Microcystins in South American aquatic ecosystems: Occurrence, toxicity and toxicological assays

The acute poisoning of chronic renal patients during hemodialysis sessions in 1996 in Caruaru City (Pernambuco State, Brazil) stimulated an intensive search for the cause of this severe complication. This search culminated in the identification of microcystins (MC), hepatotoxic cyclic heptapeptides produced by cyanobacteria, as the causative agents. More than ten years later, additional research data provides us with a better understanding of the factors related to cyanobacterial bloom occurrence and production of MC in Brazil and other South American countries. The contamination of water bodies and formation of toxic blooms remains a very serious concern, especially in countries in which surface water is used as the main source for human consumption. The purpose of this review is to highlight the discoveries of the past 15 years that have brought South American researchers to their current level of understanding of toxic cyanobacteria species and that have contributed to their knowledge of factors related to MC production, mechanisms of action and consequences for human health and the environment. (C) 2010 Elsevier Ltd. All rights reserved.

Separation of Steroids and the Determination of Estradiol Content in Transdermic Patches by Microemulsion Electrokinetic Chromatography

A novel microemulsion electrokinetic capillary chromatography (MEEKC) method has been developed which separates a range of nine steroids. A microemulsion containing ethyl acetate, butan-1-ol, sodium dodecyl sulfate, 15% (v/v) acetonitrile and 12 mmol L(-1) sodium tetraborate aqueous buffer at pH 9.2 was used with direct UV detection at 200 nm. The method was validated for the determination of 17 beta-estradiol content, a hormone steroid, in transdermal patches. Adequate sensitivity (DL = 0.88 mu g mL(-1); QL = 2.65 mu g mL(-1)) without interference from sample excipients was obtained. 17 beta-Estradiol migrates in approximately 5.4 min. Estrone was used as internal standard and acceptable precision (< 1.2% RSD), linearity (r = 0.9996; range from 40.0 to 60.0 mu g mL(-1)), and recovery (100.4 +/- A 0.9% at three concentration levels) were obtained. The principal advantage of the method is that it is rapid and avoids the need of time consuming and expensive sample pre-treatment steps.

LC-UV Methodology for Simultaneous Determination of Lamivudine and Zidovudine in Plasma by Liquid-Liquid Extraction

This study describes an accurate, sensitive, and specific chromatographic method for the simultaneous quantitative determination of lamivudine and zidovudine in human blood plasma, using stavudine as an internal standard. The chromatographic separation was performed using a C8 column (150 x 4.6 mm, 5 mu m), and ultraviolet absorbency detection at 270 nm with gradient elution. Two mobile phases were used. Phase A contained 10 mM potassium phosphate and 3% acetonitrile, whereas Phase B contained methanol. A linear gradient was used with a variability of A-B phase proportion from 98-2% to 72-28%, respectively. The drug extraction was performed with two 4 mL aliquots of ethyl acetate.

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).

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).

New Rapid Derivative Spectrophotometric and Chromatographic Methods for Assay of Loratadine in Tablets and Syrups

New rapid first-derivative spectrophotometric (UVDS) and a stability-indicating high performance liquid chromatographic (HPLC) methods were developed, validated and successfully applied in the analysis of loratadine (LT) in tablets and syrups. In the UVDS method, 0.1 M HCl was used as solvent. The measurements were made at 312.4 nm in the first order derivative spectra. The HPLC method was carried out on a RP-18 column with a mobile phase composed of methanol-water-tetrahydrofuran (50:30:20, v/v/v). UV detection was made at 247 nm. For HPLC methods the total analysis time was <3min, adequate for routine quality control of tablets and syrups containing loratadine.

Development and validation of high performance liquid chromatographic and UV-derivative spectrophotometric methods for the determination of sotalol hydrochloride in tablets

High performance liquid chromatographic (HPLC) and UV derivative spectrophotometric (UVDS) methods were developed and validated for the quantitative determination of sotalol hydrochloride in tablets. The HPLC method was performed on a C18 column with fluorescence detection. The excitation and emission wavelengths were 235 and 310nm, respectively. The mobile phase was composed of acetonitrile-water containing 0.1% trietylamine (7:93v/v) and pH adjusted to 4.6 with formic acid. The UVDS method was performed taking a signal at 239.1nm in the first derivative. The correlation coefficients (r) obtained were 0.9998 and 0.9997 for HPLC and UVDS methods, respectively. The proposed methods are simple and adaptable to routine analysis.

Capillary electrophoresis method for plasmatic determination of imatinib mesylate in chronic myeloid leukemia patients

Imatinib (IMAT) is a tyrosine kinase inhibitor that has been used for the treatment of chronic myeloid leukemia (CML). Despite the efficacy of IMAT therapy, some cases of treatment resistance have been described in CML. Developing a plasma method is important since there are several studies that provided a higher correlation between IMAT plasma concentration and response to treatment. Therefore, in this investigation we validated a method by CE as an alternative, new, simple and fast electrophoretic method for IMAT determination in human plasma. The analysis was performed using a fused silica capillary (50 mm id x 46.5 cm total length, 38.0 cm effective length); 50 mmol/L sodium phosphate buffer, pH 2.5, as BGE; hydrodynamic injection time of 20 s (50 mbar); voltage of 30 kV; capillary temperature of 35 degrees C and detection at 200 nm. Plasma samples pre-treatment involved liquid-liquid extraction with methyl-tert-butyl ether as the extracting solvent. The method was linear from 0.125 to 5.00 mg/mL. The LOQ was 0.125 mg/mL. Mean absolute recovery of IMAT was 67%. The method showed to be precise and accurate with RSD and relative error values lower than 15%. Furthermore, the application of the method was performed in the analysis of plasma samples from CML patients undergoing treatment with IMAT.

Enantioselective analysis of mirtazapine, demethylmirtazapine and 8-hydroxy mirtazapine in human urine after solid-phase microextraction

A selective and reproducible off-line solid-phase microextraction procedure was developed for the simultaneous enantioselective determination of mirtazapine (MRT), demethylmirtazapine and 8-hydroxymirtazapine in human urine. CE was used for optimization of the extraction procedure whereas LC-MS was used for method validation and application. The influence of important factors in the solid-phase microextraction efficiency is discussed, such as the fiber coatings, extraction time, pH, ionic strength, temperature and desorption time. Before extraction, human urine samples were submitted to enzymatic hydrolysis at 37 degrees C for 16 h. Then, the enzyme was precipitated with trichloroacetic acid and the pH was adjusted to 8 with 1 mol/L pH 11 phosphate buffer solution. In the extraction, the analytes were transferred from the aqueous solution to the polydimethylsiloxane-divinylbenzene fiber coating and then desorbed in methanol. The mean recoveries were 5.4, 1.7 and 1.0% for MRT, demethylmirtazapine and 8-hydroxymirtazapine enantiomers, respectively. The method was linear over the concentration range of 62-1250 ng/mL. The within-day and between-day assay precision and accuracy were lower than 15%. The method was successfully employed in a preliminary cumulative urinary excretion study after administration of racemic MRT to a healthy volunteer.

Development and Validation of a Simple Method for Routine Analysis of Ractopamine Hydrochloride in Raw Material and Feed Additives by HPLC

A simple method was optimized and validated for determination of ractopamine hydrochloride (RAC) in raw material and feed additives by HPLC for use in quality control in veterinary industries. The best-optimized conditions were a C8 column (250 x 4.6 mm id, 5.0 mu m particle size) at room temperature with acetonitrile-100 mM sodium acetate buffer (pH 5.0; 75 + 25, v/v) mobile phase at a flow rate of 1.0 mL/min and UV detection at 275 nm. With these conditions, the retention time of RAC was around 5.2 min, and standard curves were linear in the concentration range of 160-240 mu g/mL (correlation coefficient >= 0.999). Validation parameters, such as selectivity, linearity, limit of detection (ranged from 1.60 to 2.05 mu g/mL), limit of quantification (ranged from 4.26 to 6.84 mu g/mL), precision (relative standard deviation <= 1.87%), accuracy (ranged from 96.97 to 100.54%), and robustness, gave results within acceptable ranges. Therefore, the developed method can be successfully applied for the routine quality control analysis of raw material and feed additives.

Simultaneous determination of multibenzodiazepines by HPLC/UV: Investigation of liquid-liquid and solid-phase extractions in human plasma

A method for simultaneous determination of seven benzodiazepines (BZPs) (flunitrazepam, clonazepam, oxazepam, lorazepam, chlordiazepoxide, nordiazepam and diazepam using N-desalkylflurazepam as internal standard) in human plasma using liquid-liquid and solid-phase extractions followed by high-performance liquid chromatography (HPLC) is described. The analytes were separated employing a LC-18 DB column (250 mm x 4.6 mm, 5 mu m) at 35 degrees C under isocratic conditions using 5 mM KH(2)PO(4) buffer solution pH 6.0: methanol: diethyl ether (55:40:5, v/v/v) as mobile phase at a flow rate of 0.8 mL min(-1). UV detection was carried out at 245 nm. Employing LLE, the best conditions were achieved with double extraction of 0.5 mL, plasma using ethyl acetate and Na(2)HPO(4) pH 9.5 for pH adjusting. Employing SPE, the best conditions were achieved with 0.5 mL plasma plus 3 mL 0.1 M borate buffer pH 9.5, which were then passed through a C18 cartridge previously conditioned, washed for 3 times with these solvents: 3 mL 0.1 M borate buffer pH 9.5,4 mL Milli-Q water and 1 mL acetonitrile 5%, finally the BZPs elution was carried with diethyl ether: n-hexane: methanol (50:30:20). In both methods the solvent was evaporated at 40 degrees C under nitrogen flow. The validation parameters obtained in LLE were linearity range of 50-1200 ng mL(-1) plasma (r >= 0.9927), limits of quantification of 50 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15%, and recovery above 65% for all BZPs. In SPE, the parameter obtained were linearity range of 30-1200 ng mL(-1) plasma (r >= 0.9900), limits of quantification of 30 ng mL(-1) plasma, within-day and between-day CV% and E% for precision and accuracy lower than 15% and recovery above 55% for all BZPs. These extracting procedures followed by HPLC analysis showed their suitable applicability in order to examine one or more BZPs in human plasma. Moreover, it could be suggested that these procedures might be employed in various analytical applications, in special for toxicological/forensic analysis. (c) 2008 Elsevier B.V. All rights reserved.

Influence of N-Hexane Inhalation on the Enantioselective Pharmacokinetics and Metabolism of Verapamil in Rats

Verapamil (VER) is commercialized as a racemic mixture of the (+)-(R)-VER and (-)-(S)-VER enantiomers. VER is biotransformed into norverapamil (NOR) and other metabolites through CYP-dependent pathways. N-hexane is a solvent that can alter the metabolism of CYP-dependent drugs. The present study investigated the influence of n-hexane (nose-only inhalation exposure chamber at concentrations of 88, 176, and 352 mg/m(3)) on the kinetic disposition of the (+)-(R)-VER, (-)-(S)-VER, (R)-NOR and (S)-NOR in rats treated with a single dose of racemic VER (10 mg/kg). VER and NOR enantiomers in rat plasma was analyzed by LC-MS/MS (m/z = 441.3 > 165.5 for the NOR and m/z 455.3 > 165.5 for the VER enantiomers) using a Chiralpak (R) AD column. Pharmacokinetic analysis was performed using a monocompartmental model. The pharmacokinetics of VER was enantioselective in control rats, with higher plasma proportions of the (-)-(S)-VER eutomer (AUC(0-infinity) = 250.8 vs. 120.4 ng/ml/h; P <= 0.05, Wilcoxon test). The (S)-NOR metabolite was also found to accumulate in plasma of control animals, with an S/R AUC(0-infinity) ratio of 1.5. The pharmacokinetic parameters AUC(0-infinity), Cl/F, Vd/F, and t(1/2) obtained for VER and NOR enantiomers were not altered by nose-only exposure to n-hexane at concentrations of 88, 176, or 352 mg/m(3) (P > 0.05, Kruskal-Wallis test). However, the verapamil kinetic disposition was not enantioselective for the animals exposed to n-hexane at concentrations equal to or higher than the TLV-TWA. This finding is relevant considering that the (-)-(S)-VER eutomer is 10-20 times more potent than R-(+)-VER in terms of its chronotropic effect on atrioventricular conduction in rats and humans. Chirality 22:29-34, 2010. (C) 2009 Wiley-Liss, Inc.

Hepatic disposition and metabolite kinetics of a homologous series of diflunisal esters

The hepatic disposition and metabolite kinetics of a homologous series of diflunisal O-acyl esters (acetyl, butanoyl, pentanoyl, anti hexanoyl) were determined using a single-pass perfused in situ rat liver preparation. The experiments were conducted using 2% BSA Krebs-Henseleit buffer (pH 7.4), and perfusions were performed at 30 mL/min in each liver. O-Acyl esters of diflunisal and pregenerated diflunisal were injected separately into the portal vein. The venous outflow samples containing the esters and metabolite diflunisal were analyzed by high performance liquid chromatography (HPLC). The normalized outflow concentration-time profiles for each parent ester and the formed metabolite, diflunisal, were analyzed using statistical moments analysis and the two-compartment dispersion model. Data (presented as mean +/- standard error for triplicate experiments) was compared using ANOVA repeated measures, significance level P < 0.05. The hepatic availability (AUC'), the fraction of the injected dose recovered in the outflowing perfusate, for O-acetyldiflunisal (C2D = 0.21 +/- 0.03) was significantly lower than the other esters (0.34-0.38). However, R-N/f(u), the removal efficiency number R-N divided by the unbound fraction in perfusate f(u), which represents the removal efficiency of unbound ester by the liver, was significantly higher for the most lipophilic ester (O-hexanoyldiflunisal, C6D = 16.50 +/- 0.22) compared to the other members of the series (9.57 to 11.17). The most lipophilic ester, C6D, had the largest permeability surface area (PS) product (94.52 +/- 38.20 mt min-l g-l liver) and tissue distribution value VT (35.62 +/- 11.33 mL g(-1) liver) in this series. The MTT of these O-acyl esters of diflunisal were not significantly different from one another. However, the metabolite diflunisal MTTs tended to increase with the increase in the parent ester lipophilicity (11.41 +/- 2.19 s for C2D to 38.63 +/- 9.81 s for C6D). The two-compartment dispersion model equations adequately described the outflow profiles for the parent esters and the metabolite diflunisal formed from the O-acyl esters of diflunisal in the liver.

Isolated limb perfusion with melphalan for human melanoma xenografts in the hindlimb of nude rats: A surviving animal model

We have established a surviving model of isolated limb perfusion using xenografts of the human melanoma cell line MM 96L injected subcutaneously into the hindlimb of a nude rat, The femoral artery and vein were cannulated via the left renal artery and vein and the hind limb was isolated using tourniquets. The limb was perfused with Krebs Heinseleit buffer at 37 degrees C containing 4.7% bovine serum albumin at a constant flow rate of 4 mi per min for 30-60 min with 100% survival of the animals, Tumour vascularization and blood flow were demonstrated using vascular casts and [Cr-51]-microspheres. Following the addition of melphalan (15 or 100 mu g/ml), drug concentrations in the perfusate, tissues and systemic circulation were determined using high pressure liquid chromatography (HPLC), Systemic leakage, assessed using [I-125]albumin and melphalan and detected by a gamma-counter and HPLC respectively, was <0.5%. The melphalan concentration and tissue flow rate in the tumour deposits were 40 and 30% respectively, when compared with the surrounding subcutaneous tissue, At a dose of 15 mu g/ml, melphalan caused a reduction in tumour growth after 60 min perfusion, and a significant reduction in tumour size was seen when the melphalan dose was 100 mu g/ml. The surviving nude rat model of isolated limb perfusion for recurrent melanoma will allow examination of optimal perfusion conditions, along with the pharmacokinetics, pharmacodynamics and efficacy of melphalan and other drugs.