ISRN Chromatography The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. Development and Validation of HPTLC Method for Simultaneous Estimation of Diosgenin and Quercetin in Fenugreek Seeds (Trigonella foenum-graceum) Thu, 10 Apr 2014 09:08:04 +0000 A sensitive, fast, and reproducible high performance thin-layer chromatographic method has been developed for simultaneous analysis of diosgenin and quercetin from fenugreek seeds, using TLC aluminium plates precoated with silica gel G60F254. Among the different combinations of mobile phases used, best separation was achieved in Toluene-ethyl acetate-formic acid (5 : 4 : 1, v/v/v). Densitometric scanning of the plates directly at 275nm was used for analysis of quercetin. While as for analysis of diosgenin, plates were scanned at 450nm after spraying with anisaldehyde-sulphuric acid reagent. The retardation factorvalue of diosgenin and quercetin was found to be 0.69 ± 0.02 and 0.57 ± 0.02, respectively. The method was validated for specificity, precision (intraday and interday), accuracy, and robustness. Accuracy of the method was checked by recovery study of three different levels with the average percentage recovery of 99.13 ± 0.26 for diosgenin and 99.63 ± 0.34 for quercetin, respectively. Dried fenugreek seed samples were found to contain diosgenin in the range of 0.113–0.135% (w/w) and quercetin in the range of 0.009–0.012% (w/w). The present method is being reported for the first time and can be used for routine quality control and quantification of these marker compounds in various plant samples, extracts, and market formulations. Omi Laila, Imtiyaz Murtaza, M. Z. Abdin, S. Ahmad, Nisar Ahmad Ganai, and Majid Jehangir Copyright © 2014 Omi Laila et al. All rights reserved. A Rapid Reversed-Phase HPLC Method for Analysis of Trans-Resveratrol in PLGA Nanoparticulate Formulation Sun, 16 Feb 2014 12:20:12 +0000 A rapid reversed-phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of trans-resveratrol (t-RVT) in PLGA nanoparticle formulation. A new formulation of t-RVT loaded PLGA nanoparticles (NPs) with potential stealth properties was prepared by nanoprecipitation method in our laboratory. The desired chromatographic separation was achieved on a Phenomenex C18 column under isocratic conditions using UV detection at 306 nm. The optimized mobile phase consisted of a mixture of methanol: 10 mM potassium dihydrogen phosphate buffer (pH 6.8): acetonitrile (63 : 30 : 7, v/v/v) at a flow rate of 1 mL/min. The linear regression analysis for the calibration curves showed a good linear correlation over the concentration range of 0.025–2.0 μg/ml, with determination coefficients, R2, exceeding 0.9997. The method was shown to be specific, precise at the intraday and interday levels, as reflected by the relative standard deviation (RSD) values, lower than 5.0%, and accurate with bias not exceeding 15% and percentage recovery was found to be in the range between 94.5 and 101.2. The limits of detection and quantification were 0.002 and 0.007 μg/ml, respectively. The method was successfully applied for the determination of t-RVT encapsulation efficiency. Gurinder Singh and Roopa S. Pai Copyright © 2014 Gurinder Singh and Roopa S. Pai. All rights reserved. Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Satranidazole from Its Formulation Sun, 12 Jan 2014 13:21:57 +0000 Satranidazole is a new nitroimidazole derivative with potent antiamoebic action and is available in market in the form of tablet and dry syrup either alone or in combination with Ofloxacin. The present study involves the development of simple, accurate, precise, and reproducible reversed phase high performance liquid chromatography (RP-HPLC) method for determination of Satranidazole from its granular dosage form. Isocratic elution at a flow rate of 1.0 mL/min was employed on BDS Hypersil C18 (250 mm × 4.6 mm, 5 μm) column at 25°C temperature. The mobile phase consists of 0.16% v/v orthophosphoric acid solution, pH 3: acetonitrile in the ratio of 60 : 40 v/v. The UV detection wavelength was 320 nm, and 20 μL sample was injected. The retention time for Satranidazole was about 4.3 minutes. The method was validated for various parameters such as system suitability, precision, recovery, robustness, and ruggedness as per ICH guidelines. The validated RP-HPLC method was found to be specific, linear, precise, and accurate and can be successfully employed for the assay of Satranidazole taste masked granules coated with Eudragit E100 and marketed tablets. Harshal Ashok Pawar and Pooja Rasiklal Joshi Copyright © 2014 Harshal Ashok Pawar and Pooja Rasiklal Joshi. All rights reserved. Stability Indicating Simultaneous Validation of Telmisartan and Cilnidipine with Forced Degradation Behavior Study by RP-HPLC in Tablet Dosage Form Tue, 31 Dec 2013 19:14:43 +0000 A simple, precise, and accurate RP-HPLC method has been developed and validated for the simultaneous assay of Telmisartan and Cilnidipine in tablets. Isocratic RP-HPLC method was developed on Waters C18  mm, 5 μm column using mobile phase as acetonitrile (ACN): buffer pH 3.0 with orthophosphoric acid (68 : 32) at a flow rate of 1.0 mL/min and the detection was carried out at 245 nm using photodiode array detector. Forced degradation study was carried out by oxidation, hydrolysis, photolysis, and heating the drug. The method was validated for specificity, linearity, precision, accuracy, robustness, and solution stability. The method was found to be linear in the concentration range of 40–160 μg/mL with correlation coefficient of 0.9990 for Telmisartan and 10–40 μg/mL with correlation coefficient of 0.9989 for Cilnidipine. Degradation products produced as a result of stress studies did not interfere with the detection of agomelatine; therefore, the assay can be considered to be stability indicating. Reema H. Rupareliya and Hitendra S. Joshi Copyright © 2013 Reema H. Rupareliya and Hitendra S. Joshi. All rights reserved. High-Performance Liquid Chromatographic Method for Analysis of Emtricitabine in Rat Plasma: Method Development, Validation and Application to a Pharmacokinetic Study Sat, 30 Nov 2013 12:09:16 +0000 A new reverse phase liquid chromatographic method for the investigation of emtricitabine in rat plasma was developed after oral administration to Wistar rats. The desired chromatographic separation was achieved on Phenomenex C18 column (250 mm × 4.6 mm I.D., 5 µm) column, under isocratic conditions using UV detection at 280 nm. The optimized mobile phase consisted of a mixture of 10 mM potassium dihydrogen phosphate buffer- (adjusted to pH 6.8) methanol-2% acetic acid in a ratio of (73 : 25 : 2, v/v/v) at a flow rate of 1 mL min−1. The system was found to produce sharp and well-resolved peaks for emtricitabine with retention time of 5.78 min. The linear regression analysis for the calibration curves showed a good linear correlation over the concentration range 0.050–3.0 µg mL−1, with determination coefficients, , exceeding 0.9970. The limits of detection (LOD) and quantitation (LOQ) were found to be 0.016 µg mL−1 and 0.049 µg mL−1, respectively. The method was successfully applied for the pharmacokinetic in rats. Emtricitabine concentration in plasma reached () was 1.357 µg mL−1 about 2 h after oral administration of 15 mg/kg/rat. The AUC0-24 was 12.175 µg mL−1* h and the apparent elimination half-life () was 8.153 h. This method was found to be suitable for examining emtricitabine concentration in rats, after oral administration of emtricitabine in a single dose. Gurinder Singh and Roopa S. Pai Copyright © 2013 Gurinder Singh and Roopa S. Pai. All rights reserved. Cefpodoxime Proxetil: A New Stability Indicating RP-HPLC Method Mon, 24 Jun 2013 08:44:58 +0000 The present work describes the development of a sensitive and economic stability indicating high performance liquid chromatographic (HPLC) method for the determination of cefpodoxime proxetil (CP) as bulk drug and as pharmaceutical formulation. Both R and S isomers of the drug were separated using Phenomenex ( mm, 5 μm particle size) ODS column with a flow rate of 1 mL min−1 and an SPD 20 A UV detector to monitor the eluate at 252 nm. The isocratic method used a mobile phase consisting of methanol and phosphate buffer of pH 4.0 in the ratio 65 : 35. The linear regression analysis data for the calibration plots showed good linear relationship with in the working concentration range of 5–100 μg mL−1. The LOD and LOQ were 53 and 160 ng mL−1, respectively. CP was subjected to stress degradation using acid, alkali, hydrogen peroxide, dry heat, wet heat, and UV light. The standard drug peaks were well resolved from the degradation products’ peaks with significantly different retention time (Rt), and the resolution factor for the R and S isomers of CP was found to be greater than 2. Ceema Mathew, M. Ajitha, and P. R. Sathesh Babu Copyright © 2013 Ceema Mathew et al. All rights reserved. Metal Species in Biology: Bottom-Up and Top-Down LC Approaches in Applied Toxicological Research Mon, 10 Jun 2013 10:42:12 +0000 Since the inception of liquid chromatography (LC) more than 100 years ago this separation technique has been developed into a powerful analytical tool that is frequently applied in life science research. To this end, unique insights into the interaction of metal species (throughout this manuscript “metal species” refers to “toxic metals, metalloid compounds, and metal-based drugs” and “toxic metals” to “toxic metals and metalloid compounds”) with endogenous ligands can be obtained by using LC approaches that involve their hyphenation with inductively coupled plasma-based element specific detectors. This review aims to provide a synopsis of the different LC approaches which may be employed to advance our understanding of these interactions either in a “bottom-up” or a “top-down” manner. In the “bottom-up” LC-configuration, endogenous ligands are introduced into a physiologically relevant mobile phase buffer, and the metal species of interest is injected. Subsequent “interrogation” of the on-column formed complex(es) by employing a suitable separation mechanism (e.g., size exclusion chromatography or reversed-phase LC) while changing the ligand concentration(s), the column temperature or the pH can provide valuable insight into the formation of complexes under near physiological conditions. This approach allows to establish the relative stability and hydrophobicity of metal-ligand complexes as well as the dynamic coordination of a metal species (injected) to two ligands (dissolved in the mobile phase). Conversely, the “top-down” analysis of a biological fluid (e.g., blood plasma) by LC (e.g., using size exclusion chromatography) can be used to determine the size distribution of endogenous metalloproteins which are collectively referred to as the “metalloproteome”. This approach can provide unique insight into the metabolism and the plasma protein binding of metal species, and can simultaneously visualize the dose-dependent perturbation of the metalloproteome by a particular metal species. The concerted application of these LC approaches is destined to provide new insight into biochemical processes which represent an important starting point to advance human health in the 21st century. Jürgen Gailer Copyright © 2013 Jürgen Gailer. All rights reserved. Profiling of Phytochemicals in Tissues from Sclerocarya birrea by HPLC-MS and Their Link with Antioxidant Activity Wed, 05 Jun 2013 08:37:24 +0000 High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was employed to investigate the differences in phytochemicals in roots, bark, and leaf of Sclerocarya birrea (marula) for methanol and water extracts that exhibited the best antioxidant activities. As many as 36 compounds were observed in the extracts of these tissues of which 27 phenolic compounds were tentatively identified. The HPLC-MS/MS results showed flavonoid glycosides were prominent in leaf extracts while the galloylated tannins were largely in bark and root extracts. Four flavonoid glycosides that were reported for the first time in the marula leaf have been identified. The HPLC-MS/MS studies also illustrated different degrees (highest degree = 3) of oligomerisation and galloylation of tannins in the bark and root extracts. Daniela Russo, Owen Kenny, Thomas J. Smyth, Luigi Milella, Mohammad B. Hossain, Moussoukhoye Sissokho Diop, Dilip K. Rai, and Nigel P. Brunton Copyright © 2013 Daniela Russo et al. All rights reserved. Development and Validation of a Stability Indicating RP-HPLC Method for the Determination of Two Sun Protection Factors (Koptrizon and Tinosorb S) in Topical Pharmaceutical Formulations Using Experimental Designs Sat, 25 May 2013 11:57:17 +0000 A novel, simple, validated stability indicating HPLC method was developed for determination of Koptrizon and Tinosorb S. Stability indicating power of the method was established by forced degradation study. The chromatographic separation was achieved with Waters X Bridge column, by using mobile phase consisting of a mixture of acetonitrile : tetrahydrofuran : water (38 : 38 : 24, v/v/v). The method fulfilled validation criteria and was shown to be sensitive, with limits of detection (LOD) and quantitation (LOQ) of 0.024 and 0.08 μg  for Koptrizon and 0.048 and 0.16 μg  for Tinosorb S, respectively. The developed method is validated for parameters like precision, accuracy, linearity, solution stability, specificity, and ruggedness as per ICH norms. Design expert with ANOVA software with linear model was applied and a 23 full factorial design was employed to estimate the model coefficients and also to check the robustness of the method. Results of the two-level full factorial design, 23 with 10 runs including two-centre-point analysis based on the variance analysis (ANOVA), demonstrated that all three factors, as well as the interactions between retention time of Koptrizon, Tinosorb S, and USP plate count for Koptrizon, are statistically significant. Chinmoy Roy and Jitamanyu Chakrabarty Copyright © 2013 Chinmoy Roy and Jitamanyu Chakrabarty. All rights reserved. Thermodynamic and Extrathermodynamic Studies of Enantioseparation of Imidazolinone Herbicides on Chiralcel OJ Column Thu, 16 May 2013 09:08:19 +0000 A homologous series of chiral imidazolinone herbicide was previously resolved on Chiralcel OJ column in high performance liquid chromatography. However, the mechanism of the chiral separation remains unclear. In this study, chromatographic behaviors of five chiral imidazolinone herbicides were characterized by thermodynamic and extrathermodynamic methods in order to enhance the understanding of the chiral separation. Thermodynamic parameters of this study were derived from equilibrium constant () that was estimated from the moment analysis of the chromatographic peak. Van't Hoff plots of ( versus ) were linear at a range of 15–50°C, only nonlinear at a range of 5–15 °C with n-hexane (0.1%, trifluoroacetic acid)-2-propanol 60/40 (v/v) mobile phase. The enantiomer retention on the chiral column was entropy-driven at a lower temperature (5°C) and enthalpy-driven at a higher temperature (10 to 50°C). Enantioseparations of four of the five imidazolinone herbicides were enthalpy-driven, only entropy-driven for imazaquin. Enantioseparation mechanisms were different in between 5–10°C and 15–50°C probably due to the conformational change of the OJ phase. Enthalpy-entropy compensation showed similar mechanisms in retention and chiral separation for the five or enantiomers. Several extrathermodynamic relationships were able to be extracted to address additivity of group contribution. Wenjian Lao Copyright © 2013 Wenjian Lao. All rights reserved. Development and Validation of High Performance Liquid Chromatographic Analysis of Residual N,N-Dimethylformamide in Spent Medium after Biodegradation by Paracoccus denitrificans SD1 Tue, 14 May 2013 19:17:38 +0000 N,N-Dimethylformamide (DMF) is a toxic organic solvent commonly found in the textile and pharmaceutical industrial effluents. The DMF degradation was successfully archived by bacterial strain Paracoccus denitrificans SD1. The study demonstrates the high performance liquid chromatographic (HPLC) approach for the estimation of residual DMF in liquid medium. The investigation mainly focuses on the method development for the detection and quantification of DMF. The bacterium is capable of utilizing DMF (1% v/v) as the sole source of carbon and nitrogen. Utilization of DMF by the bacterium was investigated at regular intervals of time to check the complete degradation at a particular period. The method was validated based on the precision, accuracy, limit of detection, and limit of quantification. Herein, the method was executed in liquid chromatographic condition which enables direct analysis of aqueous samples from the spent medium avoiding the extraction and prederivatization. This improved method allows estimation of residual DMF from the aqueous medium in adequate ranges of precision and accuracy with 99.17% and 99.43% recovery, respectively. The method was validated by investigating the limit of detection (LOD) and limit of quantification (LOQ) of 0.2 and 0.40 mg/l, respectively. The method was found to be precise for detection of DMF by using liquid chromatography. Sanjeevkumar Sanganal, Guruprasad B. Kulkarni, and Timmanagouda B. Karegoudar Copyright © 2013 Sanjeevkumar Sanganal et al. All rights reserved. Estimation of Abamectin Residues Present in Tea: High-Performance Liquid Chromatography Technique Thu, 18 Apr 2013 15:32:14 +0000 A simple, reliable, and sensitive method was based on high-performance liquid chromatography (HPLC) was developed and validated for the estimation of abamectin residues present in tea. The abamectin residues extracted with acetone-water mixture (70 : 30, v/v) and derivatised with 1-methylimidazole (1-MIM) and trifluoroacetic anhydride (TFAA) were estimated by HPLC using fluorescence detector (FLD). The technique was validated in terms of linearity, precision, recovery, specificity, limit of detection (LOD), and limit of quantification (LOQ). A good linear relationship () was absorbed in the abamectin concentration range from 0.01 to 1.0 μg mL−1. The limit of detection and limit of quantification of the method were 0.01 and 0.02 μg g−1, respectively. The average recoveries of the pesticide from black tea and dried green leaves ranged from 83.3 to 103.8% and 83.8 to 98.0%, respectively. Madasamy Kottiappan, Shanmugaselvan Veilumuthu Anandhan, and Selvaganapathi Chandran Copyright © 2013 Madasamy Kottiappan et al. All rights reserved. Quality by Design Approach for the Development and Validation of Glipizide, an Antidiabetic Drug, by RP-UPLC with Application to Formulated Forms and Urine Wed, 27 Feb 2013 10:55:12 +0000 Quality by design (QbD) refers to the achievement of certain predictable quality with desired and predetermined specifications. The objective of this study was to develop and demonstrate an integrated multivariate approach to develop and quantify the constituent concentrations of glipizide (GPZ) drug in its pure and tablet forms. The method was developed using Zorbax Extend C-18 (50 mm × 4.6 mm × 1.8 μm) column with mobile phase consisting of a mixture of phosphate buffer of pH 3.5 and acetonitrile (60 : 40 v/v). The method fulfilled validation criteria and was shown to be sensitive, with limits of detection (LOD) and quantitation (LOQ) of 0.001 and 0.005 μg mL−1, respectively. The percentage relative standard deviations for robustness and ruggedness were observed within the range of 0.1 and 0.99. The calibration graph was linear in the range of 0.005–300 μg mL−1. The applicability of the method was shown by the analysis of formulated drug and spiked urine samples. The proposed method can be used for routine analysis in quality control laboratories for its bulk and formulated product, and this is the first UPLC method reported for the assay of GPZ in bulk, formulated form and urine. Cijo M. Xavier, Kanakapura Basavaiah, K. B. Vinay, and N. Swamy Copyright © 2013 Cijo M. Xavier et al. All rights reserved. Determination of Ranitidine in Human Plasma by SPE and ESI-LC-MS/MS for Use in Bioequivalence Studies Mon, 04 Feb 2013 16:16:20 +0000 A method for determining ranitidine in human plasma by ESI-LC-MS/MS was validated, using propranolol as internal standard. The extraction method used was solid phase extraction (SPE). Chromatographic separation was performed in a Chromolith C18 (50 mm × 4.6 mm i.d.) analytical column, which provided good separation of ranitidine and propranolol peaks with an analysis time of 2.5 minutes. Extraction yields of 94.4% for ranitidine and 89.4% for the internal standard were obtained. The lower limit of quantification (LLOQ) was 3.00 ng/mL, and limit of detection (LOD) was 0.05 ng/mL, with linearity ranging from 3.00 to 500 ng/mL. The results, thus, showed that this method is suitable for application in bioequivalence studies of ranitidine in human plasma. Karini B. Bellorio, Maria Isabel R. Alves, and Nelson R. Antoniosi Filho Copyright © 2013 Karini B. Bellorio et al. All rights reserved. A Validated Stability-Indicating RP-HPLC Method for the Simultaneous Determination of Tenofovir, Emtricitabine, and a Efavirenz and Statistical Approach to Determine the Effect of Variables Mon, 28 Jan 2013 10:25:23 +0000 A simple, rapid, and stability-indicating RP-HPLC method for a combination of tenofovir disoproxil fumarate (TDF), emtricitabine (FTC), and efavirenz (EFV) was developed and validated with the help of a suitable statistical software as an application tool for the quality by design. The drugs individually, and in combination, were subjected to forced degradation (thermal, photolytic, hydrolytic, and oxidative stress conditions) and accelerated stability studies (40 ± 1°C/75 ± 3% RH for three months). Successful separation of combined drugs from degradation products was achieved by gradient elution on a reverse-phase C18 column, using a mobile phase containing phosphate buffer (pH 3.5): acetonitrile at 1.5 mL min−1 flow rate, detection wavelength 256 nm, column oven temperature 25°C, and injection volume 10 μL. Linearity was established in the range of 20–300 μg mL−1, 24.5–367.5 μg mL−1 and 60–900 μg mL−1 for FTC, TDF, and EFV, respectively. The method was successfully applied for quantifying the drugs in marketed dosage forms and on stability samples. Prashant S. Devrukhakar, Roshan Borkar, Nalini Shastri, and K. V. Surendranath Copyright © 2013 Prashant S. Devrukhakar et al. All rights reserved. Isolation and TLC Densitometric Quantification of Lysergol from the Seeds of Ipomoea muricata (Linn.) Jacq. Tue, 22 Jan 2013 15:15:46 +0000 Seeds of Ipomoea muricata, well known in Ayurveda for its purgative action, contains mainly indole alkaloids. Lysergol (major alkaloid) exhibits hypotensive, psychotropic, and uterus and intestine-stimulating properties. TLC fingerprint profile of I. muricata seeds was developed using chloroform : methanol (95 : 5 v/v) as the mobile phase. Plate was visualized under UV 254 nm and UV 366 nm and after derivatization with Van Urk reagent. Lysergol resolved at . Further, TLC-densitometric method was developed and validated for quantification of Lysergol avoiding derivatization step. Ethyl acetate :  methanol (7 : 3 v/v) was used as the mobile phase. Linear regression analysis data for the calibration curve showed a good linear relationship () in the concentration range from 20 ng to 140 ng, with respect to the peak area. The developed method was precise with RSD for intraday (range from 1.20 to 1.89) and interday (range from 1.39 to 1.92) for 60, 80, and 100 ng/spot of Lysergol. The instrumental precision was 0.67 (% RSD). The limit of detection and limit of quantification for Lysergol were 12 ng and 40 ng, respectively. The average percentage recovery was 99.68. The amount of Lysergol was found to be 0.23% w/w. To the best of our knowledge, this is the first report for the quantification of Lysergol from I. muricata seeds without derivatization. Shrikant Patil, Manish Nivsarkar, and Sheetal Anandajiwala Copyright © 2013 Shrikant Patil et al. All rights reserved. On the Combined Application of Iatroscan TLC-FID and GC-FID to Identify Total, Neutral, and Polar Lipids and Their Fatty Acids Extracted from Foods Tue, 15 Jan 2013 11:53:30 +0000 An efficient separation and quantification of the individual neutral and polar lipid classes and their constituent fatty acids was achieved by the combination of two different detection techniques: Iatroscan TLC-FID and GC-FID. The solvent composition and ratio of development system, the sample size, the fidelity, and precision were tested in order to estimate the effectiveness of separation of individual neutral and polar lipid classes and the quantitative reproducibility of the Iatroscan TLC-FID technique. GC-FID method, with a high-quality capillary column, allowed sensitive and reproducible fatty acid qualitative and quantitative analyses, separation of fatty acid structural isomers (e.g., n-C16:0, iso-C16:0 and anteiso-C16:0), positional isomers (e.g., C18:1ω-9 and C18:1ω-7), geometrical isomers (cis-trans), and homologues (e.g., C16:0, C17:0, C18:0, etc.) in standards and complex lipid samples. Seventeen (17) lipid classes and fifty-two (52) saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA) fatty acids were identified and quantified, respectively, in samples of standard lipid and fatty acid mixtures, simulating the composition of natural lipids and their fatty acid methyl esters in common foods. The wide number of applications establishes this combination of Iatroscan TLC-FID and GC-FID methods as a powerful tool for lipid class and fatty acid analysis of any fat origin. Vassilia J. Sinanoglou, Irini F. Strati, Sotirios M. Bratakos, Charalampos Proestos, Panagiotis Zoumpoulakis, and Sofia Miniadis-Meimaroglou Copyright © 2013 Vassilia J. Sinanoglou et al. All rights reserved. Implementation of Quality by Design for the Development and Validation of Pioglitazone Hydrochloride by RP-UPLC with Application to Formulated Forms Wed, 19 Dec 2012 13:02:45 +0000 Quality by Design (QbD) is a philosophy that refines the level of knowledge associated with a product that uses process understanding to deliver a product with the desired critical quality attributes. The objective of this study was to develop an integrated multivariate QbD approach to develop and quantify the constituent concentrations of pioglitazone hydrochloride (PGZ) drug in its pure and formulated forms. To facilitate studies investigating the determination of PGZ in bulk drug and its pharmaceutical formulations, a rapid UPLC method was developed and validated for the determination of PGZ accompanied by its degradation studies in different stress conditions. The method fulfilled validation criteria and was shown to be sensitive, with limits of detection (LOD) and quantitation (LOQ) of 0.01 and 0.05 μg mL−1, respectively. The percent relative standard deviations for robustness and ruggedness were observed within the range of 0.1–1.74. The calibration graph was linear in the range of 0.05–300 μg mL−1. The applicability of the method was shown by analysis of formulated drug samples and spiked human urine. The proposed method can be used for routine analysis in quality controlled laboratories for its bulk and formulated product and this is the first reported UPLC method for the assay of PGZ. Cijo M. Xavier and Kanakapura Basavaiah Copyright © 2012 Cijo M. Xavier and Kanakapura Basavaiah. All rights reserved. An Isocratic Method for Quantification of Valproic Acid and Its Related Impurities Using Ion Pair Reagent by Ultraperformance Liquid Chromatography Mon, 17 Dec 2012 13:18:49 +0000 A selective ultraperformance liquid chromatographic (UPLC) method for the quantification of valproic acid and its known related impurities using ion pair reagent has been developed. The method includes reversed-phase Acquity HSS T3 column with 100 mm × 2.1 mm i.d. and 1.7 μ particle size. The mobile phase consists of acetonitrile, 5 mM 1-hexanesulphonic acid sodium salt, flow rate is 0.6 mL/min, and UV detection is performed at 215 nm. A system suitability test (SST) was developed to govern the quality of the separation. The developed method has been validated further with respect to linearity, accuracy, precision, selectivity, LOD, LOQ, and Robustness. Rakshit Thakkar, Hitesh Saravaia, Mrunal Ambasana, Madhavi Patel, and Anamik Shah Copyright © 2012 Rakshit Thakkar et al. All rights reserved. Assessment of Pseudoaffinity Chromatography Using Textile Dyes for Isolation of Buffalo Pituitary Luteinizing Hormone Sun, 16 Dec 2012 09:24:55 +0000 Extensive investigation has been carried out to elucidate the mechanisms involved in pseudoligand affinity chromatography using textile dyes, and, empirically, it has been attributed to the chemical and steric structures of dye and protein. Possibly, a variety of interactions especially ionic and/or hydrophobic influence with a varying share in the binding and differ from protein to protein and from dye to dye. In this study, we have attempted to understand the effect of various biophysical parameters like the nature of the eluant, pH, and ionic strength on the binding of crude luteinizing hormone (LH) with various triazine-based dyes and thus predict their nature. Based on the elution patterns, cibacron and reactive brown suggested a dual electrostatic and hydrophobic nature. Reactive blue and reactive yellow reflected a major electrostatic/ionic nature with yellow offering 50-fold purification in a single step, while reactive red and reactive green had a predominant hydrophobic nature. Appreciably, reactive red was binding LH very tightly unlike other dyes, and addition of the arginine in the elution buffer substantially weakened the protein-dye interactions. pH was observed to be a principal factor assisting the protein-dye binding as well as hydrophobicity of the dye and the proteins. Taruna Arora, Pankaj Patel, and K. Muralidhar Copyright © 2012 Taruna Arora et al. All rights reserved. Using Size-Exclusion Chromatography to Monitor Variations in the Sizes of Microwave-Irradiated Gold Nanoparticles Thu, 13 Dec 2012 15:17:38 +0000 Size-exclusion chromatography (SEC) was used to evaluate gold nanoparticles (Au NPs) for variations in their sizes after microwave (MW) irradiation, with the eluted NPs monitored through diode array detection to reveal their surface plasmon absorptions. The sizes of citrate-capped Au NPs decreased upon increasing the MW irradiation temperature, consistent with digestive ripening of these NPs under the operating conditions. In contrast, Au NPs capped with sodium dodecyl sulfate increased in size upon increasing the MW irradiation temperature, consistent with Ostwald ripening. When the Au NPs were capped with 3A-amino-3A-deoxy-(2AS,3AS)--cyclodextrin (H2N--CD), however, their dimensions were barely affected by the MW irradiation temperature, confirming that H2N--CD is a good stabilizer against MW irradiation. Therefore, SEC—with its short analysis times, low operating costs, automated operation, and in situ analysis—has great potential for use in the rapid monitoring of NPs subjected to treatment under various MW irradiation conditions. Fu-Ken Liu Copyright © 2012 Fu-Ken Liu. All rights reserved. Application of HPLC for the Simultaneous Determination of Paracetamol, Chlorzoxazone, and Nimesulide in Pharmaceutical Dosage Form Tue, 11 Dec 2012 14:48:19 +0000 A simple, precise, and accurate reversed-phase liquid chromatographic method has been developed for the simultaneous determination of paracetamol (PCM), chlorzoxazone (CHZ), and nimesulide (NIM) in pharmaceutical dosage form. The chromatographic separation was achieved on a Thermo Hypersil GOLD C18 column (250 × 4.6 mm i.d., 5 μm particle size). The mobile phase consisted of water : acetonitrile (55 : 45 v/v). The flow rate was set to 1.2 mL min−1 and UV detection was carried out at 275 nm. The retention time () for PCM, CHZ, and NIM was found to be 2.69 ± 0.02, 4.61 ± 0.01, and 9.55 ± 0.02 min, respectively. The validation of the proposed method was carried out for linearity, precision, robustness, limit of detection, limit of quantitation, specificity, and accuracy. The linear dynamic ranges were 32.5–65.0 μg mL−1 for PCM, 37.5–75.0 μg mL−1 for CHZ, and 10.0–20.0 μg mL−1 for NIM. The developed method can be used for routine quality control analysis of titled drugs in pharmaceutical dosage form. Snehal J. More, Suparna S. Tandulwadkar, Ajinkya R. Nikam, Atul S. Rathore, L. Sathiyanarayanan, and Kakasaheb R. Mahadik Copyright © 2012 Snehal J. More et al. All rights reserved. Application of a Reliable LC-MS/MS Method for Determination of Rizatriptan in Healthy Subject Samples: Demonstration of Assay Reproducibility by Incurred Sample Reanalysis Mon, 10 Dec 2012 16:09:50 +0000 A reliable, rapid, and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been proposed for the determination of rizatriptan in human plasma using sumatriptan as internal standard (IS). The analyte and IS were extracted from 300 μL human plasma via liquid-liquid extraction and the chromatography was achieved on Hypurity C18 (50 mm × 4.6 mm, 5 μm) column under isocratic conditions. Detection of rizatriptan and IS was done by tandem mass spectrometry, operating in positive ionization and multiple-reaction monitoring mode. The limit of detection and lower limit of quantitation of the method were 0.04 and 0.20 ng/mL, respectively, with a linear dynamic range of 0.20–60.0 ng/mL. The intrabatch and interbatch precision (% CV) was ≤8.4% while the mean extraction recovery was >78% across quality control levels. Bench top stability, freeze and thaw stability, processed sample stability, and long-term stability in plasma were evaluated at two quality control levels. It was successfully applied to a bioequivalence study of 10 mg rizatriptan orally disintegrating tablet formulation in 40 and 32 healthy Indian male subjects under fasting and fed conditions, respectively. The reproducibility in the measurement of study data was demonstrated by reanalysis of 254 incurred samples. Dinesh S. Patel, Naveen Sharma, Mukesh C. Patel, Bhavin N. Patel, Pranav S. Shrivastav, and Mallika Sanyal Copyright © 2012 Dinesh S. Patel et al. All rights reserved. Dried Blood Spot Sampling with LC-MS Analysis for Routine Therapeutic Caffeine Monitoring in Neonates Tue, 20 Nov 2012 10:07:25 +0000 A liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the determination of therapeutic levels of caffeine in dried blood spot (DBS) samples. Caffeine is used in the treatment of Apnoea of Prematurity (AoP) in newborn children. Calibration DBS samples were prepared by spotting 15 μL of whole blood spiked with the analyte onto specimen collection cards. 3 mm disks cut from the centre of the DBS were extracted in methanol containing the internal standard. The extract was separated using a Zorbax Eclipse Plus C18 column and the MS, operated in electrospray positive ion mode, used single ion monitoring at m/z 195 for caffeine and m/z 198 for the IS. The overall extraction recovery of caffeine from spiked blood spots was demonstrated to be 44–47%. Validation of the microanalytical method showed good precision (coefficient of variation) and accuracy (relative error) and specificity and was linear within the tested calibration range 500–25000 ng/mL for caffeine. Investigation of different specimen collection papers revealed different matrix effects with significant ion suppression from the FTA Elute paper itself. Requiring only a microvolume (15 μL) blood sample for analysis, the developed DBS based microanalytical method has the potential to facilitate the routine monitoring of caffeine in neonates. Graham Lawson, Parul Patel, Hussain Mulla, and Sangeeta Tanna Copyright © 2012 Graham Lawson et al. All rights reserved. Improved Chromatographic Methods for Determination of Bioactive Compounds from Aloe vera Leaves Wed, 14 Nov 2012 10:13:29 +0000 In this work three chromatographic methods were developed to reduce the total time of the analysis of main compounds in Aloe vera extracts. The first method was developed in a regular reverse phase chromatographic system using a particulate reverse phase C-18 column. Methods already published were used as a starting point for the development of the new method. All the compounds were separated in 32 minutes. The second method was developed in a regular reverse phase chromatographic system employing a monolithic type column. Using a 4.5 mL min−1 flow, the total time of analysis was reduced to 6 minutes with very similar resolution values. The third method was developed in an ultraperformance liquid chromatographic system, and the final time for the analysis of the phenolic compounds was reduced to 4 minutes. The analytical properties of the three chromatographic methods were compared for the main compounds in the chromatograms. Robustness of the three new methods was also checked with regard to the injection volume and the amount of methanol in the sample. A fast method (4 min) is then available for bioactive compounds from Aloe vera determination. L. Azaroual, A. Liazid, G. F. Barbero, J. Brigui, M. Palma, and C. G. Barroso Copyright © 2012 L. Azaroual et al. All rights reserved. Development and Validation of Stability-Indicating RP-UPLC Method for the Determination of Methdilazine in Bulk Drug and in Pharmaceutical Dosage Form Thu, 01 Nov 2012 12:04:38 +0000 A simple, precise, and accurate, and stability-indicating isocratic Ultraperformance Liquid Chromatography (UPLC) method was developed for the determination of methdilazine hydrochloride (MDH) in bulk drug and in its tablets. The use of UPLC, with a rapid 5-minute-reversed-phase isocratic separation on a 1.7 μm reversed-phase packing material to provide rapid ‘‘high throughput’’ support for MDH, is demonstrated. The method was developed using Waters Acquity BEH C18 column (100 mm × 2.1 mm, 1.7 μm) with mobile phase consisting of a mixture of potassium dihydrogenorthophosphate and 1-pentane sulphonic acid buffer of pH 4.0 and acetonitrile (60 : 40 v/v). The eluted compound was detected at 254 nm with a UV detector. The standard curve of mean peak area versus concentration showed an excellent linearity over a concentration range 0.5–80 μg mL−1 MDH with regression coefficient () value of 0.9999. The limit of detection () was 0.2 μg mL−1 and the limit of quantification () was 0.5 μg mL−1. Forced degradation of the bulk sample was conducted in accordance with the ICH guidelines. Acidic, basic, hydrolytic, oxidative, thermal, and photolytic degradations were used to assess the stability indicating power of the method. The drug was found to be stable in acidic, basic, thermal, hydrolytic, and photolytic stress conditions and showed slight degradation in oxidative stress condition. Madihalli S. Raghu, Kanakapura Basavaiah, Cijo M. Xavier, and Kudige N. Prashanth Copyright © 2012 Madihalli S. Raghu et al. All rights reserved. Development and Validation of RP-HPLC Method for the Determination of Ganciclovir in Bulk Drug and in Formulations Sun, 08 Jul 2012 08:18:30 +0000 A simple, rapid, accurate, and precise gradient reversed-phase HPLC (RP-HPLC) method has been developed for the determination of ganciclovir (GNC) in pharmaceuticals. Chromatographic separation was carried out on inertsil ODS C18 (4.6 mm i.d×250 mm, 5.0 μm) LC column using ammonium acetate buffer, sodium salt of hexane sulfonic acid as ion-pairing reagent in 1000 mL water, and acetonitrile (90 : 10) (v/v) as mobile phase at a flow rate of 1.0 mL min−1 and with UV detection at 245 nm at column temperature (30°C). The runtime under these chromatographic conditions was 10 min. The method was linear over the range of 0.02–75 μg mL−1. The limits of detection (LOD) and quantification (LOQ) values were 4.1 and 20 ng mL−1, respectively. The method was successfully extended to study the effect on GNC upon treatment with 2 N NaOH, 2N HCl, and 5% H2O2 for 2 hrs at 80°C and upon exposure to UV (1200 K lux hrs) for 72 hrs and thermal (105°C) for 5 hrs. The proposed method was further applied to the determination of GNC in pharmaceuticals, with good percent recovery. The accuracy and the precision of the method were validated on intraday and interday basis in accordance with ICH guidelines. P. J. Ramesh, K. Basavaiah, K. B. Vinay, and Cijo M. Xavier Copyright © 2012 P. J. Ramesh et al. All rights reserved. Chromatographic Removal of Endotoxins: A Bioprocess Engineer's Perspective Thu, 05 Jul 2012 10:17:45 +0000 Gram-negative bacteria are widely used for the production of gene-based products such as DNA vaccines and bio-drugs, where endotoxin contamination can occur at any point within the process and its removal is of great concern. In this article, we review the structures of endotoxin and the effects that it causes in vivo. The endotoxin removal strategies are also discussed in the light of the different interaction mechanisms involved between endotoxins and bioproducts particularly plasmid DNA and proteins. For most cases, endotoxin removal is favoured at a highly ionic or acidic condition. Various removal methods particularly chromatography-based techniques are covered in this article according to the relevant applications. Clarence M. Ongkudon, Jia Han Chew, Boyin Liu, and Michael K. Danquah Copyright © 2012 Clarence M. Ongkudon et al. All rights reserved. Optimization of SPE/GC/HPLC Analytical Procedure for Determination of Phenol, Quinones, and Carboxylic Acids in Water Samples Mon, 18 Jun 2012 10:12:20 +0000 Chromatographic techniques are among the most useful analytical methods. Gas and liquid chromatography were used in the analysis of some organic compounds: phenol, hydroquinone, benzoquinone, and maleic and fumaric acids. The analytical way for the determination of these compounds in water samples was investigated. Solid-phase extraction (SPE) technique was used on the sample preparation step, different divinylbenzene-based sorbents were applied. Calibration curves of given compounds were linear over the ranges: 50–500 μg/mL for phenol and its acetic derivatives, 50–1500 μg/mL for benzoquinone in GC analysis, and 50–250 μg/mL for phenol, 40–1000 μg/mL for hydroquinone, and 4–4500 μg/mL for carboxylic acids in HPLC analysis. The LOD and LOQ of proposed analytical procedure were in the ranges of LOD: 0.042–23.83 μg/mL; LOQ: 0.138–78.64 μg/mL. Katarzyna Bielicka-Daszkiewicz, Monika Hadzicka, and Adam Voelkel Copyright © 2012 Katarzyna Bielicka-Daszkiewicz et al. All rights reserved. A Novel Application of Natural Peat for Solid-Phase Extraction of Pyrimethanil, Flumetralin, and Kresoxim-Methyl in Water Using Gas Chromatography-Mass Spectrometry Thu, 14 Jun 2012 15:01:22 +0000 Natural peat was tested for extraction of pyrimethanil, flumetralin, and krexosim-methyl from water, with analysis using gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring mode (SIM). Experiments were carried out at one fortification level (0.1 μg L−1) and resulted in recoveries in the range 41–96%, with RSD values between 6.8 and 12.6% for natural peat as sorbent. Detection and quantification limits ranged from 0.02 to 0.05 μg L−1 and from 0.07 to 0.1 μg L−1, respectively, for the different pesticides studied. The method developed was linear over the range tested (0.07–4.0 μg L−1), with correlation coefficients ranging from 0.9919 to 0.9989. Comparison between peat and commercial sorbents (C18-bonded silica, ENVI-Carb, Florisil, silica gel, ENVI-Carb/LC-NH2) showed better performance of peat sorbent for flumetralin and kresoxim-methyl. Alex S. M. S. J. Santos, Adriano Aquino, Luciane P. C. Romão, and Sandro Navickiene Copyright © 2012 Alex S. M. S. J. Santos et al. All rights reserved.