Simple Rapid Validated RP-LC Method for the Estimation of Flurbiprofen in Rabbit Serum and Aqueous Humor

Abstract

New reversed-phase liquid chromatographic methods, with UV detection, were developed for the quantitative estimation of flurbiprofen in rabbit blood serum and aqueous humor. The mobile phase and other chromatographic conditions were optimized to minimize interference from biological matrix and at the same time provide sufficient sensitivity for the method to be adopted for in vivo studies of ophthalmic formulations of flurbiprofen. Acetonitrile was used to precipitate proteins from serum or aqueous humor during sample preparation. A mobile phase of methanol: acetonitrile: phosphate buffer pH 5.6 (40:20:40) was employed with UV detection at 248 nm for estimation of drug in both the biological matrix. The retention time and asymmetry factor for the proposed method of estimation in serum and aqueous humor was found to be 3.1312±0.0101 min and 1.1310±0.0091 respectively. The linear regression equations obtained by least square regression method, were Area (µV sec) = 52.27 × Conc. (in ng/ml)–1618.70 in serum and Area (µV sec) = 61.79 × Conc. (in ng/ml) ? 783.24 in aqueous humor. The results of analysis were treated statistically, as per ICH guidelines for validation of analytical procedures, USP-2003, and by recovery studies. The results were found to be accurate, reproducible and free from interference. The developed methods were further used for estimation of flurbiprofen in rabbit serum and aqueous humor following single topical administration of in-house aqueous drop and market formulation to rabbit eye.     

Ascorbic Acid Determination by Hydrophobic Liquid Chromatography of the Osazone Derivative. Application to the Analysis of Aqueous Humor

Abstract

The dinitrophenylhydrazine colorimetric method for the analysis of ascorbic acid has been converted to a high-performance liquid chromatography (HPLC) procedure. The bis-(dinitrophenyl) hydrazone resulting from the reaction of ascorbic acid and dinitrophenylhydrazine is separated on a reversed-phase system from other components in the reaction mixture and used for quantitation purposes. The specificity of the analysis is thus greatly improved and its sensitivity is brought down to the picomol level. The HPLC procedure described here is therefore specially suitable for the analysis of very small volume samples when high specificity and sensitivity are of prime importance. With this method, as in the original colorimetric procedure, it is also possible to specify the contribution of the reduced and oxidized forms of ascorbic acid to the total amount present in the sample. Basically, the same approach could be advantageously used to improve other colorimetric methods of analysis. Human aqueous humor samples taken from senile cataract patients at the time of surgery show variable but significant amounts of oxidized ascorbic acid. This oxidized traction, therefore, should not be neglected as is frequently done when the ascorbic acid system of the aqueous humor is studied.     

Hydride Generation Graphite Furnace Atomic Absorption Determination of Bismuth in Clinical Samples with in Situ Preconcentration

A sensitive method for the determination of bismuth in clinical samples using hydride generation/trapping and atomization in a graphite furnace is described. Addition of Pd-Ir to the furnace tube surface before hydride trapping leads to great improvement in the sensitivity of the method. Calibration is achieved with simple aqueous standards carried through this same procedure. An absolute detection limit (3σ) of 100 pg and a concentration detection limit of 20 ng/liter are obtained using 5-ml sample volumes. Corresponding precisions of 8-12% are typical for analyses of these samples. Microwave-assisted sample digestion procedures using HNO₃ and H₂O₂ were used to decompose clinical materials. The method was used to determine Bi in human blood, serum, urine, and tissue before and after intake of therapeutic doses of colloidal bismuth citrate.     

An HPLC Procedure for the Analysis of Furosemide in Pharmaceuticals-Analysis of Furosemide Tablets and Furosemide Injection

Abstract

A simple and specific procedure was developed for the analysis of furosemide from tablets and injections. The procedure consists of extracting furosemide into aqueous sodium hydroxide, addition of the internal standard, appropriate dilution and injection onto a u Bondapak C₁₈ reversed phase column. The mobile phase consisted of a solvent containing acetonitrile and aqueous sodium acetate and the eluate was monitored by either U.V. absorption or spectrofluorimetry. A standard linear calibration curve was obtained for direct standard solutions containing 75 ng to 500 ng on column. This procedure was successfully used to analyze furosemide tablets (individual assay) and injections.     

Development of an HPLC method for determining the alpha 2-adrenergic receptor agonist brimonidine in blood serum and aqueous humor of the eye

A procedure for determining brimonidine [5-bromo-6-(2-imidazolidinylideneamino) quinoxaline] in biological samples using a reversed-phase isocratic HPLC method is described. The application in blood serum and eye aqueous humor of patients treated with the Alphagan ophthalmic solution was carried out by enrichment of samples in brimonidine with solid-phase liquid extraction. Brimonidine reached maximum levels in aqueous humor and serum within 2-2.5 h, whereafter a declining pattern was obtained. An approximate 50% level of brimonidine was identified in serum at 12 h after ocular administration, whereas in aqueous humor this percentage was determined after a period of 4-5 h.     

Chromatography of Uranium on High-Performance Ion Exchangers

Abstract

The potential of high-performance liquid chromatography (HPLC) for the determination of U(VI) in ground waters and urine has been examined under a variety of HPLC experimental conditions. Conventional cross-linked and bonded-phase ion exchangers, both cation and anion, were studied with aqueous mobile phases containing tartrate, citrate, or α-hydroxyisobutyrate. The best chromatography was obtained on bonded-phase cation exchangers with an α-hydroxyisobutyrate eluent. The metal ions were detected either by visible spectrophotometry after a post-column reaction with a complexing reagent, or with a polarographic detector. Dectection after post-column reaction gave the best sensitivity; the detection limit (2 × baseline noise was 6 ng or 60 ng.ml^?1 for 100 μl samples. In-line trace enrichment was used to decrease detection limits and linear calibration curves were observed in the ranges studied; 0.5 to 50 ng.mL^?1 for ground waters and 25 to 400 ng.mL^?1 for artificial urine.     

Quantification of Levosulpiride in Human Plasma by High‐Performance Liquid Chromatography

Abstract

An analytical procedure was developed and validated for the quantification of levosulpiride in human plasma. After subjecting a plasma sample to a two‐step extraction procedure, an aliquot of the aqueous phase was injected onto an high‐performance liquid chromatography system equipped with a fluorescence detector. The detector response was linear for levosulpiride concentrations in the range of 2.5 to 500 ng/ml. The intra‐ and inter‐day precision was below 15.4 and 10.1%, and the accuracy was in a range from 89.7 to 109.4%. The method is applicable for use in the pharmacokinetic characterization of levosulpiride after a 75‐mg oral dose in humans.     

Simultaneous Determination of Chloroquine and Desethylchloroquine in Blood,Plasma and Urine by High-Performance Liquid Chromatography

Abstract

A high-performance liquid chromatographic method is described for the determination of chloroquine and its major metabolite desethylchloroquine in blood, plasma and urine. The procedure employs reversed-phase chromatography, with ultraviolet detection, and chlorpheniramine as an internal standard. One milliliter samples of biologic fluid are extracted in a single step with ether. The method has a sensitivity limit of 5 ng/ml for chloroquine and its metabolite. The applicability of the method is demonstrated by the analysis of blood and plasma samples obtained from rabbits following intravenous administration of chloroquine.     

The determination of chromium-50 in human blood and its utilization for blood volume measurements

Possible relationships between insufficient blood volume increases during pregnancy and infant mortality could be established with an adequate measurement procedure. An accurate and precise technique for blood volume measurements has been found in the isotope dilution technique using chromium-51 as a label for red blood cells. However, in a study involving pregnant women, only stable isotopes can be used for labeling. Stable chromium-50 can be determined in total blood samples before and after dilution experiments by neutron activation analysis (NAA) or mass spectrometry. However, both techniques may be affected by insufficient sensitivity and contamination problems at the inherently low natural chromium concentrations to be measured in the blood. NAA procedures involving irradiations with highly thermalized neutrons at a fluence rate of 2·10¹³ n·cm^–2·s^–1 and low background gamma spectrometry are applied to the analysis of total blood. Natural levels of chromium-50 in human and animal blood have been found to be <0.1 ng/ml; i.e., total chromium levels of <3 ng/ml. Based on the NAA procedure, a new approach to the blood volume measurement via chromium-50 isotope dilution has been developed which utilizes the ratio of the induced activities of chromium-51 to the iron-59 in three blood samples taken from each individual, namely blank, labeled and diluted labeled blood.     

Dexamethasone delivery to posterior segment of the eye

Due to anatomic barriers and lacrimal drainage it is difficult to obtain therapeutic drug concentrations in the posterior part of the eye after topical drug administrations. Lipophilic cyclodextrins, such as randomly methylated β-cyclodextrin (RMβCD), are known to act both as solubilizers of water-insoluble drugs in aqueous solutions and as penetration enhancers that reduce the barrier function of lipophilic membranes. The purpose of this study was to investigate the effects of RMβCD on dexamethasone delivery from aqueous eye drop solution into rabbit eyes. Dexamethasone (0.5 and 1.5% w/v) drops (50 μl) were administered to the left eye of rabbits (n = 6) and the drug levels measured in different eye tissues 2 h after administration. In aqueous humor dexamethasone levels were 1,190 ± 110 and 1,670 ± 630 ng/g (mean ± SD) after administration of the 0.5 and 1.5% dexamethasone eye drops, respectively. In the retina the levels were 33 ± 7 and 66 ± 49 ng/g, and in optic nerve 41 ± 12 and 130 ± 50 ng/g, respectively. In a previous study the dexamethasone concentration in aqueous humor after topical administration of 1.3% (w/v) dexamethasone eye drops in aqueous 2-hydroxypropyl-β-cyclodextrin (HPβCD) solution was determined to be 320 ± 230 ng/g and 66 ± 20 ng/g after administration of Maxidex^® eye drops. Both the hydrophilic HPβCD and the lipophilic RMβCD enhance topical dexamethasone delivery into the eye, but of the two, the lipophilic RMβCD results in higher dexamethasone concentrations.     

Hydrotrope induced synthesis of 1,8-dioxo-octahydroxanthenes in aqueous medium

Abstract

Hydrotrope induced synthesis of 1,8-dioxo-octahydroxanthenes from aromatic aldehydes and dimedone/1,3-cyclohexadione in aqueous medium is reported. The remarkable features of the new procedure are high conversions, shorter reaction times, cleaner reaction profiles, and simple experimental and work-up procedures.     

A Selective Pharmaceutical Analysis Technique with Sensitive Piezoelectric Crystal Quartz Sensors

Abstract

The frequency shift of a piezoelectric crystal sensor showed a linear dependence on the concentration for drugs which can be hydrolyzed in base aqueous solutions, at a given reacting time after the addition of the base, when the base concentration is basically constant during the entire measurement period. The sensitivity of the analysis method was improved, if crystal responses at different reacting times were recorded and utilized to estimate component concentration, when used for determining atropine sulfate in a pharmaceutical compound. The analytical results indicated that the selective and sensitive analytical procedure could be easily applied in true low concentration pharmaceutical analysis.     

Mahganese Determination In Blood Serum Using Electrothermal Atomic Absorption Spectrometry

Abstract

Twelve procedures of sample pretreatment for manganese determination in blood serum were tested in comparable instrumental conditions. the best results were obtained by dilution of serum with diluted nitric acid, which leads to formation of an internal matrix modifier composed of calcium and phosphoric acid. Other procedures may also give satisfactory results, but then as standards aqueous albumin solutions should be used.     

Resorcinol as Fluorimetric Reagent for the Determination of Nitrate

Abstract

Sensitive fluorometric procedure has been developed for the determination of nitrate with resorcinol. Reaction conditions, sensitivity of the method and the effect of nitrite are discussed. It is possible to determine 5 to 70 ng of nitrate-nitrogen by the recommended method.     

Spectrofluorimetric determination of europium and samarium with mixtures of 2-thenoyltrifluoroacetone and tri-n-octylphosphine oxide in aqueous solutions containing triton x-100

The europium or samarium complex with thenoyltrifluoroacetone and tri-n-octylphosphine oxide in aqueous 0.2% Triton X-100 solution at pH 3.6 is used to determine europium (1.5–760 ng ml^-1) or samarium (15–1500 ng ml^-1). The excitation wavelength is 352 or 372 nm; the emission wavelength is 613 or 561 nm. The procedures give better sensitivity than the corresponding extraction methods. A standard addition method is used to overcome interferences.     

Liquid Chromatographic Analysis of Ciprofloxacin and Ciprofloxacin Metabolites in Body Fluids

Abstract

An isocratic HPLC assay procedure for analysis of ciprofloxacin and three metabolites was developed. The procedure requires only dilution of bile, saliva, and urine samples prior to reverse-phase chromatography on a polystyrene-divinylbenzene (PSDVB) column; analysis of serum samples requires a cleanup step on a PSDVB cartridge prior to chromatography. The dependence of chromatographic efficiency on flow rate and temperature was investigated and the accuracy, precision, selectivity, and sensitivity of the procedure were evaluated. The developed procedure was also compared to a modified version of a published ciprofloxacin procedure that requires an octadecyl-silane (ODS) column for chromatographic separation. Similar efficiency, precision, and accuracy were observed with both procedures and both were used for analysis of clinical samples. However, the procedures were used for different purposes. The PSDVB procedure, because of more favorable column selectivity, was used to assay ciprofloxacin and its metabolites in bile, urine and saliva samples. The ODS procedure, because of a simpler serum preparation step, was used t o assay ciprofloxacin in serum samples.     

HPLC Determination of Methylphenidate in Human Plasma

Abstract

A simple, rapid and sensitive method for measuring methylphenidate in human plasma by HPLC has been developed. After the addition of the internal standard, ethylphenidate, the two compounds are extracted under basic conditions. The residue obtained is resuspended in acetonitrile and analysed on an ODS reversed phase column with detection by UV absorbance at 192 nm. The limit of sensitivity is 5 ng/ml and the procedure is linear over the 5–50 ng/ml concentration range.     

Detection of Cocaine Using the Flow Immunosensor

Abstract

A continuous flow immunosensor has been designed for the detection of cocaine in aqueous samples. The continuous flow immunosensor relies on the displacement of fluorophore-labeled antigen from immobilized monoclonal antibody. The sensitivity and accuracy of the flow immunosensor were investigated while varying the parameters of immobilized antibody density, flow rate, amount of antibody-coated Sepharose used in each column, and the saturation of antibody binding sites with fluorophore-labeled antigen. Using a low density of immobilized anti-benzoylecgonine antibody, as little as 5 ng/ml cocaine could be detected. Small amounts of antibody-coated Sepharose could be used repeatedly and the lifetime of the column was proportional to the amount of Sepharose used. Results were obtained in less than a minute and cross-reactivity against various other drugs was negligible.     

Determination of bisphenol A in foods as 2,2-bis-(4-(isopropoxycarbonyloxy)phenyl)propane by gas chromatography/mass spectrometry

A procedure has been developed for the determination of bisphenol A (BPA) in foods by gas chromatography/mass spectrometry as 2,2-bis-(4-(isopropoxycarbonyloxy)phenyl)propane formed in the reaction with isopropyl chloroformate. Optimal conditions have been found for BPA derivatization, providing its quantitative conversion into diether derivative in aqueous media. The concentration of BPA has been determined in some samples of canned foods and beverages (from 2.15 to 42.91 ng/g). The detection limit is 0.05–0.1 ng/g.     

Magnetic solid‐phase extraction for determination of sulpiride in human urine and blood using high‐performance liquid chromatography

A novel and efficient sample preconcentration technique based on the Fe₃O₄ magnetic nanoparticles (Fe₃O₄ MNPs) coated with silica (SiO₂) has been developed for extraction and determination of sulpiride. The functionalized MNPs showed excellent dispersibility in aqueous solution and were applied to magnetic solid‐phase extraction of sulpiride from human urine and blood prior to high‐performance liquid chromatography analysis. The separation, preconcentration and desorption procedure was completed in 10 min. Optimal experimental conditions, including sample pH, the amount of the MNPs, eluent type and volume, and the ultrasonication time were studied and established. The method showed good linearity for the determination of sulpiride in the concentration range of 10–1000 ng/mL in urine and blood. The recovery of the method was in the range between 91.2 and 97.5%, and the limit of detection was 2 ng/mL for sulpiride in human blood and urine. The results indicated that the present procedure is a suitable pretreatment method for biological samples. Copyright © 2015 John Wiley & Sons, Ltd.