A graphene tip coupled with liquid chromatography tandem mass spectrometry for the determination of four synthetic adulterants in slimming supplements.

Slimming supplements were popularly sold online driven by the increasement of obesity and the development of social networking platform. However, events of drug abuse in slimming supplements were also frequently reported. In this study, a graphene tip solid-phase extraction (Gtip SPE) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was established for determining fenfluramine, phenolphthalein, bumetanide, and sibutramine in slimming supplements. It was validated in terms of linearity (0.9985-0.9995), LOD (1.8ngmL-1), LOQ (5.6ngmL-1), intra-day precision (<5.1%), inter-day precision (<7.3%), and recovery (82.9-95.2%). Sibutramine is the most commonly used drug, which was detected in Bihais, Galong, and Aolist, with content 12.4, 3.6, 20.3mgg-1, respectively. Phenolphthalein was also found with content lower than 5.2mgg-1. The successful application of Gtip SPE and UPLC-MS/MS method indicated its advantage in analyzing low level of contaminates resulted from violation of regulation.

In vivo effects of bumetanide at brain concentrations incompatible with NKCC1 inhibition on newborn DGC structure and spontaneous EEG seizures following hypoxia-induced neonatal seizures.

Neonatal seizures caused by perinatal asphyxia and hypoxic-ischemic encephalopathy can be refractory to conventional anticonvulsants. This may be due to the depolarizing effects of gamma-aminobutyric acid (GABA) achieved by the activity of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). The aim of this study is to evaluate the long-term effects of bumetanide, a NKCC1 inhibitor, on hippocampal neurogenesis and seizure susceptibility in hypoxia-induced neonatal seizure model. Wistar rats were subjected to hypoxia-induced neonatal seizures at postnatal day 10 (P10). Following acute seizures, the rats were treated with intraperitoneal injection (i.p.) of bumetanide at a dose of 0.5mg/kg for 3 weeks. In later adulthood, hypoxia-induced seizures increased the number of newborn dentate gyrus cells (DGCs), promoted mossy fiber sprouting (MFS) and reduced the apical dendritic complexity of newborn DGCs 1 month after the insults. In addition, these seizures resulted in long-lasting consequences, such as spontaneous electroencephalography (EEG) seizures, though spatial learning impairments were not seen. Bumetanide treatments significantly enhanced cell proliferation and dendritic development of newborn DGCs after neonatal seizures, accompanied by the decreased seizure activity. However, systemic administration of bumetanide resulted in much lower brain concentrations, and was incompatible with NKCC1 inhibition in blood-brain barrier (BBB)-protected brain tissue. Our results suggested that bumetanide might have long-term effects in suppressing seizure activity, and altering the neurogenesis after neonatal seizures. These effects of bumetanide may be mediated by the targets outside the BBB-protected central nerve system (CNS) or CNS-located target(s) other than NKCC1.

Sensitive isotope dilution liquid chromatography/tandem mass spectrometry method for quantitative analysis of bumetanide in serum and brain tissue.

We have developed and validated a simple and sensitive stable isotope dilution liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for the quantification of bumetanide in human serum. Samples were prepared with a simple acetonitrile based protein precipitation. The supernatant was then analyzed directly using LC-MS/MS. Chromatographic separation was achieved on a C18 reversed phase column using a methanol and water gradient. The detection was performed in selected reaction monitoring (SRM) mode via a positive electrospray ionization (ESI) interface. The method had a lower limit of quantification (LLOQ) of 1 ng/mL, linearity up to 1250 ng/mL, intra- and inter-day precision less than 10%, and accuracy within ±10%. This method was also demonstrated to be suitable for the analysis of bumetanide in rat serum and brain tissue. Bumetanide concentrations in rat serum and brain were determined for samples collected at several intervals following intraperitoneal (i.p.) injection of bumetanide, and were used to calculate bumetanide permeability through the blood-brain barrier.

Disease-modifying effects of phenobarbital and the NKCC1 inhibitor bumetanide in the pilocarpine model of temporal lobe epilepsy.

Accumulating evidence suggests that changes in neuronal chloride homeostasis may be involved in the mechanisms by which brain insults induce the development of epilepsy. A variety of brain insults, including status epilepticus (SE), lead to changes in the expression of the cation-chloride cotransporters KCC2 and NKCC1, resulting in intracellular chloride accumulation and reappearance of immature, depolarizing synaptic responses to GABA(A) receptor activation, which may critically contribute to the neuronal hyperexcitability underlying epileptogenesis. In the present study, it was evaluated whether prolonged administration of the selective NKCC1 inhibitor, bumetanide, after a pilocarpine-induced SE modifies the development of epilepsy in adult female rats. The antiepileptic drug phenobarbital, either alone or in combination, was used for comparison. Based on pharmacokinetic studies with bumetanide, which showed extremely rapid elimination and low brain penetration of this drug in rats, bumetanide was administered systemically with different dosing protocols, including continuous intravenous infusion. As shown by immunohistochemistry, neuronal NKCC1 expression was markedly upregulated shortly after SE. Prophylactic treatment with phenobarbital after SE reduced the number of rats developing spontaneous seizures and decreased seizure frequency, indicating a disease-modifying effect. Bumetanide did not exert any significant effects on development of spontaneous seizures nor did it enhance the effects of phenobarbital. However, combined treatment with both drugs counteracted several of the behavioral consequences of SE, which was not observed with single drug treatment. These data do not indicate that bumetanide can prevent epilepsy after SE, but the disease-modifying effect of this drug warrants further studies with more lipophilic prodrugs of bumetanide.

[Simultaneous determination of five illegal drugs in weight control foods with solid phase extraction-high performance liquid chromatography].

OBJECTIVE: To develop a method for simultaneous determination of hydrochlorothiazide, furosemide, clopamide, bumetanide and sibutramine hydrochloride in weight control foods with solid phase extraction-high performance liquid chromatography. METHODS: The analytes in the samples were extracted with 2% phosphoric acid-methanol (1:1, V/V) solution ultrasonically and centrifuged. The extracts were clean-up with Osis MCX SPE columns, concentrated under weak N2 stream, and reconstituted with 2% phosphoric acid-methanol (1:1, V/V) solution, vortex mixing and centrifugation at 12,000 r/min. The high performance liquid chromatography was performed with Phenomenex C18 (250 x 4.60 mm, 5 microm) as separation column, 0.02 mol/L acetonitrile potassium dihydrogen phosphate buffer as mobile phase, gradient elution of 1.0 mL/min for the flow rate, and 40 degrees C for the column temperature. The standard curve method was used for the quantitative analysis. RESULTS: A good linear range appeared for the five analytes from 0.25 to 100 microg/mL (r > or = 0.999). The detection limits were 5.2-108 microg/kg. The average recoveries were 86.5%-113.1%, with the relative standard deviations of 1.6%-8.9%. CONCLUSION: The proposed method is a reliable method with high selectivity and high sensitivity for the detection of the five illegal chemicals in the weight control foods.

An environmentally friendly reflectometric method for bumetanide determination in pharmaceuticals.

This paper describes a green analytical procedure for the determination of bumetanide using diffuse reflectance spectroscopy. The proposed method is based on reflectance measurements of a violet compound produced from a spot test reaction between bumetanide and p-dimethylaminocinnamaldehyde (p-DAC) in an acid medium, using filter paper as a solid support. The best conditions for the reaction have been found by experimental design methodologies. All reflectance measurements were carried out at 525 nm, and the linear range was from 1.37 x 10(-4) to 1.37 x 10(-3) mol L(-1), with a correlation coefficient of 0.998. The detection limit was estimated to be 3.98 x 10(-5) mol L(-1). Five commercial medicines containing bumetanide were analyzed by the proposed method. No interferences were observed from the common excipients present in pharmaceutical formulations. The results were favorably compared with those obtained by the United States Pharmacopoeia procedure at 95% confidence level.

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.

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).

Pharmacokinetics and pharmacodynamics of bumetanide in neonates treated with extracorporeal membrane oxygenation.

Eleven term neonates treated with extracorporeal membrane oxygenation received bumetanide to treat volume overload. All patients had stable renal function, no history of prior diuretic therapy, and no overt evidence of hepatobiliary disease or hypoalbuminemia. Pretreatment creatinine clearance was 35.2 +/- 4.5 ml/min per 1.73 m2 (range, 20.3 to 57.5). Bumetanide, 0.095 +/- 0.003 mg/kg, was administered for 2 minutes into the postmembrane side of the extracorporeal membrane oxygenation circuit. Serial plasma and urine samples were collected for measurement of bumetanide and electrolyte concentrations. Total plasma and renal clearances for bumetanide were 0.63 +/- 0.11 and 0.16 +/- 0.04 ml/min per kilogram, respectively. The steady-state volume of distribution (0.44 +/- 0.03 L/kg) and the elimination half-life (13.2 +/- 3.8 hours) were greater than similar values reported in previous studies of bumetanide disposition in premature and term neonates who were not treated with extracorporeal membrane oxygenation. At observed rates of bumetanide excretion, the diuretic, natriuretic, and kaliuretic responses were linear. Significant diuresis, natriuresis, and kaliuresis were observed, although the duration of these effects was less than expected given the prolonged renal elimination of bumetanide. Nonrenal elimination of bumetanide was variable (47.2% to 96.9%) but higher than expected; this may explain the relatively brief diuretic and kaliuretic response.

Investigation of Fe(III) chloride as a colour reagent for the spectrophotometric determination of bumetanide in the pure form and in preparations.

The formation and stability of the complex between bumetanide (3-butylamino-4-phenoxy-5-sulphamoylbenzoic acid) and Fe(III) ion was studied. It has been found that bumetanide reacts with Fe(III) chloride in the presence of ammonium thiocyanate at the pH 1.83-1.92 to form a violet complex soluble in chlorophorm, with the maximum absorbance at 514.5 nm. At the optimum experimental conditions the conditional stability constant of the complex was found to be log K' = 7.34. The Beer's law was obeyed up to the 160 microg x cm(-3) and the relative standard deviation varied from 0.406% to 0.475% (n = 10). The proposed method was found to be suitable for the accurate and sensitive analysis of bumetanide as a pure compound and from tables and ampoules.

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

A new high-performance liquid chromatographic (HPLC) method was developed for the analysis of bumetanide in plasma and urine. A reversed-phase column was fitted to the instrument and fluorescent (excitation lambda = 338 nm, emission lambda = 433 nm) and UV (254 nm) detectors were utilized to monitor simultaneously bumetanide and the internal standard, acetophenone, respectively. The assay is rapid, sensitive, and specific. Plasma bumetanide concentrations can be detected as low as 5 ng/ml using a 0.20-ml sample. Time-consuming extraction and/or derivatization steps are not required. The only clean-up procedure involved is the precipitation of plasma proteins with acetonitrile.

Bumetanide: radioimmunoassay and pharmacokinetic profile in humans.

A simple, specific, and sensitive radioimmunoassay was developed for the determination of the diuretic bumetanide in plasma and urine. Antiserum to bumetanide was obtained from rabbits immunized with an immunogen prepared by covalently coupling the glycine conjugate of bumetanide to bovine serum albumin. Following extraction of the sample at pH 5.5 with ether, radioimmunoassay of the residue from the ether extract allows for the determination of bumetanide with a limit of sensitivity of about 1 ng/ml using 0.1 ml of plasma or urine. The specificity of the radioimmunoassay was established by comparison with specific radiometric and spectrofluorometric techniques. The pharmacokinetic profile of bumetanide in eight human subjects receiving single 2-mg oral doses of the drug was elucidated using the radioimmunoassay. The peak plasma levels ranged from 39 to 50 ng/ml at 1-4 hr after administration and declined with a mean apparent half-life of 1.17 hr. The mean plasma clearance rate was calculated to be 255 ml/min. During the first 24 hr, a mean of 43% of the bumetanide dose was excreted in the urine as intact drug.