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.

Development and Validation of a Mice liar Electrokinetic Chromatography Method for Quantitative Determination of Butenolides in Piper malacophyllum (C. Presl) C. DC.

Introduction - A large number of natural and synthetic compounds having butenolides as a core unit have been described and many of them display a wide range of biological activities. Butenolides from P. malacophyllum have presented potential antifungal activities but no specific, fast, and precise method has been developed for their determination. Objective - To develop a methodology based on micellar electrokinetic chromatography to determine butenolides in Piper species. Methodology - The extracts were analysed in an uncoated fused-silica capillaries and for the micellar system 20 mmol/L SDS, 20% (v/v) acetonitrile (ACN) and 10 mmol/L STB aqueous buffer at pH 9.2 were used. The method was validated for precision, linearity, limit of detection (LOD) and limit of quantitation (LOQ) and the standard deviations were determined from the standard errors estimated by the regression line. Results - A micellar electrokinetic chromatography (MEKC) method for determination of butenolides in extracts gave full resolution for 1 and 2. The analytical curve in the range 10.0-50.0 mu g/mL (r(2) = 0.999) provided LOD and LOQ for 1 and 2 of 2.1/6.3 and 1.1/3.5 mu g/mL, respectively. The RSD for migration times were 0.12 and 1.0% for peak area ratios with 100.0 +/- 1.4% of recovery. Conclusions - A novel high-performance MEKC method developed for the analysis of butenolides 1 and 2 in leaf extracts of P. malacophyllum allowed their quantitative determined within an analysis time shorter than 5 min and the results indicated CE to be a feasible analytical technique for the quantitative determination of butenolides in Piper extracts. Copyright (C) 2010 John Wiley & Sons, Ltd.

Development of an Analytical Methodology for Quantification of Strong Acid in Diesel Oil

Many factors can affect the quality of diesel oil, in particular the degradation processes that are directly related to some organosulfur compounds. During the degradation process, these compounds are oxidized into their corresponding sulfonic acids, generating a strong acid content during the process. p-Toluene sulfonic acid analysis was performed using the linear sweep voltammetry technique with a platinum ultramicroelectrode in aqueous solution containing 3 mol L(-1) potassium chloride. An extraction step was introduced prior to the voltammetric detection in order to avoid the adsorption of organic molecules, which inhibit the electrochemical response. The extraction step promoted the transference of sulfonic acid from the diesel oil to an aqueous phase. The method was accurate and reproducible, with detection and quantification limits of 5 ppm and 15 ppm, respectively. Recovery of sulfonic acid was around 90%.

Development and validation of a simple and rapid capillary zone electrophoresis method for determination of nnrti nevirapine in pharmaceutical formulations

A simple and fast capillary zone electrophoresis (CZE) method has been developed and validated for quantification of a non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine, in pharmaceuticals. The analysis was optimized using 10 mmol L-1 sodium phosphate buffer pH 2.5, +25 kV applied voltage, hydrodynamic injection 0.5 psi for 5 s and direct UV detection at 200 µm. Diazepam (50.0 µg mL-1) was used as internal standard. Under these conditions, nevirapine was analyzed in approximately less than 2.5 min. The analytical curve presented a coefficient of correlation of 0.9994. Limits of detection and quantification were 1.4 µg mL-1 and 4.3 µg mL-1, respectively. Intra- and inter-day precision expressed as relative standard deviations were 1.4% and 1.3%, respectively and the mean recovery was 100.81%. The active pharmaceutical ingredient was subjected to hydrolysis (acid, basic and neutral) and oxidative stress conditions. No interference of degradation products and tablet excipients were observed. This method showed to be rapid, simple, precise, accurate and economical for determination of nevirapine in pharmaceuticals and it is suitable for routine quality control analysis since CE offers benefits in terms of quicker method development and significantly reduced operating costs.

Development and validation of a fast RP-HPLC method to determine the analogue of the thyroid hormone, 3,5,3 '-triiodothyroacetic acid (TRIAC), in polymeric nanoparticles

The objective of this work was to develop and validate a rapid Reversed-Phase High-Performance Liquid Chromatography method for the quantification of 3,5,3 '-triiodothyroacetic acid (TRIAC) in nanoparticles delivery system prepared in different polymeric matrices. Special attention was given to developing a reliable reproductive technique for the pretreatment of the samples. Chromatographic runs were performed on an Agilent 1200 Series HPLC with a RP Phenomenex (R) Gemini C18 (150 x 4, 6 mm i.d., 5 mu m) column using acetonitrile and triethylamine buffer 0.1% (TEA) (40 : 60 v/v) as a mobile phase in an isocratic elution, pH 5.6 at a flow rate of 1 ml min(-1). TRIAC was detected at a wavelength of 220 nm. The injection volume was 20 mu l and the column temperature was maintained at 35 degrees C. The validation characteristics included accuracy, precision, specificity, linearity, recovery, and robustness. The standard curve was found to have a linear relationship (r(2) - 0.9996) over the analytical range of 5-100 mu g ml(-1) . The detection and quantitation limits were 1.3 and 3.8 mu g ml(-1), respectively. The recovery and loaded TRIAC in colloidal system delivery was nearly 100% and 98%, respectively. The method was successfully applied in polycaprolactone, polyhydroxybutyrate, and polymethylmethacrylate nanoparticles.

Optimization and validation of a LIBS method for the determination of macro and micronutrients in sugar cane leaves

Laser induced breakdown spectrometry (LIBS) was applied for the determination of macro (P, K, Ca, Mg) and micronutrients (B, Cu, Fe, Mn and Zn) in sugar cane leaves, which is one of the most economically important crops in Brazil. Operational conditions were previously optimized by a neuro-genetic approach, by using a laser Nd:YAG at 1064 nm with 110 mJ per pulse focused on a pellet surface prepared with ground plant samples. Emission intensities were measured after 2.0 mu s delay time, with 4.5 mu s integration time gate and 25 accumulated laser pulses. Measurements of LIBS spectra were based on triplicate and each replicate consisted of an average of ten spectra collected in different sites (craters) of the pellet. Quantitative determinations were carried out by using univariate calibration and chemometric methods, such as PLSR and iPLS. The calibration models were obtained by using 26 laboratory samples and the validation was carried out by using 15 test samples. For comparative purpose, these samples were also microwave-assisted digested and further analyzed by ICP OES. In general, most results obtained by LIBS did not differ significantly from ICP OES data by applying a t-test at 95% confidence level. Both LIBS multivariate and univariate calibration methods produced similar results, except for Fe where better results were achieved by the multivariate approach. Repeatability precision varied from 0.7 to 15% and 1.3 to 20% from measurements obtained by multivariate and univariate calibration, respectively. It is demonstrated that LIBS is a powerful tool for analysis of pellets of plant materials for determination of macro and micronutrients by choosing calibration and validation samples with similar matrix composition.

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).

Development and Validation of Stability-Indicating HPLC Methods for Quantitative Determination of Pravastatin, Fluvastatin, Atorvastatin, and Rosuvastatin in Pharmaceuticals

High-performance liquid-chromatographic (HPLC) methods were validated for determination of pravastatin sodium (PS), fluvastatin sodium (FVS), atorvastatin calcium (ATC), and rosuvastatin calcium (RC) in pharmaceuticals. Two stability-indicating HPLC methods were developed with a small change (10%) in the composition of the organic modifier in the mobile phase. The HPLC method for each statin was validated using isocratic elution. An RP-18 column was used with mobile phases consisting of methanol-water (60:40, v/v, for PS and RC and 70:30, v/v, for FVS and ATC). The pH of each mobile phase was adjusted to 3.0 with orthophosphoric acid, and the flow rate was 1.0mL/min. Calibration plots showed correlation coefficients (r)0.999, which were calculated by the least square method. The detection limit (DL) and quantitation limit (QL) were 1.22 and 3.08 mu g/mL for PS, 2.02 and 6.12 mu g/mL for FVS, 0.44 and 1.34 mu g/mL for ATC, and 1.55 and 4.70 mu g/mL for RC. Intraday and interday relative standard deviations (RSDs) were 2.0%. The methods were applied successfully for quantitative determination of statins in pharmaceuticals.

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.

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.

Development and validation of HPLC method for imiquimod determination in skin penetration studies

A simple, rapid and sensitive analytical procedure for the measurement of imiquimod in skin samples after in vitro penetration studies has been developed and validated. In vitro penetration studies were carried out in Franz diffusion cells with porcine skin. Tape stripping technique was used to separate the stratum corneum (SC) from the viable epidermis and dermis. Imiquimod was extracted from skin samples using a 7:3 (v/v) methanol:acetate buffer (100 mm, pH 4.0) solution and ultrasonication. Imiquimod was analyzed by H-PLC using C(8) column and UV detection at 242 ran. The mobile phase used was acetonitrile:acetate buffer (pH 4.0, 100 mM):diethylamine (30:69.85:0.15, v/v) with flow rate 1 mL/min. Imiquimod eluted at 4.1 min and the running time was limited to 6.0 min. The procedure was linear across the following concentration ranges: 100-2500 ng/mL for both SC and tape-stripped skin and 20-800 ng/mL for receptor solution. Intra-day and inter-day accuracy and precision values were lower than 20% at the limit of quantitation. The recovery values ranged from 80 to 100%. The method is adequate to assay imiquimod from skin samples, enabling the determination of the cutaneous penetration profile of uniquimod by in vitro studies. Copyright (C) 2008 John Wiley & Sons, Ltd.

Validation of a gas chromatographic method to quantify sesquiterpenes in copaiba oils

Copaifera species (Leguminoseae) are popularly known as ""copaiba"" or ""copaiva"". The oleoresins obtained from the trunk of these species have been extensively used in folk medicine and are commercialized in Brazil as crude oil and in several pharmaceutical and cosmetic products. This work reports a complete validated method for the quantification of beta-caryophyllene, alpha-copaene, and alpha-humulene in distinct copaiba oleoresins available commercially. Thus, essential oil samples (100 mu L) were dissolved in 20 mL of hexanes containing internal standard (1,2,4,5-tetramethylbenzene, 3.0 mM) in a 25 mL glass flask. A 1 mu L aliquot was injected into the GC-FID system. A fused-silica capillary column HP-5, coated with 5% phenylmethylsiloxane was used for this study. The developed method gave a good detection response with linearity in the range of 0.10-18.74 mM. Limits of detection and quantitation variety ranged between 0.003 and 0.091 mM. beta-Caryophyllene, alpha-copaene, and alpha-humulene were recovered in a range from 74.71% to 88.31%, displaying RSD lower than 10% and relative errors between -11.69% and -25.30%. Therefore, this method could be considered as an analytical tool for the quality control of different Copaifera oil samples and its products in both cosmetic and pharmaceutical companies. (C) 2010 Elsevier B.V. All rights reserved.

Validation of a Spectrophotometric Method for Quantification of Carboxyhemoglobin

Universidade de São Paulo - LIM[40/HC-FM-USP]