.7% for solid-liquid extraction. The method developed has a linear concentration range from 50 to 499 ng/mL with a reproducibility in terms of relative standard deviation of 1.73% and 3.85% for a concentration level of 70 ng/mL and 237 ng/mL, respectively, and a detection limit of 0.25 ng/mL (3:1 signal-to-noise ratio). The method is applied to the determination of bumetanide in pharmaceutical formulations and urine obtained from hypertensive patients and healthy volunteers after the ingestion of a therapeutic dose of Fordiuran (1 mg bumetanide).

Mild metabolic alkalosis impairs the natriuretic response to bumetanide in normal human subjects.

1. This study was designed to test the hypothesis that acid-base status affects the response to a loop diuretic in human subjects. The renal responses to bumetanide (1 mg intravenously) were studied in eight normal subjects consuming a constant diet supplemented for 3 days on three separate occasions with equivalent quantities of NaCl, NaHCO3 (metabolic alkalosis) or NH4Cl (metabolic acidosis). 2. A significant (P < 0.025) reduction in bumetanide-induced diuresis (-40%), natriuresis (-21%), and chloruresis (-25%) was observed during NaHCO3 compared with NaCl. The renal response was unaltered during NH4Cl. 3. The creatinine and para-aminohippurate clearances were unchanged during NaHCO3, as were the blood pressure and plasma levels of renin activity, aldosterone and noradrenaline, and the plasma volume. 4. Bumetanide excretion was increased during NaHCO3 compared with NaCl (2.13 +/- 0.18 versus 1.76 +/- 0.17 micrograms/min, P < 0.025) but was not changed during NH4Cl (1.68 +/- 0.26 micrograms/min; not significant). 5. Plasma aldosterone concentration was increased 3-fold during acidosis and the kaliuretic response to bumetanide was enhanced significantly. 6. In conclusion, compared with NaCl, NaHCO3 reduces the diuretic, natriuretic and chloruretic response to bumetanide without significant changes in renal haemodynamics, plasma volume, the renin-angiotensin-aldosterone axis or the sympathetic nervous system, and despite increasing renal bumetanide excretion. NH4Cl enhances aldosterone secretion and diuretic-induced kaliuresis.

The pharmacokinetics of bumetanide in the newborn infant.

This study characterizes the pharmacokinetics of bumetanide after an intravenous dose of 0.05 or 0.10 mg/kg to 14 neonates (weight range 820-4,000 g; gestational age 26-40 weeks) during the first week of life. Blood samples and urine were collected for up to 12 h after dosing. Estimated serum clearance was 0.2-1.0 ml/min.kg (range), volume of distribution was 0.22 l/kg (range 0.11-0.32 l/kg), and the harmonic mean half-life was 6-7 h (range of 4-19 h). Nonrenal clearance accounted for 58-97% of the serum clearance with the presence of certain oxidative metabolites of bumetanide in the urine. These findings suggest higher dosing requirements and prolonged intervals as compared to adults. Utilizing these pharmacokinetic data, pharmacodynamic and ototoxicity studies should be conducted to establish a safe and effective neonatal dose.

Arterial and venous blood sampling in pharmacokinetic studies: bumetanide in rabbits.

The pharmacokinetics of bumetanide were evaluated simultaneously using both arterial and venous plasma data in 4 rabbits after a rapid 5 sec intravenous (iv) bolus dosing. Initial arterial to venous concentration ratios at 5 sec after injection were the highest with the values of 1410, 246, 4.25, and 351 for rabbits 1-4, respectively. Both curves decayed paralleling each other at the terminal phase with the higher venous levels than the arterial levels by 21.6, 48.2, 17.0, and 47.9% for rabbits 1-4, respectively. An exponential term with a negative coefficient was used to account for the short and steep rising phase of venous plasma levels after injection. Detailed analysis showed apparent volume of distribution at steady state (VSS) and mean residence time (MRT) values calculated from venous plasma data were higher than those form arterial plasma data. A plot of 1/Q (urine flow rate) versus 1/CLR (renal clearance) of bumetanide yielded a straight line in 4 rabbits, indicating that the CLR of bumetanide is urine flow dependent in rabbits.

Direct determination of diuretic drugs in urine by capillary zone electrophoresis using fluorescence detection.

Four diuretic drugs banned in sport (amiloride, triamterene, bendroflumethiazide and bumetanide) have been separated by capillary zone electrophoresis (CZE) and detected using conventional fluorescence spectrometry. The effect of pH on electrophoretic parameters such as migration time, peak efficiency and peak height is discussed. Complete separation of the four drugs is achieved in less than 8 min at pH 8. No interference due to endogenous urine components is observed and thus direct urine analysis is feasible. Analytical figures of merit including precision and limits of detection are presented. Limits of detection range between 0.5 fmol for triamterene and 21.6 fmol for bumetanide.

Pharmacokinetics of bumetanide in critically ill infants.

OBJECTIVE: Define the pharmacokinetics of bumetanide after single intravenous doses in volume-overloaded critically ill infants. METHODS: A prospective, open-label study was carried out in a group of 58 infants aged 0 to 6 months who required diuretic therapy. Each patient received a single dose of intravenous bumetanide. Doses selected in sequential order ranged from 0.005 to 0.10 mg/kg. Hematologic and serum chemistry studies were performed before and at 6 and 24 hours after bumetanide administration. Determinations of urine volume and chemistries were performed before (collected from -2 to -4 hours to time 0) and at 1, 2, 3, 4, 6, and 12 hours after bumetanide dosing. Serum samples collected at time 0 and at 5, 15, 30, 60, 120, 180, 240, 360, and 480 minutes and urine collected at time 0 and at 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 6, and 6 to 12 hours were analyzed for bumetanide concentration. Data were evaluated by standard noncompartmental pharmacokinetic techniques. RESULTS: Peak serum bumetanide concentrations occurred at 5 minutes after bumetanide administration. Area under the curve and peak serum bumetanide concentrations showed linear increases over the twentyfold dose range; whereas beta volume of distribution, volume of distribution at steady state, clearance, renal clearance, half-life, and mean residence time values were independent of dose. Peak urinary excretion rates of bumetanide increased linearly with increasing doses. The mean percent of bumetanide recovered in the urine from 0 to 12 hours was 40% +/- 15% of the administered dose. CONCLUSIONS: Distribution and elimination kinetics of bumetanide were similar in all patients. Elimination kinetics were first order over the dose range of 0.005 to 0.10 mg/kg. Pharmacokinetic parameter estimates (beta volume of distribution, volume of distribution at steady state, clearance, renal clearance, half-life, and mean residence time) were independent of the dose of bumetanide administered. Single doses of bumetanide up to 0.10 mg/kg appear to be well tolerated in acutely ill volume-overloaded infants aged 0 to 6 months.

Dose-ranging evaluation of bumetanide pharmacodynamics in critically ill infants.

OBJECTIVES: Determine the diuretic effects of single intravenous doses of bumetanide in volume-overloaded critically ill infants. METHODS: A prospective, open-label study was carried out in 56 infants aged 0 to 6 months who required diuretic therapy. Each patient received a single intravenous dose of bumetanide. Doses selected in sequential order ranged from 0.005 to 0.10 mg/kg. Determinations of urine volume, electrolytes, creatinine levels, and osmolality were performed before (collected from -2 to -4 hours to time 0) and at 1, 2, 3, 4, 6, and 12 hours after bumetanide dosing. Serum samples collected at time 0 and at 5, 15, 30, 60, 120, 180, 240, 360, and 480 minutes and urine aliquots collected at time 0, 0 to 1, 1 to 2, 2 to 3, 3 to 4, 4 to 6, and 6 to 12 hours were analyzed for bumetanide concentration. Individual changes in urine flow rate and electrolyte excretion were plotted against corresponding bumetanide excretion rates, taken as the effective dose of the drug. RESULTS: Peak bumetanide excretion rates increased linearly with increasing doses of drug. Time course patterns for urine flow rate and electrolyte excretion were similar for all dosage groups. Urine flow rate and electrolyte excretion increased linearly up to a bumetanide excretion rate of approximately 7 micrograms/kg/hr and either plateaued (urine flow rate) or declined at a bumetanide excretion rate of > 10 micrograms/kg/hr. Diuretic efficiency of bumetanide was maximal at doses of 0.005 to 0.010 mg/kg but decreased at higher doses. CONCLUSIONS: Maximal diuretic responses occurred at a bumetanide excretion rate of about 7 micrograms/kg/hr, corresponding to doses of 0.035 to 0.040 mg/kg. Higher doses produced a proportionately higher bumetanide excretion rate but no increased diuretic effect. Lower doses of bumetanide had the greatest diuretic efficiency, suggesting that continuous infusion of low doses of bumetanide or intermittent low-dose boluses may produce optimal diuretic responses in critically ill infants.

Immunoassay detection of drugs in racing horses: detection of ethacrynic acid and bumetanide in equine urine by ELISA.

We have raised antibodies and developed one-step enzyme-linked immunosorbent assays (ELISA) for the diuretics ethacrynic acid and bumetanide as part of a panel of pre- and post-race tests for high potency drugs in racing horses. These ELISA tests are rapid (completed within one hour), sensitive, and can be read by eye. The ELISA detects ethacrynic acid at a drug concentration for half-maximal inhibition (I-50) of about 2.5 ng/mL for the parent drug. After dosing horses intravenously with 5 mg ethacrynic acid per horse, the parent drug or its metabolites are detectable in urine for at least 8 hours. The bumetanide ELISA has an I-50 for the parent drug of about 2.0 ng/mL and will detect bumetanide or its metabolites for about 8 hours in urine after intravenous administration of a 1.7-mg dose per horse. Both antibodies are relatively specific for each drug and do not cross-react with other commonly used diuretics or other acidic compounds often found in post-race equine urine samples. Ethacrynic acid and bumetanide are potent diuretics suspected of being illegally substituted for furosemide in certain racing jurisdictions. Development of these rapid, sensitive, and simple tests for these agents will allow more effective pre- and post-race control of the use of these agents in racing horses. Both tests have recently uncovered several "positives" for these medications in a midwestern racing jurisdiction.

Measurement of bumetanide in plasma and urine by high-performance liquid chromatography and application to bumetanide disposition.

A high-performance liquid chromatographic method for the measurement of bumetanide in plasma and urine is described. Following precipitation of proteins with acetonitrile, bumetanide was extracted from plasma or urine on a 1-ml bonded-phase C18 column and eluted with acetonitrile. Piretanide dissolved in methanol was used as the internal standard. A C18 Radial Pak column and fluorescence detection (excitation wavelength 228 nm; emission wavelength 418 nm) were used. The mobile phase consisted of methanol-water-glacial acetic acid (66:34:1, v/v) delivered isocratically at a flow-rate of 1.2 ml/min. The lower limit of detection for this method was 5 ng/ml using 0.2 ml of plasma or urine. Nafcillin, but not other semi-synthetic penicillins, was the only commonly used drug that interfered with this assay. No interference from endogenous compounds was detected. For plasma, the inter-assay coefficients of variation of the method were 7.6 and 4.4% for samples containing 10 and 250 ng/ml bumetanide, respectively. The inter-assay coefficients of variation for urine samples containing 10 and 2000 ng/ml were 8.1 and 5.7%, respectively. The calibration curve was linear over the range 5-2000 ng/ml.

Screening for diuretics in human urine by gas chromatography-mass spectrometry with derivatisation by direct extractive alkylation.

A rapid, sensitive and reliable gas chromatographic-mass spectrometric (GC-MS) screening procedure for diuretics in human urine has been developed. The procedure uses derivatisation by extractive methylation directly from the urine. The suitability of a number of phase transfer reagents and solvents were studied for the detection of sixteen diuretics. The results obtained indicate that the screening procedure employing tetrahexylammonium hydrogensulphate at pH 12 with methyl iodide in toluene at room temperature was the most effective. This method gives selectivity and sensitivities down to 0.03-0.1 microgram/ml and provides a substrate suitable for GC-MS confirmation without further manipulation. The application of the method is demonstrated by the screening of urine for bumetanide, ethacrynic acid, acetazolamide, chlorothiazide and hydrochlorothiazide.

Analysis of bumetanide in human urine by high-performance liquid chromatography with fluorescence detection and gas chromatography/mass spectrometry.

Bumetanide is a potent diuretic. Its concentration in urine after therapeutic doses is in the low ng/mL range. A procedure using reversed-phase HPLC with fluorescence detection is described. Using this procedure, the authors have been able to measure bumetanide in a urine sample at 10 ng/mL with a signal-to-noise ratio of 7. Therefore, the detection limit is lower than this concentration. Confirmation is by a GC/MS procedure after methylating the diuretic. Derivatization is by extractive alkylation with tetrabutylammonium hydrogen sulfate as the phase transfer reagent and iodomethane as the methylating agent.

Metabolism of the diuretic bumetanide in healthy subjects and patients with renal impairment.

The pharmacokinetics and metabolism of orally administered bumetanide were studied in five healthy subjects and in five patients with renal insufficiency. Healthy subjects excreted 51% of the dose as unchanged drug in the urine, whilst the patients with renal insufficiency excreted only 11% of the dose as bumetanide. Similarly the urinary excretion of the gamma-hydroxybutyl metabolite was reduced from 6% in healthy subjects to 2.3% in patients with renal impairment. In both groups of subjects the mean elimination half-life of the metabolite was greater than for bumetanide. The results indicate a possible accumulation of bumetanide and metabolite in patient with renal failure.

Kinetics, dynamics, and bioavailability of bumetanide in healthy subjects and patients with chronic renal failure.

Six patients with chronic renal failure (CRF group) and four healthy subjects (HS group) were given 5 mg oral and intravenous doses of bumetanide in a random, crossover design. The CRF group had significantly lower plasma and renal clearances, resulting in a five-to sixfold reduction in the fractional urinary excretion of the drug. The percent free drug in plasma for the CRF group was more than double that for the HS group, and significant correlations were observed for volume of distribution at steady state vs. percent free (r = 0.661; P less than 0.05), nonrenal clearance vs. percent free (r = 0.796; P less than 0.01), and renal clearance vs. creatinine clearance (r = 0.995; P less than 0.001). Although bioavailability was relatively consistent among the HS (0.664 +/- 0.112) and CRF (0.689 +/- 0.149) groups, the absorption-time profiles were more irregular for both groups. Cumulative sodium excretion and overall efficiency of response to bumetanide did not differ significantly between the two routes of administration in either group.

Pharmacokinetics of bumetanide following intravenous, intramuscular, and oral administrations to normal subjects.

The pharmacokinetics of bumetanide was studied in 12 normal subjects after 1-mg intravenous, intramuscular, oral solution, and tablet administrations in a random four-treatment crossover design. Plasma and urine concentrations of intact bumetanide were analyzed by a sensitive and specific RIA. The pharmacokinetics of bumetanide after intravenous administration was characterized by a biexponential equation, including an initial disposition phase (t 1/2, alpha = 5.1 min), followed by a slower elimination phase (t 1/2, beta = 44 min). Bumetanide pharmacokinetics after intramuscular and oral administration could be described by a biexponential equation with first-order absorption and elimination. Bumetanide is rapidly absorbed via the intramuscular and oral routes, with mean +/- SD maximum plasma concentrations of 38.2 +/- 9.8 (intramuscular), 34.0 +/- 10.6 (oral solution), and 30.9 +/- 14.6 ng/mL (tablet) achieved within 0.34 +/- 0.23, 0.76 +/- 0.27, and 1.8 +/- 1.2 h after dosing, respectively. The drug is rapidly eliminated from the body after intravenous, intramuscular, oral solution, and oral tablet administrations, with half-lives ranging from 24-86, 47-139, 27-71, and 26-99 min, respectively. Approximately 70% of a parenteral dose and 60% of an oral dose are excreted as intact drug in urine taken 0-24 h after administration. The extent of bioavailability of bumetanide from the tablet and oral solution dosage forms are equivalent, and the absolute bioavailability of the intramuscular and oral preparations are approximately 100 and 80%, respectively. This is consistent with the predicted limited extent of first-pass metabolism after complete absorption of an oral dose.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of effect-compartment model to bumetanide-indomethacin interaction in dogs.

Analyses of bumetanide's dose-response relationship have been complicated by the hysteresis observed between the drug's urinary excretion rate and its sodium excretion. This apparent time lag reflects the disequilibrium between the urine concentration and effect compartment (biophase) which occurs during the early distribution phase. In the present article, an expanded pharmacodynamic model has been introduced in which the hypothetical effect compartment is linked, by a first-order process (Kue), to the urine compartment. Drug dissipation from the effect compartment occurs by means of the first-order rate constant, Keo. This representation accommodates bumetanide's luminal site of action in the kidney tubule as well as the drug's temporal component. Application of this model to the bumetanide-indomethacin interaction in dogs is examined.