Fatty acid profile and qualitative characteristics of meat from zebu steers fed with different oilseeds

The objective of this study was to evaluate the effects of dietary addition of ground oilseed sources on the quality, fatty acid profile, and CLA content of meat from zebu steers. Thirty-one zebu steers with an initial average age of 23 mo and an initial BW of 365 kg were used in this study. The experimental period was 84 d, which was preceded by an adaption period of 28 d. The diet was provided ad libitum with a forage: concentrate ratio of 40:60. Corn silage was used as the forage source. Four different concentrates were formulated for each treatment: without additional lipids (control) or with ground soybeans (SB), ground cottonseed (CS), or ground linseed (LS). The SB, CS, and LS diets were formulated to have 6.5% ether extract on a total dietary DM basis. The experiment was set up as a completely randomized design. After slaughter, samples were taken from the longissimus thoracis muscle for the measurement of fatty acid concentration and the evaluation of meat quality. The luminosity index was greater in the control and LS diets (P < 0.01). The greatest percentages of myristic acid (C14:0), palmitic acid (C16:0), trans octadecenoic acid (C18:1 trans-10, trans-11, or trans-12), and SFA in the subcutaneous fat were observed in the CS treatment (P < 0.01). Moreover, the least percentages of oleic acid (C18:1 cis-9) and total unsaturated fatty acids in the subcutaneous fat were observed in the CS diet (P < 0.01). The meat linoleic acid and a-linolenic acid percentages were greatest in the SB and LS treatments, respectively (P < 0.001). The unsaturated fatty acid: SFA ratio was smallest for the CS diet (P < 0.01). A gradual increase in oxidation was observed as a function of storage time; however, the diets did not affect the rancidity of the meat (P > 0.05). The fatty acid profile of subcutaneous fat was impaired by the addition of CS. Supplying ground oilseeds did not increase the content of CLA in the meat.

Diet-induced milk fat depression: Association with changes in milk fatty acid composition and fluidity of milk fat

This experiment was designed to examine changes in milk fatty acids during fish oil-induced milk fat depression (MFD) and to test the theory that these changes are related to milk fat fluidity. The experiment was divided into three periods: 1) Baseline: all cows (n = 12) received a high fiber diet without fish oil (FO) for 12 days; 2) Treatment: 4 cows/group received the following treatments for 21 days: a) Low fiber diet without FO (LF), b) High fiber diet+FO (HF+FO) and c) Low fiber diet+FO (LF+FO); 3) Post-treatment: cows returned to the baseline diet and were monitored for 12 days. FO was included at 1.6% DM and HF and LF diets had 40 and 26% NDF, respectively. Milk fat content and yield were unchanged by the LF diet, but were reduced by FO diets at both dietary fiber levels and recovered in the post-treatment period. FO diets caused a pronounced reduction in stearic and oleic acid concentrations in milk fat and an equally pronounced increase in trans-18:1 fatty acid concentrations. Milk fat mean melting point (MMP) was correlated with MFD (r=0.73) and with milk oleic acid concentration (r=-0.92). The ratio of oleic:stearic in milk fat increased gradually and consistently in response to FO. Trans-C18:1 isomers with double bounds at carbon :<= 10 increased with greater MFD and those with double bonds at carbon ! I I decreased with greater MFD. Trans-9 cis-11 CLA explained more than 80% of MFD and was strongly correlated with trans-10 C18:1. Maintenance of MMP below 39-40 degrees C suggests that the mammary gland was able to secrete only milk fat with adequate fluidity and that MFD could be an adaptation mechanism to prevent secretion of milk with higher MMP. (C) 2007 Elsevier B.V. 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.

Simultaneous Determination of Choline Citrate and Acetylmethionine in Injectable Solutions by High Performance Liquid Chromatography

Choline citrate (CC) and acetylmethionine (AM) are lipotropic drugs used in several pharmaceutical formulations. The objective of this research was to develop and validate a high performance liquid chromatographic (HPLC) method for simultaneous determination of CC and AM in injectable solutions, aiming its application in routine analysis for quality control of these pharmaceutical formulations. The method was validated using a Shim-Pack (R) C18 (250 x 4.6 mm, 5 mu m) column. The mobile phase was constituted of 25 mM potassium phosphate buffer solution, pH 5.7, adjusted with 10 % orthophosphoric acid, acetonitrile and methanol (88:10:2, v/v/v). The flow rate was 1.1 mL.min(-1) and the UV detection was made at 210 nm. The analyses were made at room temperature (25 +/- 1 degrees C). The method is precise, selective, accurate and robust, and was successfully applied for simultaneous quantitative determination of CC and AM in injectables.

Simultaneous determination of econazole nitrate, main impurities and preservatives in cream formulation by high performance liquid chromatography

A reversed-phase high performance liquid chromatographic (RP-HPLC) method for determination of econazole nitrate, preservatives (methylparaben and propylparaben) and its main impurities (4-chlorobenzl alcohol and alpha-(2,4-dicholorophenyl)-1H-imidazole-1-ethanol) in cream formulations, has been developed and validated. Separation was achieved on a column Bondclone (R) C18 (300 mm x 3.9 mm i.d., 10 mu m) using a gradient method with mobile phase composed of methanol and water. The flow rate was 1.4 mL min(-1), temperature of the column was 25 C and the detection was made at 220 nm. Miconazole nitrate was used as an internal standard. The total run time was less than 15 min, The analytical curves presented coefficient of correlation upper to 0.99 and detection and quantitation limits were calculated for all molecules. Excellent accuracy and precision were obtained for econazole nitrate. Recoveries varied from 97.9 to 102.3% and intra- and inter-day precisions, calculated as relative standard deviation (R.S.D), were lower than 2.2%. Specificity, robustness and assay for econazole nitrate were also determined. The method allowed the quantitative determination of econazole nitrate, its impurities and preservatives and could be applied as a stability-indicating method for econazole nitrate in cream formulations. (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.

UV-Derivative Spectrophotometric and Stability-Indicating High-Performance Liquid Chromatographic Methods for Determination of Simvastatin in Tablets

A stability-indicating high-performance liquid chromatographic (HPLC) and a second-order derivative spectrophotometric (UVDS) analytical methods were validated and compared for determination of simvastatin in tablets. The HPLC method was performed with isocratic elution using a C18 column and a mobile phase composed of methanol:acetonitrile:water (60:20:20, v/v/v) at a flow rate of 1.0 ml/min. The detection was made at 239 nm. In UVDS method, methanol and water were used in first dilution and distilled water was used in consecutive dilutions and as background. The second-order derivative signal measurement was taken at 255 nm. Analytical curves showed correlation coefficients > 0.999 for both methods. The quantitation limits (QL) were 2.41 mu g/ml for HPLC and 0.45 mu g/ml for UVDS, respectively. Intra and inter-day relative standard deviations were < 2.0 %. Statistical analysis with t- and F-tests are not exceeding their critical values demonstrating that there is no significant difference between the two methods at 95 % confidence level.

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.

LC-MS-MS Identification and Determination of the Flavone-C-Glucoside Vicenin-2 in Rat Plasma Samples Following Intraperitoneal Administration of Lychnophora Extract

The flavone C-glucoside, vicenin-2, in semi-purified extracts of the leaves of Lychnophora ericoides was quantified in rat plasma samples using a method based on reversed-phase high performance liquid chromatography coupled to tandem mass spectrometry. Vicenin-2 was analyzed on a LiChrospher (R) RP18 column using an isocratic mobile phase consisting of a mixture of methanol: water (30:70, v/v) plus 2.0% glacial acetic acid at a flow rate of 0.8 mL min(-1). Genistein was used as internal standard. The mass spectrometer was operated in positive ionization mode and analytes were quantified by multiple reaction monitoring at m/z 595 > 457 for vicenin-2 and m/z 271 > 153 for internal standard. Prior to the analysis, each rat plasma sample was acidified with 200 mu L of 50 mmol L(-1) acetic acid solution and extracted by solid-phase extraction using a C18 cartridge. The absolute recoveries were reproducible and the coefficients of variation values were lower than 5.2%. The method was linear over the 12.5 - 1500 ng mL(-1) concentration range and the quantification limit was 12.5 ng mL(-1). Within-day and between-day assay precision and accuracy were studied at three concentration levels (40, 400 and 800 ng mL(-1)) and were lower than 15%. The developed and validated method seems to be suitable for analysis of vicenin-2 in plasma samples obtained from rats that receive a single i.p. dose of 200 mg kg(-1) vicenin-2 extract.

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.

A HPLC method to evaluate the influence of photostabilizers on cosmetic formulations containing UV-filters and vitamins A and E

This paper reports a simple and reliable HPLC method to evaluate the influence of two currently available photostabilizers on cosmetic formulations containing combined UV-filters and vitamins A and E. Vitamins and UV-filters, widely encountered in products of daily use have to be routinely evaluated since photoinstability can lead to reductions in their efficacy and safety. UV-irradiated formulation samples were submitted to a procedure that included a reliable, precise and specific HPLC method employing a C18 column and detection at 325 and 235 nm. Methanol, isopropanol and water were the mobile phases in gradient elution. The method precision was between 0.28 and 5.07. The photostabilizers studied [diethylhexyl 2,6-naphthalate (DEHN) and benzotriazolyl dodecyl p-cresol (BTDC)], influenced the stability of octyl methoxycinnamate (OMC) associated with vitamins A and E. BTDC was considered the best photostabilizer to vitamins and OMC when the UV-filters were combined with both vitamins A and E. (C) 2010 Elsevier B.V. All rights reserved.

Methylmercury and inorganic mercury determination in blood by using liquid chromatography with inductively coupled plasma mass spectrometry and a fast sample preparation procedure

Despite the necessity to differentiate chemical species of mercury in clinical specimens, there area limited number of methods for this purpose. Then, this paper describes a simple method for the determination of methylmercury and inorganic mercury in blood by using liquid chromatography with inductively coupled mass spectrometry (LC-ICP-MS) and a fast sample preparation procedure. Prior to analysis, blood (250 mu L) is accurately weighed into 15-mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCI was added to the samples following sonication for 15 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of mercury species was accomplished in less than 5 min on a C18 reverse-phase column with a mobile phase containing 0.05% (v/v) mercaptoethanol, 0.4% (m/v) L-cysteine, 0.06 mol L(-1) ammonium acetate and 5% (v/v) methanol. The method detection limits were found to be 0.25 mu g L(-1) and 0.1 mu Lg L(-1) for inorganic mercury and methylmercury, respectively. Method accuracy is traceable to Standard Reference Material (SRM) 966 Toxic Metals in Bovine Blood from the National Institute of Standards and Technology (NIST). The proposed method was also applied to the speciation of mercury in blood samples collected from fish-eating communities and from rats exposed to thimerosal. With the proposed method there is a considerable reduction of the time of sample preparation prior to speciation of Hg by LC-ICP-MS. Finally, after the application of the proposed method, we demonstrated an interesting in vivo ethylmercury conversion to inorganic mercury. (C) 2009 Elsevier B.V. All rights reserved.

A fast sample preparation procedure for mercury speciation in hair samples by high-performance liquid chromatography coupled to ICP-MS

A simple method for mercury speciation in hair samples with a fast sample preparation procedure using high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry is proposed. Prior to analysis, 50 mg of hair samples were accurately weighed into 15 mL conical tubes. Then, an extractant solution containing mercaptoethanol, L-cysteine and HCl was added to the samples following sonication for 10 min. Quantitative mercury extraction was achieved with the proposed procedure. Separation of inorganic mercury (Ino-Hg), methylmercury (Met-Hg) and ethylmercury (Et-Hg) was accomplished in less than 8 min on a C18 reverse phase column with a mobile phase containing 0.05% v/v mercaptoethanol, 0.4% m/v L-cysteine, 0.06 mol L(-1) ammonium acetate and 5% v/v methanol. The method detection limits were found to be 15 ng g(-1), 10 ng g(-1) and 38 ng g(-1), for inorganic mercury, methylmercury and ethylmercury, respectively. Sample throughput is 4 samples h(-1) (duplicate). A considerable improvement in the time of analysis was achieved when compared to other published methods. Method accuracy is traceable to Certified Reference Materials (CRMs) 85 and 86 human hair from the International Atomic Energy Agency (IAEA). Finally, the proposed method was successfully applied to the speciation of mercury in hair samples collected from fish-eating communities of the Brazilian Amazon.

Stereoselective analysis of carvedilol in human plasma and urine using HPLC after chiral derivatization

An enantioselective high-performance liquid chromatographic method for the analysis of carvedilol in plasma and urine was developed and validated using (-)-menthyl chloroformate (MCF) as a derivatizing reagent. Chloroform was used for extraction, and analysis was performed by HPLC on a C18 column with a fluorescence detector. The quantitation limit was 0.25 ng/ml for S(-)-carvedilol in plasma and 0.5 ng/ml for R(+)-carvedilol in plasma and for both enantiomers in urine. The method was applied to the study of enantioselectivity in the pharmacokinetics of carvedilol administered in a multiple dose regimen (25mg/12h) to a hypertensive elderly female patient. The data obtained demonstrated highest plasma levels for the R(+)-carvedilol(AUCSS 75.64 vs 37.29ng/ml). The enantiomeric ratio R(+)/S(-) was 2.03 for plasma and 1.49 0 - 12 for urine (Aeo-12 17.4 vs 11.7 pg). Copyright (c) 2008 John Wiley & Sons, Ltd.

Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom

An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65 kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAC-cDNA of 1548 bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58 kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Elp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents. (C) 2009 Elsevier Masson SAS. All rights reserved.