In vitro acaricidal activity of neem (Azadirachta indica) seed extracts with known azadirachtin concentrations against Rhipicephalus microplus

The effect of four extracts from neem seeds (Azadirachta indica) containing 2000, 5000, 9000 and 10,000 ppm of azadirachtin A (AZA), quantified by high-performance liquid chromatography (HPLC) and diluted to 1.25%; 2.5%; 5.0%; 10.0% and 12.8% was verified by in vitro tests with engorged females and larvae of the cattle tick Rhipicephalus micro plus. The results from the bioassays with the engorged females showed that the main toxic effect of the extracts was reduction of the reproductive parameters, with a sharp drop in the number of eggs laid and the hatching rate, mainly when the extracts were diluted to 10.0% and 12.8%. The product effectiveness (PE) calculations for all the solutions tested showed that the AZA solution at 10,000 ppm (N10) was the most effective. However, statistical analysis of the PE data obtained for the proportional AZA concentrations in the different diluted extracts showed significance (P<0.05) of the effects included in the model (extract dilution, principle effect (classificatory) of the assay (extract) and the interaction between the two), indicating significant variations due to the dilution, the test and the interaction between the two factors in the tests with engorged females. For solutions N2, N5, and N9, it was not possible to estimate LC(90) values in the dilution range tested. The lowest LC(50) was observed for extract N5, and although extract N10 was the only extract for which the LC(90) could be estimated within the range tested, the LC(50) was higher than for N5 and N9. These results suggest that substances other than AZA present in the extracts influenced the efficacy, especially up to a certain LC range. In the tests with larvae, no mortality was observed, indicating zero effectiveness of all the extracts tested. The results of the tests with engorged females showed that the neem extracts had acaricide activity, inhibiting egg laying and the larval hatching rate. Complementary studies are necessary to develop new methods to isolate and/or identify other substances besides AZA contained in this plant, to enable using products made from it as acaricides. (C) 2011 Elsevier B.V. All rights reserved.

Determination of amfepramone hydrochloride, fenproporex, and diazepam in so-called natural capsules used in the treatment of obesity

A simple and rapid method was developed for the determination of amfepramone hydrochloride, fenprorex, and diazepam in capsules using high performance liquid chromatography (HPLC) with UV detection. This procedure provided conditions for the separation of the active ingredient from the complex matrices of the dosage forms by extraction in methanol. Isocratic reversed phase chromatography was performed using acetonitrile, methanol, and aqueous 0,1% ammonium carbonate (50:10:40) as a mobile phase, LiChrospher 100 RP 18 column (125 x 5 mm id, 5 mu m), a column temperature of 25 +/- 1 degrees C and detection at 230 nm.The calibration curves were linear over a wide concentration range (20-2000 mu g.mL(-1) to amfepramone hydrochloride, 8-800 mu g.mL(-1) to fenproporex, and 4-200 mu g.mL(-1) to diazepam) and good analytical recovery (87.1 to 107.8%) was obtained. The method is accurate and precise, as well as having advantages such as simplicity and short duration of analysis. Twenty samples of pharmaceutical preparations labelled as natural products were analysed. Anorectics and diazepam, were detected in 40% of the samples.

Application of an HPLC method for analysis of propolis extract

Propolis obtained from honeybee hives has been widely used in medicine, cosmetics, and industry due to its versatile biological activities (antioxidant, antimicrobial, fungicidal, antiviral, antiulcer, immunostimulating, and cytostatic). These activities are mainly attributed to the presence of flavonoids in propolis, which points out the interest in quantifying these constituents in propolis preparations, as well as validation of analytical methodologies. High-performance liquid chromatography (HPLC) methods have been reported to quantify isolated flavonoids or these compounds in complex biological matrices, such as herbal raw materials and extractive preparations. An efficient, precise, and reliable method was developed for quantification of propolis extractive solution using HPLC with UV detection. The chromatograms were obtained from various gradient elution systems (GES) tested in order to establish the ideal conditions for the analysis of propolis extractive solution, using methanol and water: acetonitrile (97.5 : 2.5, v/v) as mobile phase. Gradient reversed phase chromatography was performed using a stainless steel column (250 x 4.6 mm i.d., 5 mum) filled with Chromsep RP 18 (Varian), column temperature at 30.0 +/- 0.1degreesC and detection at 310 nm. The main validation parameters of the method were also determined. The method showed linearity for chrysin in the range 0.24-2.4 mug mL(-1) with good correlation coefficients (0.9975). Precision and accuracy were determined. The obtained results demonstrate the efficiency of the proposed method. The analytical procedure is reliable and offers advantages in terms of speed and cost of reagents.

Determination of oxamniquine in capsules by HPLC

A sensitive, accurate, reliable and easy method was developed for the quantification of oxamniquine in capsules using high-performance liquid chromatography (HPLC) with UV detection. This technique provided conditions for the separation of the active ingredient from the dosage form by extraction in methanol. Isocratic reversed phase chromatography was performed using methanol, water, and triethanolamine (60:40:0.099, v/v/w) (System C) or methanol, acetonitrile, water and formic acid (40:30:30:0.083, v/v/w) (System D) as mobile phase, a stainless steel column (125 x 4 mm i.d., 5 mum) filled with LiChrospher 100 RP-18 (Merck), column temperature of 28 +/- 2 degreesC and detection at 260 nm. The calibration curves were linear over a wide concentration range (1.0-20.0 mug ml(-1) of oxamniquine) to the Systems C and D with good correlation factor (0.9990 and 0.9982, respectively). The average content obtained were 100.1 +/- 1.5% (System C) and 102.4 +/- 0.8% (System D). The presence of lactose, starch, magnesium stearate and sodium laurylsulphate did not interfere in the results of the analysis. The above findings showed the proposed method to be both simple and added advantage of allowing for fast analysis. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Development and validation of HPLC method for analysis of dexamethasone acetate in microemulsions

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Enrofloxacin assay validation and pharmacokinetics following a single oral dose in chickens

The pharmacokinetics of enrofloxacin (ENRO), a fluoroquinolone antimicrobial agent, was studied in male broiler chickens (Cobb) after single oral administration of 10 mg of ENRO/kg b.w. A high-performance liquid chromatography-photodiode array detector (DAD) (HPLC-DAD) method was developed and validated and used for quantitation of ENRO and its major metabolite ciprofloxacin in plasma. The HPLC analyses were carried out using a cationic-octadecyl mixed column and 0.05 mol/L phosphate buffer (pH 2.5)/acetonitrile as mobile phase. The sample preparation of plasma consisted of the precipitation of proteins followed by solid phase extraction on cationic-octadecyl mixed cartridges. The method was validated considering linear range, linearity, selectivity, sensitivity, limit of detection (LOD), limit of quantitation (LOQ), intra- and inter-day precisions and accuracy. The LOD and LOQ for both fluoroquinolones were 60 and 200 ng/mL for plasma. The plasma concentration vs. time graph was characteristic of a two-compartment open model. The maximal plasma concentration of 1.5 +/- 0.2 mg/mL was achieved at 9 +/- 2 h. The elimination half-life and the mean residence time of ENRO were 1.5 +/- 0.2 and 15.64 h, respectively. The area under the concentration-time curve was calculated as 35 +/- 4 mg(.)h/mL.

Development of an HPLC-UV/Vis method for the determination of dyes in a gasoline sample employing different pre-treatments

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identificação de alguns constituintes químicos de Indigofera hirsuta Linn. (Fabaceae) por CLAE-IES-EM (TOF) e avaliação da atividade antirradicalar

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Correlation between acidic ninhydrin and HPLC methods to evaluate fraudulent addition of whey in milk

The acidic ninhydrin spectrophotometric method (ANSM) for quantitative determination of free and bound sialic acid of milk glycoprotein has been proved to be fast and efficient for routine detection of fraudulent addition of rennet whey to fluid milk. In this research the ANSM was compared with the high performance liquid chromatography (HPLC) method, internationally recommended for caseinomacropeptide (CMP) determination, which besides its high accuracy is more sophisticated and requires trained personnel. For several sample conditions (raw milk and milk with variable added amounts of rennet cheese whey), the methods showed an excellent linear correlation, with r = 0.981 when milk was deproteinized with a 120 g.L-1 final concentration of trichloroacetic acid (TCA) concentration. The best correlations could be seen with final concentrations of 100 g.L-1 and 80 g.L-1 TCA; respectively, r = 0.992 and 0.993.

Dimorphandra mollis: an alternative as a source of flavonoids with antioxidant action

Dimorphandra mollis: An Alternative as a Source of Flavonoids with Antioxidant Action. Dimorphandra molls fruits are rich in flavonoids rutin and quercetin, which are compounds with high antioxidant activity and can be used to prevent diseases caused by free radicals. The aim of this study was to obtain an extract rich in flavonoids from fruits of D. mollis. The extract was analyzed using the methods of determination by spectrophotometry and high performance liquid chromatography. The presence of rutin and quercetin was identified in the extract, which showed a total flavonoids content of 33.71 To. The extract also showed antioxidant activity as scanvanger of DPPH and ABTS radicals. It was possible to conclude that D. mollis fruits are a rich source of flavonoids with antioxidant action.

HPLC determination of flumethrin, deltamethrin, cypermethrin, and cyhalothrin residues in the milk and blood of lactating dairy cows

A procedure to determine residue concentrations of synthetic pyrethroid insecticides (flumethrin, deltamethrin, cypermethrin and cyhalothrin) in the milk and blood of lactating dairy cows was developed. Extraction was performed with acetonitrile, n-hexane partitioning, and silica gel column cleanup with n-hexane and diethyl ether. Analysis was carried out by high- performance liquid chromatography and ultraviolet detection. Recovery of the four pyrethroids averaged 78 to 91% with a minimum detectable concentration of 0.001 mg/kg. The method was reproducible and sensitive.

A simplified spectrophotometric method for routine analysis of saccharin in commercial noncaloric sweeteners

A simple, rapid, and sensitive spectrophotometric method for routine analysis of saccharin in commercial noncaloric sweeteners is proposed. This method is based on the reaction of saccharin with tetrachloro-p-benzoquinone (p-chloranil) accelerated by hydrogen peroxide and conducted in an ethanol:acetone (4:1) medium, producing a violet-red compound (γ max = 550 nm). Beer's law is obeyed in a concentration range of 2.05 × 10 -4 to 3.00 × 10 -3 M with an excellent correlation coefficient (r = 0.9998). The detection limit was 1.55 × 10 -5 M, and the effect of interferences on the spectrophotometric measurements was evaluated. The proposed procedure was applied successfully to the determination of saccharin in noncaloric sweeteners. Recoveries were within 99.2-104.3% with standard deviations ranging from to 0.5-1.6%. Results of the proposed method compare very favorably with those given by the high-performance liquid chromatography method recommended by the Food and Drug Administration.

Deposition and leaching of tebuthiuron on sugar cane straw applied with and without alkyl polyglycoside adjuvant

A laboratory experiment was carried out aiming to study the effects of an alkyl polyglycoside adjuvant (APG) on deposition and leaching of the herbicide tebuthiuron applied on sugar cane straw. Tebuthiuron, at concentration of 1200 mg L-1, was applied separately and in tank mix with the APG adjuvant, at concentrations of 0.07 and 0.09% (wt v-1), using a spraying volume of 204 L ha-1. A precipitation equivalent to 20 mm of rain was simulated, 24 h after the applications, to evaluate the herbicide leaching. The quantification of tebuthiuron was carried out by the high performance liquid chromatography (HPLC). It was observed that the addition of APG adjuvant at 0.07% (wt v-1) provided an increase of 11.5% in the deposition of tebuthiuron on straw, reduction of 50.4% in the drift of the herbicide and it did not affect significantly the leached amount (68.5%), when compared with the treatment where tebuthiuron was applied alone (70.8%). At the concentration of 0.09% (wt v-1), the APG adjuvant caused an increase of 22.7% in the deposition; it reduced the drift of the herbicide by 99.9% and reduced the leached amount by 7.6% thereby increasing the retention of the herbicide by straw.

Intravenous versus nebulized ceftazidime in ventilated piglets with and without experimental bronchopneumonia: Comparative effects of helium and nitrogen

Background: Lung deposition of intravenous cephalosporins is low. The lung deposition of equivalent doses of ceftazidime administered either intravenously or by ultrasonic nebulization using either nitrogen-oxygen or helium-oxygen as the carrying gas of the aerosol was compared in ventilated piglets with and without experimental bronchopneumonia. Methods: Five piglets with noninfected lungs and 5 piglets with Pseudomonas aeruginosa experimental bronchopneumonia received 33 mg/kg ceftazidime intravenously. Ten piglets with noninfected lungs and 10 others with experimental P. aeruginosa bronchopneumonia received 50 mg/kg ceftazidime by ultrasonic nebulization. In each group, the ventilator was operated in half of the animals with a 65%/35% helium-oxygen or nitrogen-oxygen mixture. Animals were killed, and multiple lung specimens were sampled for measuring ceftazidime lung tissue concentrations by high-performance liquid chromatography. Results: As compared with intravenous administration, nebulization of ceftazidime significantly increased lung tissue concentrations (17 ± 13 vs. 383 ± 84 μg/g in noninfected piglets and 10 ± 3 vs. 129 ± 108 μg/g in piglets with experimental bronchopneumonia; P < 0.001). The use of a 65%/35% helium-oxygen mixture induced a 33% additional increase in lung tissue concentrations in noninfected piglets (576 ± 141 μg/g; P < 0.001) and no significant change in infected piglets (111 ± 104 μg/g). Conclusion: Nebulization of ceftazidime induced a 5- to 30-fold increase in lung tissue concentrations as compared with intravenous administration. Using a helium-oxygen mixture as the carrying gas of the aerosol induced a substantial additional increase in lung deposition in noninfected piglets but not in piglets with experimental bronchopneumonia. © 2005 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc.

Utilization of different methodologies for the characterization of Hb Hasharon heterozygotes

Hb Hasharon has an electrophoretic mobility similar to that of Hb S in cellulose acetate and a mobility between Hb S and C at acid pH. In high-performance liquid chromatography, Hb Hasharon shows a distinct chromatographic profile and retention time. The origin of this variant is a mutation in codon 47 (GAC → CAC) of the α2-globin gene, resulting in the replacement of asparagine by histidine during the translation process. Ten blood samples from individuals suspected of being Hb Hasharon carriers were analyzed. In addition to classic laboratory tests and high-performance liquid chromatography, molecular analysis by polymerase chain reaction with restriction fragment length polymorphism designed in the laboratory was performed to confirm this mutation. The study of these cases showed that a combination of classical and molecular methodologies is necessary in the diagnosis of hemoglobinopathies for a correct hemoglobin mutant identification. The accurate identification of hemoglobin variants is essential for genetic counseling and choice of therapy. ©FUNPEC-RP.