Isocratic HPLC Method Research Articles

Development and Validation of Stability Indicating LC-PDA Method for Mycophenolate Mofetil in Presence of Mycophenolic Acid and Its Application for Degradation Kinetics and pH Profile Study

Factorial design tool applied for development of isocratic reversed-phase stability-indicating HPLC method for the analysis of mycophenolate mofetil (MMF) and its degradation products. MMF stress degradation products mycophenolate acid (MPA) and DP3 (USP impurity H) were isolated and used for quantitation. Separation achieved on a Symmetry C18 (250 mm × 4.6 mm, 5.0 μ) column using a methanol: acetate buffer (75 : 25 v/v), pH 6.0 (adjusted with acetic acid), at 0.5 mL flow rate, column maintained at 55°C, and data integrated at 251 nm. MMF is subjected to hydrolysis, oxidation, heat degradation, and so forth; under all these conditions degraded products are well separated. The method validation characteristics included accuracy, precision, linearity, range, specificity, and sensitivity. Robustness testing is conducted to evaluate the effect of minor changes to the chromatographic conditions and to establish appropriate system suitability parameters. The proposed method is used to investigate kinetics of acid, alkali hydrolysis and oxidation process. Major degradation products MPA and DP3 were isolated and quantitated. Characterization of MPA by NMR and LC-MS/MS and other degraded products by LC-MS/MS is attempted successfully. The method is used successfully for the quality assessment of three MMF drug commercial formations and its acid, alkali, and oxidative degradation kinetics study.

A Comparative Validation Study of Fluconazole by HPLC and UPLC with Forced Degradation Study

The simplest stability indicating reversed phase Isocratic HPLC and UPLC methods has been developed and validated for the determination of fluconazole in bulk and solid pharmaceutical dosage form. A SunFire C18 (250 × 4.5 mm, 5 μm particle size) column has been used for HPLC and BEH C18 (100 × 2.1 mm, 1.7 μm particle size) column used for UPLC. The Mobile phase consisted of Methanol : Water (70 : 30) for HPLC and Methanol : Water (55 : 45 v/v) for UPLC. Isocratic flow was set at 1 mL/min and 0.30 mL/min, respectively, for HPLC and UPLC. For both HPLC and UPLC system detection has been performed at 211 nm with 30°C column oven temperature (good elution was obtained at 30°C) and injection volume, respectively, 2 μL and 20 μL for HPLC and UPLC.

DETERMINATION OF RESIDUAL CHLORIDE IN SEVELAMER CARBONATE USING HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Sevelamer carbonate is the active ingredient of Renvela tablets; a cross-linked polymeric amine that binds phosphate. It was developed as a pharmaceutical alternative to sevelamer hydrochloride (Renagel). Renvela is indicated for the control of hyperphosphatemia in patients with end-stage renal disease (ESRD). Sevelamer carbonate is manufactured from sevelamer hydrochloride, in which carbonate replaces chloride as the counter ion. Therefore, chloride is one of the impurities present in sevelamer carbonate. A number of suitable analytical techniques are available for chloride estimation, including titration, colorimetry, electrochemical chromatography, indirect photochemical detection, and ion chromatography. Limits of detection range from 50 g/L for colorimetry to 5 mg/L for titration. The specification limit of chloride content in sevelamer carbonate is not more than 0.05%. As a result, accurate determination of this residual chloride in sevelamer carbonate is not possible with colorimetry and titration. In the case of electrochemical detectors, the electrode response could drift with time. Indirect photometric detection is a more time consuming method. A novel isocratic reverse phase HPLC method has been developed in order to detect and quantify the residual chloride. This method involves mixing the drug (polymer) with water, filtering off the solution, and quantitating the water soluble chloride by HPLC using refractive index detector. The method was validated with respect to specificity, precision, linearity, and robustness. The presented method provides equal sensitivity with ion chromatography, specific, and inexpensive analytical procedure and, therefore, is highly suitable for detection and quantification of the residual chloride in sevelamer carbonate API.

Determination of egg yolk xanthophylls by isocratic high-performance liquid chromatography

An isocratic HPLC method was developed for the determination of eight xanthophylls (lutein, capsanthin, zeaxanthin, canthaxanthin, β-apo-8′-carotenal, ethyl-8′-apo-β-carotene-8′-oate, citranaxanthin and β-cryptoxanthin; registered as additives in poultry feeding) in egg yolks. Optimum separation of all-E-isomers of these xanthophylls was achieved in less than 18min on a ProntoSIL C30 column at 27°C using acetone–methanol–0.5M triethylammonium acetate buffer pH 7 14:5:1 (v/v) as the mobile phase with a flow rate of 1mL/min using spectrophotometric detection at 450nm. Other mobile phases were also found suitable, including acetone–water 93:7 (v/v) and acetone–methanol 1:4 (v/v) and the influences of column temperature on the separation and addition of triethylammonium acetate buffer pH 7 to the mobile phase on enhancement of the peak areas were evaluated. Preparation of test solution from yolks included a short vortexing of 0.5g of yolk in 10mL of acetone, followed by 15min magnetic stirring under nitrogen and centrifugation. The method was validated for 5 analytes. The calibration range was between 0.04 and 2μg/mL and the mean recovery of the analytes (95%) and the intra-day precision of the method (RSD less than 5%) on three levels in triplicates were obtained. Analyses of eggs from four husbandry classes showed the presence of up to four xanthophylls (lutein, zeaxanthin, canthaxanthin and ethyl-8′-apo-β-carotene-8′-oate) and traces of β-cryptoxanthin.

Development and application of an HPLC method for erlotinib protein binding studies.

The aim of the present study was to develop a simple and rapid reversed-phase high performance liquid chromatographic method with UV detection for erlotinib hydrochloride quantification, which is applicable for protein binding studies. Ultrafilteration method was used for protein binding study of erlotinib hydrochloride. For sample analysis a simple and rapid reversed-phase high performance liquid chromatographic method with UV detection at 332 nm was developed. The mobile phase was a mixture of methanol, acetonitril and potassium dihydrogen phosphate buffer (15:45:40 %v/v) set at flow rate of 1.3 ml/min. The run time for erlotinib hydrochloride was approximately 6 minutes. The calibration curve was linear over the range of 320-20000 ng/ml with acceptable intra- and inter-day precision and accuracy. The intra-day and inter-day precisions were less than 10% and the accuracies of intra and inter-day assays were within the range of 97.20-104.83% and 98.8-102.2% respectively. Based on the obtained results, a simple, accurate and precise reversed-phase isocratic HPLC method with UV detection has been optimized and validated for the determination of erlotinib hydrochloride in biological samples.

Application of an isocratic methanol-based HPLC method for the determination of iohexol concentrations and glomerular filtration rate in patients with cirrhosis

Glomerular filtration rate (GFR) and mortality is more accurately determined by gold standard measures than serum creatinine-based estimates in cirrhosis. No formal validation of any gold standard method has been reported. An isocratic methanol-based method incorporating the reference standard iohexol-related compound C was developed and validated in 12 patients with cirrhosis by simultaneously determining GFR using iohexol and chromium-51 labelled ethylenediamine tetraacetic acid ((51)Cr-EDTA) clearance. Iohexol pharmacokinetics was also studied with the collection of blood and ascitic fluid at intervals following an iohexol bolus. Triplicate assays produced a linear calibration curve (R (2)=0.99, N=5) over an iohexol concentration range of 23.6-755 µg/L. Mean (range) extraction recovery of iohexol from serum was greater than 95% (94-97%), with an intra-day coefficient of variation less than 3%. Twelve patients with cirrhosis with mean Child-Pugh score of 9 displayed a mean difference (bias) -1.3 mL/min/1.73 m(2) (-18 to +16) comparing iohexol with (51)Cr-EDTA. Iohexol equilibrated between blood and ascitic compartments after 4 h. A simple, cheap, and accurate isocratic, methanol-based method for the determination of iohexol concentrations is described, validated according to Food and Drug Administration guidance. Iohexol demonstrated comparable performance with (51)Cr-EDTA in determining GFR. Delayed equilibrium of iohexol between blood and ascitic compartments suggests sampling beyond 4 h would improve accuracy of GFR determinations in patients with cirrhosis.

Development and Validation of a HPLC Method for Dissolution and Stability Assay of Liquid-Filled Cyclosporine Capsule Drug Products

To assay the dissolution samples of a drug product from several sources, a simple but broadly applicable analytical method is always desired. For the liquid-filled cyclosporine capsules, while analyzing the dissolution samples, the current compendial and literature HPLC methods have been found to be inadequate to provide satisfactory separation of the drug and the excipient peaks. Accordingly, a suitable isocratic reverse-phase HPLC method was developed for the analysis of dissolution samples of liquid-filled cyclosporine capsules. The method successfully separated the cyclosporine peak from the interfering chromatographic peaks of the excipients. The method was validated according to the ICH and FDA guidelines. Specificity, selectivity, linearity, accuracy, precision, and robustness were established over 3 days as part of method validation. Additionally, the degradation kinetics of cyclosporine in dissolution media was determined. Cyclosporine degradation followed a zero-order kinetics in the dissolution media with the respective rate constants of -3.5, -1.5, and -0.3%/h at 37°C, 25°C, and 10°C.

A rapid stability-indicating, fused-core HPLC method for simultaneous determination of β-artemether and lumefantrine in anti-malarial fixed dose combination products

BackgroundArtemisinin-based fixed dose combination (FDC) products are recommended by World Health Organization (WHO) as a first-line treatment. However, the current artemisinin FDC products, such as β-artemether and lumefantrine, are inherently unstable and require controlled distribution and storage conditions, which are not always available in resource-limited settings. Moreover, quality control is hampered by lack of suitable analytical methods. Thus, there is a need for a rapid and simple, but stability-indicating method for the simultaneous assay of β-artemether and lumefantrine FDC products.MethodsThree reversed-phase fused-core HPLC columns (Halo RP-Amide, Halo C18 and Halo Phenyl-hexyl), all thermostated at 30°C, were evaluated. β-artemether and lumefantrine (unstressed and stressed), and reference-related impurities were injected and chromatographic parameters were assessed. Optimal chromatographic parameters were obtained using Halo RP-Amide column and an isocratic mobile phase composed of acetonitrile and 1mM phosphate buffer pH 3.0 (52:48; V/V) at a flow of 1.0 ml/min and 3 μl injection volume. Quantification was performed at 210 nm and 335 nm for β-artemether and for lumefantrine, respectively. In-silico toxicological evaluation of the related impurities was made using Derek Nexus v2.0®.ResultsBoth β-artemether and lumefantrine were separated from each other as well as from the specified and unspecified related impurities including degradants. A complete chromatographic run only took four minutes. Evaluation of the method, including a Plackett-Burman robustness verification within analytical QbD-principles, and real-life samples showed the method is suitable for quantitative assay purposes of both active pharmaceutical ingredients, with a mean recovery relative standard deviation (± RSD) of 99.7 % (± 0.7%) for β-artemether and 99.7 % (± 0.6%) for lumefantrine. All identified β-artemether-related impurities were predicted in Derek Nexus v2.0® to have toxicity risks similar to β-artemether active pharmaceutical ingredient (API) itself.ConclusionsA rapid, robust, precise and accurate stability-indicating, quantitative fused-core isocratic HPLC method was developed for simultaneous assay of β-artemether and lumefantrine. This method can be applied in the routine regulatory quality control of FDC products. The in-silico toxicological investigation using Derek Nexus® indicated that the overall toxicity risk for β-artemether-related impurities is comparable to that of β-artemether API.

SIMPLE AND FAST RP-HPLC METHOD FOR THE DETERMINATION OF PREDNISOLONE SODIUM PHOSPHATE, PREDNISOLONE, ATROPINE, AND HOMATROPINE AS RESIDUALS IN CLEANING VALIDATION OF INDUSTRIAL PHARMACEUTICAL EQUIPMENT

Determining traces of pharmaceuticals in equipment is an important step for controlling cross-contamination during the production cycle. Time-consuming analysis is not applicable because it of incremental costs, especially in evaluating residuals as steroids and alkaloids that require a fast and specific technique. Here, an isocratic reversed-phase HPLC method was developed for the trace analysis of Prednisolone Sodium Phosphate (PSP), Homatropine (HOM), Atropine (ATR), and Prednisolone (PRE) in cleaning samples from pharmaceutical equipment. For this, a mobile phase mixture of 35/65 (V/V) acetonitrile/potassium dibasic phosphate 10 m mol L−1, pH 6.9 at 1.0 mL min−1, with a phenyl-hexyl column, were used. These parameters enabled quantifying the analytes within 7.0 min, detecting ATR and HOM at 210 nm, while PSP and PRE were monitored at 240 nm, being lineal between 1–100 µg mL−1 in all cases. The quantification and detection limits were 0.04 and 0.02 µg mL−1 for PSP, ATR, and HOM and 0.02 and 0.01 µg mL−1 for PRE. The method proved its worth and specificity even in presence of other formulation components of the final dosage forms without extraction or concentration procedures.

Development and Application of a Validated HPLC Method for the Determination of Clindamycin Palmitate Hydrochloride in Marketed Drug Products: An Optimization of the Current USP Methodology for Assay

A simple efficient isocratic reversed-phase HPLC method was developed and validated for the determination of clindamycin palmitate hydrochloride (CPH) and its commercially available oral solution products. Separation was achieved on a Phenomenex Zorbax (Luna) cyano column (150 × 4.6 mm, 5 μm) with a Phenomenex cyano guard cartridge (4 × 3.0 mm) on Agilent 1050 series HPLC system. CPH and its resolution standard lincomycin were eluted isocratically at a flow rate of 1 mL/min with a simplified mobile phase (potassium phosphate buffer (5 mM, pH 3.0)—acetonitrile—tetrahydrofuran (20:75:5, v/v/v)) and detected at 210 nm. The column was maintained at 25?C. The method was validated according to USP category I requirements. Robustness and forced degradation studies were also conducted. CPH marketed drug products were obtained from a drug distributor and assayed for potency using the validated method. Validation acceptance criteria were met in all cases. The analytical range for CPH was 15 - 500 μg/mL and the linearity was r2 > 0.999 over three days. The method was determined to be specific and robust. Both accuracy (92.0% - 103.8%) and precision (0.67% - 1.52%) were established across the analytical range for low, intermediate and high QC concentrations. Method applicability was demonstrated by analyzing two marketed products of CPH, in which results showed potency >98%. The method was determined to be an enhancement over the current USP methodology for assay as a result of increased efficiency, reduced organic solvents and the elimination of matrix modifiers. This method was successfully applied for the quality assessment of: 1) currently marketed drug products and 2) will in future assess the product quality of novel dosage forms of CPH for pediatric use.

A rapid and validated reverse phase liquid chromatographic method for determination of imiquimod from topical cream formulations

A Novel, rapid, accurate and precise reverse phase isocratic HPLC method has been developed for the determination of Imiquimod content in Imiquimod cream which is widely used for treatment of precancerous skin lesions known as actinic keratosis, a certain type of skin cancer and treatment of genital warts, known as Human Papilloma Virus (HPV). A mixture of 10 mM monobasic phosphate containing 0.1% triethylamine adjusted to pH 2.45 and acetonitrile in the ratio of 70:30 (v/v) was used as mobile phase. C-18 column of dimension 250 × 4.6 mm, 5 μ from Inertsil was used for analysis. Detector wavelength was kept at 245 nm and flow rate was kept 1.4 mL min−1. The retention time was observed at about 3.0 ± 0.1 min. The method has been validated as per ICH guidelines, with respect to specificity, linearity, accuracy, precision, robustness, solution stability and filter compatibility. Results for all the parameters were found within predetermined limits. The proposed method was applied to determine the content of Imiquimod in Imiquimod creams from different manufacturers. Content of the imiquimod was in good agreement with label claim of Imiquimod creams. This showed that proposed method is rapid, simple, precise, linear, robust, rugged and accurate which is useful and economic for routine analysis of Imiquimod content in Imiquimod cream.

Isocratic RP-HPLC method for separation and simultaneous determination of ternary mixture of omeprazole, tinidazole and doxycycline in their raw materials and combined capsules

A simple, sensitive and rapid isocratic HPLC method has been developed for simultaneous estimation of a ternary mixture of omeprazole, tinidazole and doxycycline in both their raw state and combined in capsules, to be used for treatment of Helicobacter pylori . The analysis was performed on a C18 column (250 mm × 4.6 mm i.d., 5 μm particle size) with a mobile phase consisting of methanol and 0.06 M sodium dihydrogen phosphate (65 : 35, v/v) adjusted to pH 4.5. The UV detector was operated at 260 nm and the effluents were pumped with a flow rate of 1.0 mL min−1 using floctafenine as internal standard. The run time under the optimum chromatographic conditions is less than 6 min. Linearity, accuracy and precision were found to be acceptable over the concentration range of 2–20 μg mL−1 for omeprazole, 5–60 μg mL−1 for tinidazole and 5–30 μg mL−1 for doxycycline. The sensitivity of the method allows the determination of the studied drugs with a limit of quantification of 1.2 μg mL−1, 1.96 μg mL−1 and 1.88 μg mL−1 for omeprazole, tinidazole and doxycycline respectively. The proposed method was fully validated according to ICH guidelines. The high sensitivity and the simplicity of the proposed method allow the successful determination of such a ternary mixture in a combined capsule with a percentage recovery of 99.61% ± 0.79 for omeprazole, 99.69% ± 0.65 for tinidazole and 99.62% ± 0.94 for doxycycline.

Validation of Quetiapine Fumarate in Pharmaceutical Dosage by Reverse‐Phase HPLC with Internal Standard Method

A rapid, specific, and accurate isocratic HPLC method was developed and validated for the assay of quetiapine fumarate in pharmaceutical dosage forms. The assay involved an isocratic‐elution of quetiapine fumarate in Grace C18 column using mobile‐phase composition of 0.1% ortho phosphoric acid with triethyl amine as modifier buffer and acetonitrile in the ratio of 50 : 50 (v/v).The wavelength of detection is 294 nm. The method showed good linearity in the range of 2.0–50.2 × 10−3 g/Lt. The runtime of the method is 5 mins. The developed method was applied to directly and easily analyse of the pharmaceutical tablet preparations. The percentage recoveries were near 100% for given methods. The method was completely validated and proven to be rugged. The excipients did not interfere in the analysis. The results showed that this method can be used for rapid determination of quetiapine fumarate in pharmaceutical tablet with precision, accuracy, and specificity.

Analysis of Anabaena vaginicola and Nostoc calcicola from Northern Iran, as rich sources of major carotenoids

Four major carotenoids of high nutritional significance, including β-carotene, lycopene, lutein and zeaxanthin were determined in three isolates of heterocystous cyanobacteria, belonging to the genera Anabaena and Nostoc, isolated from Iranian terrestrial and aquatic ecosystems, for the first time. The ultrasonically extracted carotenoids were identified and quantified by a rapid and sensitive isocratic HPLC method and identification was further confirmed by spiking authentic standards and the pattern of the UV–Vis spectra obtained from photo-diode array detector. The results showed that these isolates contain large amounts of four major carotenoids, especially lycopene (up to 24570μg/g dry weight, DW) which appears to be the highest reported amount until present; and β-carotene (up to 8133μg/g DW) which is comparable with the best natural sources of β-carotene. Meanwhile, they are rich in the cis-isomers of lycopene and β-carotene which is important in their bioavailability and health benefits.

Enantioseparation of Nadifloxacin by High performance liquid Chromatography

A rapid isocratic chiral HPLC method has been developed for the separation of R-Nadifloxacin from S-Nadifloxacin. Good resolution viz. Rs> 4.0 between R- and S- forms of Nadifloxacin was achieved by RP-HPLC using endcappedC18 stationary phase and chiral mobile phase. Chirality to the mobile phase was imparted with addition of β-CD in phosphate buffer with EDTA.Column temperature was 45<SUP>°</SUP>C and flow rate was kept 1.5 mL min<SUP>-1</SUP>.The elution was monitored by UV-vis detector at λ-290 nm. The calibration curve showed excellent linearity over concentration range 0.040-20 µg mL<SUP>-1</SUP>.This method was further used to determine the amount of R-Nadifloxacin in pure and active pharmaceutical ingredient of S-Nadifloxacin and is capable to quantitate and detect R-Nadifloxacin to the levels of 0.040µg mL<SUP>-1</SUP>and 0.020µg mL<SUP>-1 </SUP>respectively. The average recovery of R-Nadifloxacin was 99.09 %. The method is better than the already reported one for the enantioseparation of the Nadifloxacin.

Development of an isocratic HPLC method for catechin quantification and its application to formulation studies

The aim of this study was to develop a simple, rapid and accurate isocratic HPLC analytical method to qualify and quantify five catechin derivatives, namely (+)-catechin (C), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG), (−)-epicatechin (EC) and (−)-epigallocatechin gallate (EGCG). To validate the analytical method, linearity, repeatability, intermediate precision, sensitivity, selectivity and recovery were investigated. The five catechin derivatives were completely separated by HPLC using a mobile phase containing 0.1% TFA in Milli-Q water (pH 2.0) mixed with methanol at the volume ratio of 75:25 at a flow rate of 0.8ml/min. The method was shown to be linear (r2>0.99), repeatable with instrumental precision<2.0 and intra-assay precision<2.5 (%CV, percent coefficient of variation), precise with intra-day variation<1 and inter-day variation<2.5 (%CV, percent coefficient of variation) and sensitive (LOD<1μg/mL and LOQ<3μg/mL) over the calibration range for all five derivatives. Derivatives could be fully recovered in the presence of niosomal formulation (recovery rates>91%). Selectivity of the method was proven by the forced degradation studies, which showed that under acidic, basic, oxidation temperature and photolysis stresses, the parent drug can be separated from the degradation products by means of this analytical method. The described method was successfully applied in the in vitro release studies of catechin-loaded niosomes to manifest its utility in formulation characterization. Obtained results indicated that the drug release from niosomal formulations was a biphasic process and a diffusion mechanism regulated the permeation of catechin niosomes.

An Isocratic HPLC Method for the Determination of Sorbitol and Glycerol in Pharmaceutical Formulations

Sorbitol and glycerol, along with other inactive ingredients such as preservatives and dyes, are commonly used in various pharmaceutical and personal care products. To accurately assess the effectiveness of various formulations containing sorbitol and/or glycerol, their quantitative determination is essential. In the current study, two types of detectors (a Varian evaporative light scattering detector and an Agilent ultraviolet-visible detector) are evaluated for the assay of working sample solutions. The two detection techniques are complimentary, and a comparison of the results obtained using the two detectors is presented here. The current method is shown to be stability-indicating and free from interference from any of the formulation excipients and potential degradation products. The method is reproducible, accurate, sensitive and selective. It provides enhanced detection sensitivity for sorbitol and comparable sensitivity for glycerol versus similar methods reported in the literature that utilize a refractive index detector for the analysis of either of the two polyols.

Abstract 5709: Formulation development and evaluation of nanostructured lipid carriers for topical delivery of honokiol

Abstract Purpose: Honokiol, a plant lignan isolated and purified from Magonila Officinalis has been shown to have chemopreventive effects in chemically induced and UV-B induced skin tumor development in SKH-1 mice. However poor solubility of honokiol greatly limits its clinical application. The key objectives of the present investigation are to improve the solubility and stability of honokiol by encapsulating in nanostructured lipid nanoparticles. Methods: Honokiol was incorporated into nanostructured lipid nanoparticles and cream using hot homogenization method. Compritol 888 ATO, Precirol ATO 5, and miglyol 812 as lipid matrices and poloxamer 188 as emulsifier. Lipid nanoparticles were characterized for size, zeta potential, shape and morphology using photon correlation spectroscopy (PCS) and atomic force microscopy (AFM). Honokiol was quantified by isocratic HPLC method using mobile phase of 60:40 (%v/v, acetonitrile:water) at a flow rate of 1 ml/min at a wavelength of 254 nm. Permeation studies were carried out using porcine dermatome skin in Franz diffusion cell. Honokiol concentration corresponding to 0.5 mg/ml in sesame oil, cream and lipid nanoparticles was dispersed in PBS pH 7.4. Permeation studies were performed till 24hrs; the samples (200 µl) were withdrawn at regular time intervals and replaced with fresh medium. Amount of honokiol permeated across porcine skin was quantified using HPLC method. Stability of cream and lipid nanoparticle formulations was studied at 30° C and 65 % relative humidity in a stability chamber for one month. Results: The size of the optimized honokiol loaded lipid nanoparticles was 145±7 nm with a polydispersity index of 0.24±0.02. The zeta potential and encapsulation efficiency was 0.34±0.02 mV and 97±2 %. The particle size of lipid nanoparticles measured in AFM was very close to size measured by PCS. In case of precirol cream, particle size was more than 20 µm with drug content of 89±5 %. Honokiol loaded nanostructured lipid nanoparticles, and cream were stable for about 30 days without any significant changes in particle size, and drug content at 30° C and 65 % relative humidity. Skin treated with honokiol entrapped in nanoparticles and cream has shown more retention of honokiol in the skin compared to plain honokiol in sesame oil. Proposed lipid cream and nanoparticles formulation has shown effective skin penetration of honkiol. From the data it is evident that nanoparticles has shown higher amount of honokiol in skin compared to cream formulation. Conclusions: Preliminary results of this study have shown the potential of lipid based formulations for topical delivery of honokiol. Chemopreventive potential and efficacy of honokiol encapsulated in lipid based formulations will be evaluated in UV-B induced skin cancer model. Acknowledgements: Supported by Translational Cancer Research Center funded by the South Dakota Governor's Office of Economic Development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5709. doi:1538-7445.AM2012-5709

A SIMPLE ISOCRATIC HPLC METHOD FOR THE SIMULTANEOUS DETERMINATION OF ANTIPSYCHOTIC INDOLE ALKALOIDS IN RAUWOLFIA TETRAPHYLLA

A simple isocratic HPLC method has been developed for the simultaneous quantitation of three antipsychotic indole alkaloid (IA), α-yohimbine (1), isoreserpiline (2), and 10-methoxy tetrahydroalstonine (3) in Rauwolfia tetraphylla leaf. Samples were analyzed by reverse-phase chromatography on a Waters spherisorb ODS2 column using isocratic elution with acetonitrile containing 0.1% TEA and water containing 0.1% TFA (35:65, v/v) at a flow rate of 1 mL/min, a column temperature of 30°C, and UV detection at 210 nm. The method was validated and applied for quantification of individual alkaloids in various leaf extracts of R. tetraphylla. The method allowed simultaneous determination of alkaloids, 1, 2, and 3 in the plant. The limits of detection and quantification were 1.30, 1.16, 1.01, and 4.35, 3.88, and 3.39 µg/mL (20 µL injection) for compounds 1, 2, and 3, respectively. The method is simple, accurate, and precise which may be recommended for the routine quality control analysis of R. tetraphylla leaf extract containing these three IA as antipsychotic principles in the herb.

Association between the plasma proteome and plasma alphatocopherol concentrations in humans

Vitamin E has been inversely associated with chronic disease; however, the biological processes regulated by this vitamin have not been fully elucidated. The objective of the present study was to examine the association between α‐tocopherol and the plasma proteome. Subjects are from the Toronto Nutrigenomics and Health Study and include men and women (n=1,022) who completed a general health and lifestyle questionnaire and 196‐ item food frequency questionnaire and provided a fasting blood sample. Levels of α‐tocopherol were analyzed using a reversed‐phase isocratic HPLC method with fluorescent detection. Fifty‐four plasma proteins were assayed by a mass spectrometry‐based multiple reaction monitoring method. Analysis of covariance was used to compare mean concentrations of plasma proteins across tertiles of α‐tocopherol. Levels of apolipoprotein C‐III, fibrinogen alpha, beta, and gamma chains, fibronectin and fibrinopeptide A were significantly and positively associated to plasma α‐tocopherol, while intermediate levels of α‐tocopherol were significantly associated with higher levels of alpha‐1B‐glycoprotein (all P&lt;0.0009). In conclusion, circulating levels of α‐tocopherol were significantly associated with plasma proteins from several physiological pathways. This research was funded by the Advanced Foods and Materials Network.