Enzymatic-spectrophotometric determination of paraquat in urine samples: A method based on its toxic mechanism

Paraquat is a broad-spectrum contact herbicide that has been encountered worldwide in several cases of accidental, homicidal, and suicidal poisonings. The pulmonary toxicity of this compound is related to the depletion of NADPH in the pneumocytes, which is continuously consumed by the reduction/oxidation of paraquat and reductase enzyme systems in the presence of O(2) (redox cycling). Based on this mechanism, an enzymatic-spectrophotometric method was developed for the determination of paraquat in urine samples. The velocity of NADPH consumption was monitored at 340 nm, every 10 s during 15 min. The velocity of NADPH oxidation correlated with the paraquat levels found in samples. The enzymatic-spectrophotometric method showed to be sensitive, making possible the detection of paraquat in urine samples at concentrations as low as 0.05 mg/L.

Ascorbic acid concentration is reduced in the secondary aqueous humour of glaucomatous patients

P>Background: We aimed to evaluate the ascorbic acid concentration in secondary aqueous humour (AH) from glaucomatous patients and to compare it with primary AH from primary open-angle glaucoma patients and non-glaucomatous patients. Methods: Primary AH samples were prospectively obtained from clinically uncontrolled primary open-angle glaucoma patients and senile cataract patients (controls) prior to trabeculectomy and cataract surgery. Secondary AH samples were obtained from eyes with previous intraocular surgery, prior to trabeculectomy or cataract surgery. AH (0.1 mL) was aspirated by inserting a 26-gauge needle into the anterior chamber just before surgery and then immediately stored at -80 degrees C. The ascorbic acid concentration was determined in a masked fashion by high-pressure liquid chromatography. Results: A total of 18 patients with senile cataract, 16 glaucomatous patients with primary AH (no previous intraocular surgery) and 11 glaucomatous patients with secondary AH (previous intraocular surgery) were included. There was no difference in mean age between groups (P = 0.15). The mean +/- standard deviation concentration of ascorbic acid in the secondary AH from glaucomatous patients (504 +/- 213 mu mol/L [95% confidence interval {CI}, 383-624]) was significantly lower than the concentration of ascorbic acid found in the primary aqueous of primary open-angle glaucoma (919 +/- 427 mu mol/L [95% CI, 709-1128]) and control patients (1049 +/- 433 mu mol/L [95% CI, 848-1249]; P < 0.01, Kruskal-Wallis test). Conclusions: The ascorbic acid concentration in secondary AH of glaucomatous patients was approximately twofold lower in comparison with primary AH of glaucomatous and cataract patients. The implications of a reduced concentration of ascorbic acid in the secondary AH deserve further investigation.

(1)H NMR determination of beta-N-methylamino-L-alanine (L-BMAA) in environmental and biological samples

A nuclear magnetic resonance ((1)H NMR) method for the determination of beta-N-methylamino-L-alanine (L-BMAA) in environmental aqueous samples was developed and validated. L-BMAA is a neurotoxic modified amino acid that can be produced by cyanobacteria in aqueous environments. This toxin was extracted from samples by means of solid-phase extraction (SPE) and identified and quantified by (1)H NMR without further derivatization steps. The lower limit of quantification (LLOQ) was 5 mu g/mL Good inter and intra-assay precision was also observed (relative standard deviation <8.5%) with the use of 4-nitro-DL-phenylalanine as an internal standard (IS). This method of 1H NMR analysis is not time consuming and can be readily utilized to monitor L-BMAA and confirm its presence in environmental and biological samples. (C) 2008 Elsevier Ltd. All rights reserved.

Development of a Stability-Indicating LC Assay Method for Determination of Chloroquine

A simple and rapid development of a stability-indicating LC method for determination of chloroquine diphosphate in the presence of its hydrolysis, oxidative and photolysis degradation products is described. Stress testing showed that chloroquine diphosphate was degraded under basic conditions and by photolytic treatment but was stable under the other stress conditions investigated. Separation of the drug from its degradation products was achieved with a Nova Pack C18 column, 0.01 M PIC B7 and acetonitrile (40:60 v/v) pH 3.6, as mobile phase. Response was linear over the range 0.08-5.70 mu g mL(-1) (r = 0.996), with limits of detection and quantification (LOD and LOQ) of 0.17 and 0.35 mu g mL(-1), respectively.

Bioequivalence assay between orally disintegrating and conventional tablet formulations in healthy volunteers

The purpose of this study was to evaluate bioequivalence of two commercial 8 mg tablet formulations of ondansetrona available ill the Brazilian market. In this study, a simple, rapid, sensitive and selective liquid chromarography-tandem mass spectrometry method is described for the determination of ondansetron in human plasma samples. The method was validated over a concentration range of 2.5-60 ng/ml and used in a bioequivalence trial between orally disintegrating and conventional tablet ondansetron formulations, to assess its usefulness in this kind of Study. Vonau flash (R) (Biolab Sanus Farmaceutica, Brazil, as test formulations) and Zofran (R) (GlaxoSmithKline, Brazil, as reference formulation) were evaluated following a single 8 mg close to 23 healthy volunteers of both genders. The dose was administered after an overnight fast according to a two-way crossover design. Bioequivalence between the products was determinated by Calculating 90% confidence interval (90% CI) for the ratio of C(max), AUC(0-t) and AUC(0-(sic)) values for the test and reference products, using logarithmically transformed data. The 90% confidence interval for the ratio of C(max) (87.5-103.8%), AUC(0-t) (89.3-107.2%) and AUC(0--(sic)) (89.7-106.0%) values for the test and reference products is Within the 80-125% interval, proposed by FDA, EMEA and ANVISA. It was concluded that two ondansetron formulations are bioequivalent ill their rate and extent of absorption. (C) 2008 Elsevier B.V. All rights reserved.

Micromethod for Quantification of Carbamazepine, Phenobartital and Phenytoin in Human Plasma by HPLC-UV Detection for Therapeutic Drug Monitoring Application

A simple, rapid, selective and sensitive analytical method by HPLC with UV detection was developed for the quantification of carbamazepine, phenobarbital and phenytoin in only 0.2 mL of plasma. A C18 column (150 x 3.9 mm, 4 micra) using a binary mobile phase consisting of water and acetonitrile (70:30, v/v) at a flow rate of 0.5 mL/min were proposed. Validation of the analytical method showed a good linearity (0.3 to 20.0 mg/L for CBZ, 0.9 to 60.0 mg/L for PB and 0.6 to 40.0 mg/L for PHT), high sensitivity (LOQ: 0.3, 0.9 and 0.6 mg/L respectively). The method was applied for drug monitoring of antiepileptic drugs (AED) in 27 patients with epilepsy under polytherapy.

Study of the interaction between hydroxymethylnitrofurazone and 2-hydroxypropyl-beta-cyclodextrin

Chagas disease is a serious health problem in Latin America. Hidroxymethylnitrofurazone (NFOH) is a nitrofurazone prodrug more active than nitrofurazone against Trypanosoma cruzi. However, NFOH presents low aqueous solubility, high photodecomposition and high toxicity. The present work is focused on the characterization of an inclusion complex of NFOH in 2-hydroxypropyl-beta-cyclodextrin (HP-beta-CD). The complexation with HP-beta-CD was investigated using reversed-phase liquid chromatography, solubility isotherms and nuclear magnetic resonance. The retention behavior was analyzed on a reversed-phase C-18 column, using acetonitrile-water (20/80, v/v) as the mobile phase, in which HP-beta-CD was incorporated as a mobile phase additive. The decrease in the retention times with increasing concentrations of HP-beta-CD enables the determination of the apparent stability constant of the complex (K = 6.2 +/- 0.3 M-1) by HPLC. The solubility isotherm was studied and the value for the apparent stability constant (K = 7.9 +/- 0.2 M-1) was calculated. The application of continuous variation method indicated the presence of a complex with 1:1 NFOH:HP-beta-CD stoichiometry. The photostability study showed that the formation of an inclusion complex had a destabilizing effect on the photodecomposition of NFOH when compared to that of the ""free"" molecule in solution. The mobility investigation (by NMR longitudinal relaxation time) gives information about the complexation of NFOH with HP-beta-CD. In preliminary toxicity studies, cell viability tests revealed that inclusion complexes were able to decrease the toxic effect (p < 0.01) caused by NFOH. (c) 2008 Elsevier B.V. All rights reserved.

Development and validation of a method for quantitative determination of econazole nitrate in cream formulation by capillary zone electrophoresis

A simple, fast, inexpensive and reliable capillary zone electrophoresis (CZE) method for the determination of econazole nitrate in cream formulations has been developed and validated. Optimum conditions comprised a pH 2.5 phosphate buffer at 20 mmol L(-1) concentration, +30 kV applied voltage in a 31.5 cm x 50 mu m I.D. capillary. Direct UV detection at 200 nm led to an adequate sensitivity without interference from sample excipients. A single extraction step of the cream sample in hydrochloric acid was performed prior to injection. Imidazole (100 mu g mL(-1)) was used as internal standard. Econazole nitrate migrates in approximately 1.2 min. The analytical curve presented a coefficient of correlation of 0.9995. Detection and quantitation limits were 1.85 and 5.62 mu g mL(-1), respectively. Excellent accuracy and precision were obtained. Recoveries varied from 98.1 to 102.5% and intra- and inter-day precisions, calculated as relative standard deviation (RSD), were better than 2.0%. The proposed CZE method presented advantageous performance characteristics and it can be considered suitable for the quality control of econazole nitrate cream formulations. (c) 2008 Elsevier B.V. All rights reserved.

Development and validation of sensitive methods for determination of sibutramine hydrochloride monohydrate and direct enantiomeric separation on a protein-based chiral stationary phase

Sibutramine hydrochloride monohydrate, chemically 1-(4-chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl) hydrochloride monohydrate (SB center dot HCl center dot H2O), was approved by the U.S. Food and Drug Administration for the treatment of obesity. The objective of this study was to develop, validate, and compare methods using UV-derivative spectrophotometry (UVDS) and reversed-phase high-performance liquid chromatography (HPLC) for the determination of SB center dot HCl center dot H2O in pharmaceutical drug products. The UVDS and HPLC methods were found to be rapid, precise, and accurate. Statistically, there was no significant difference between the proposed UVDS and HPLC methods. The enantiomeric separation of SB was obtained on an alpha-1 acid glycoprotein column. The R- and S-sibutramine were eluted in < 5 min with baseline separation of the chromatographic peaks (alpha = 1.9 and resolution = 1.9).

HPLC-UV determination of metformin in human plasma for application in pharmacokinetics and bioequivalence studies

In this study, a simple, rapid and sensitive HPLC method with UV detection is described for determination of metformin in plasma samples from bioequivalence assays. Sample preparation was accomplished through protein precipitation with acetonitrile and chromatographic separation was performed on a reversed-phase phenyl column at 40 degrees C. Mobile phase consisted of a mixture of phosphate buffer and acetonitrile at flow rate of 1.0 ml/min. Wavelength was set at 236 nm. The method was applied to a bioequivalence study of two drug products containing metformin, and allowed determination of metformin at low concentrations with a higher throughput than previously described methods. (c) 2007 Elsevier B.V. All rights reserved.

Validation of an HPLC analytical method for determination of pancuronium bromide in pharmaceutical injections

Pancuronium bromide is used with general anesthesia in surgery for muscle relaxation and as an aid to intubation. A high performance liquid chromatographic method was fully validated for the quantitative determination of pancuronium bromide in pharmaceutical injectable solutions. The analytical method was performed on an amino column (Luna 150mm4.6mm, 5m). The mobile phase was composed of acetonitrile:water containing 50mmol L-1 of 1-octane sulfonic acid sodium salt (20:80v/v) with a flow rate of 1.0mL min-1 and ultraviolet (UV) detection at 210nm. The proposed analytical method was compared with that described in the British Pharmacopoeia.

Determination of Vecuronium Bromide in Pharmaceuticals: Development, Validation and Comparative Study of HPLC and CZE Analytical Methods

Vecuronium bromide is a neuromuscular blocking agent used for anesthesia to induce skeletal muscle relaxation. HPLC and CZE analytical methods were developed and validated for the quantitative determination of vecuronium bromide. The HPLC method was achieved on an amino column (Luna 150 x 4.6 mm, 5 mu m) using UV detection at 205 nm. The mobile phase was composed of acetonitrile:water containing 25.0 mmol L(-1) of sodium phosphate monobasic (50:50 v/v), pH 4.6 and flow rate of 1.0 mL min(-1). The CZE method was achieved on an uncoated fused-silica capillary (40.0 cm total length, 31.5 cm effective length and 50 mu m i.d.) using indirect UV detection at 230 nm. The electrolyte comprised 1.0 mmol L(-1) of quinine sulfate dihydrate at pH 3.3 and 8.0% of acetonitrile. The results were used to compare both techniques. No significant differences were observed (p > 0.05).

The effects of enzymatic interesterification on the physical-chemical properties of blends of lard and soybean oil

The main goal of the present research effort was to evaluate the physical-chemical properties of blends of lard and soybean oil following enzymatic interesterification catalyzed by an immobilized lipase from Thermomyces lanuginosa (Lipozyme (TM) TL IM). Lipase-catalyzed interesterification produced new tri-acylglycerols that changed the physical-chemical properties of the fat blends under study. Solid fat content (31.3 vs 31.5 g/100 g), consistency (104.7 vs 167.6 kPa), crystallized area (0.6 vs 11.8) and softening point (31.8 vs 32.2 degrees C) of lard increased after interesterification, and this was mostly due to the increase of SSS (saturated) + SSU (disaturated-monounsaturated) triacylglycerols. These contents (SSU + SSS) increased in lard after interesterification from 42.9 to 46.7 g/100 g. The interesterified blends exhibited lower values for the physical properties when compared with their counterparts before enzymatic interesterification. The interesterification of blends of lard with soybean oil increased the amounts of UUU (triunsaturated) and SSS triacylglycerols and reduced the amounts of UUS (diunsaturated-monosaturated) triacylglycerols. The interesterified blends of lard and soybean oil demonstrated physical properties and chemical composition similar to human milk fat and they could be used for the production of a human milk fat substitute. (C) 2009 Elsevier Ltd. All rights reserved.

Microbiological Assay for Apramycin Soluble Powder

The aim of this study was to validate an agar diffusion method through the parameters linearity, precision and accuracy, to quantify apramycin in soluble powder. The calibration curve of apramycin was constructed by plotting log of concentrations (mu g ml(-1)) versus zone diameter (mm) and shows good linearity in the range of 1.0-4.0 mu g.ml(-1). The precision of the assay was determined by assaying samples at the same day (repeatability - R.S.D. = 2.00%) and on different days (intermediate precision - R.S.D. = 5.06%) and indicate good precision. The accuracy expresses the agreement between the accepted value and the value found. The mean recovery was found to be 100.49 % for apramycin soluble powder. The results indicated that the microbiological assay proposed in this work hold linearity, precision and accuracy being an acceptable alternative method for routine quality control of apramycin in the pharmaceutical dosage form studied.