Chromatography Research International The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. Development and Validation of a Rapid Chemometrics Assisted RP-HPLC with PDA Detection Method for the Simultaneous Estimation of Pyridoxine HCl and Doxylamine Succinate in Bulk and Pharmaceutical Dosage Form Wed, 23 Apr 2014 14:13:40 +0000 Simple, rapid, precise, and accurate RP-HPLC method was developed and optimized with the help of chemometric tool for the simultaneous estimation of pyridoxine HCl and doxylamine succinate in bulk and pharmaceutical dosage form. Optimization was done by central composite design in response surface methodology. Based on the trial and error, percentage of organic phase (methanol) in mobile phase, flow rate, and molarity of the buffer were selected as factors. Resolution and retention time were used for the estimation of system response during the optimization procedure. The optimized condition was used and the separation was carried out on phenomenex C18 column (150 × 4.6 mm; i.d, 5 μ particle size) using the mobile phase containing 49.37% of methanol and 50.63% of phosphate buffer (45.14 mM) at a flow rate of 1 mL/min. Retention time was found to be 1.884 minutes for pyridoxine HCl and 3.959 minutes for doxylamine succinate. The calibration curves were found to be linear from 10 to 70 μg/mL and 10 to 90 μg/mL for pyridoxine HCl and doxylamine succinate with their correlation coefficient values 0.9995 and 0.9997. LOD and LOQ were found to be 23.5 ng/mL and 71.1 ng/mL for pyridoxine HCl and 99.9 ng/mL and 302.6 ng/mL for doxylamine succinate. P. Giriraj and T. Sivakkumar Copyright © 2014 P. Giriraj and T. Sivakkumar. All rights reserved. A Stability-Indicating High Performance Liquid Chromatographic Assay for the Simultaneous Determination of Pyridoxine, Ethionamide, and Moxifloxacin in Fixed Dose Combination Tablets Wed, 23 Apr 2014 10:02:33 +0000 Stability indicating reversed phase HPLC method was developed and validated for the simultaneous quantitation of antitubercular drugs, ethionamide (ETH), and moxifloxacin (MOX) with commonly coprescribed vitamin, pyridoxine (PYR) in tablet dosage form. The method was found rapid, precise and accurate. The separation was performed in Hibar 150-4.6, Purospher STAR, RP-18e (5 μm) column, using mobile phase A (0.03 M sodium citrate adjusted to pH 5 with glacial acetic acid) and mobile phase B (100% methanol), ran at variable proportions at flow rate of 1.0 mL/min. The detection was carried out at 320 nm. The method was observed linearly in the range of 2.5–17.5 μg/mL for PYR, 25–175 μg/mL for ETH, and 40–280 μg/mL for MOX with respective limits of detection/quantitation of 0.125 μg/mL/1.28 μg/mL, 0.25 μg/mL/2.56 μg/mL, and 0.35 μg/mL/3.65 μg/mL. The drugs were also subjected to oxidative, hydrolytic, photolytic, and thermal degradation; the degradation products showed interference with the detection of PYR, ETH, and MOX. The proposed method was observed to be effective to quantitate MOX (400 mg), ETH (250 mg), and PYR (25 mg) in fixed dose combination tablet formulation. Munib-ur-Rehman, Rabia Ismail Yousuf, and Muhammad Harris Shoaib Copyright © 2014 Munib-ur-Rehman et al. All rights reserved. Development and Validation of a Novel RP-HPLC Method for Estimation of Losartan Potassium in Dissolution Samples of Immediate and Sustained Release Tablets Wed, 09 Apr 2014 11:36:01 +0000 A simple, rapid, selective, and reproducible reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for the estimation of Losartan potassium in dissolution samples of Losartan potassium immediate and sustained release tablets. Analysis was performed on an Agilent, Zorbax Eclipse XDB C18 column (150 mm × 4.6 mm, 5 μm) with the mobile phase consisting of orthophosphoric acid (0.1% v/v)—acetonitrile (55 : 45, v/v) at a flow rate of 1.0 mL/min. UV detection was performed at 225 nm and the retention time for Losartan was about 2.6 minutes. The calibration curve was linear (correlation coefficient = 0.999) in the selected range of analyte. The optimized dissolution conditions include the USP apparatus 2 at a paddle rotation rate of 50 rpm and 900 mL of pH 6.8 phosphate buffer as dissolution medium, at ∘C. The method was validated for precision, linearity, specificity, accuracy, limit of quantitation, and ruggedness. The system suitability parameters, such as theoretical plate, tailing factor and relative standard deviation (RSD) between five standard replicates, were well within the limits. The stability result shows that the drug is stable in the prescribed dissolution medium. Harshal A. Pawar and K. G. Lalitha Copyright © 2014 Harshal A. Pawar and K. G. Lalitha. All rights reserved. Stability Indicating Liquid Chromatographic Method for Estimation of Trihexyphenidyl Hydrochloride and Risperidone in Tablet Formulation: Development and Validation Consideration Wed, 19 Mar 2014 08:40:26 +0000 This paper describes validated reverse phase high-performance liquid chromatographic (RP-HPLC) method for simultaneous estimation of trihexyphenidyl hydrochloride (THP) and risperidone (RSP) in the pure powder form and in combined tablet dosage form. The HPLC separation was achieved on a core shell C18 (100 mm length × 4.6 mm, 2.6 μm particle size) using methanol : ammonium acetate buffer 1% (85 : 15 v/v; pH-6.5) as mobile phase and delivered at flow rate of 0.8 mL/min. The calibration plot showed good linear relationship with r2 = 0.997 ± 0.001 for THP and r2 = 0.998 ± 0.001 for RSP in concentration range of 50–175 μg/mL and 50–175 μg/mL, respectively. LOD and LOQ were found to be 0.40 and 1.29 μg/mL for THP and 1.24 and 3.92 μg/mL for RSP. Assay of THP and RSP was found to be 100.16 ± 0.03% and 99.83 ± 0.02%, respectively. THP and RSP were subjected to different stress conditions (acidic, basic, oxidative, thermal, and photolytic degradation). The degraded product peaks were well resolved from the pure drug peak. The method was successfully validated as per the ICH guidelines. The developed RP-HPLC method was successfully applied for the estimation of THP and RSP in tablet dosage form. Patel Bhaumik, Gopani Mehul, Vikani Kartik, Patel Rashmin, and Patel Mrunali Copyright © 2014 Patel Bhaumik et al. All rights reserved. Estimation of Diafenthiuron Residues in Cardamom (Elettaria cardamomum (L.) Maton) Using Normal Phase HPLC: Dissipation Pattern and Safe Waiting Period in Green and Cured Cardamom Capsules Mon, 24 Feb 2014 08:08:09 +0000 Diafenthiuron is an effective insecticide used for pest management in cardamom. Residues of diafenthiuron and its degradation/dissipation pattern in cardamom were determined to work out safe waiting period. Samples were collected after three sprays of diafenthiuron @ 400 and 800 g a.i ha−1 and the residues extracted in acetonitrile and quantified in normal phase HPLC in UV detector. Diafenthiuron was detected in  min. The limits of detection (LOD) and limits of quantification (LOQ) were determined to be 0.01 and 0.05 μgmL−1. The initial deposits were found to be 3.82 and 4.10 μg g−1 after sprays of diafenthiuron @ 400 g a.i ha−1 in the first and second experiments, respectively. Nearly cent percent of residues dissipated at 10 days after treatment in the recommended dose of diafenthiuron 400 g a.i ha−1 and the half life varied from 2.0 to 2.8 days with a waiting period of 5.5 to 6.7 days in green capsules of cardamom. The waiting period was 5.4 to 7.0 days in cured capsules of cardamom. With harvest being the focal point for enforcement of residue tolerances, the suggested waiting period of seven days is safe without the problem of pesticide residues in harvestable produce. Johnson Stanley, Subramanian Chandrasekaran, Gnanadhas Preetha, Sasthakutty Kuttalam, and R. Sheeba Jasmine Copyright © 2014 Johnson Stanley et al. All rights reserved. Quantification of Gymnemagenin and -Sitosterol in Marketed Herbal Formulation by Validated Normal Phase HPTLC Method Thu, 06 Feb 2014 06:47:09 +0000 This research study describes development and validation of new, rapid, accurate, robust, and precise, high performance thin layer chromatographic (HPTLC) method for concurrent quantitative determination of gymnemagenin and β-sitosterol in herbal formulation with densitometric detection. Chromatographic separation was achieved on Merck aluminum HPTLC plates precoated with silica gel 60 F254. The optimized solvent system consisted of toluene : ethyl acetate : methanol (6.5 : 2.5 : 1.4, v/v/v). Developed plates were derivatized with 5% sulphuric acid reagent followed by heating at 110°C for 4 min in a preheated oven and scanned at 423 nm in reflectance-absorbance mode. The retention factor for gymnemagenin and β-sitosterol was found to be and , respectively. The proposed densitometric method was validated according to ICH Q2 (R1) guidelines. Results were found to be linear over a range of 100–1200 ng band−1 and 200–1200 ng band−1 for gymnemagenin and β-sitosterol, respectively. The percent content of gymnemagenin and β-sitosterol in the marketed polyherbal formulation was found to be 0.0405% and 0.1377%, respectively. The proposed HPTLC method can be used for quantification of gymnemagenin and β-sitosterol in marketed polyherbal formulation used in the study in quality-control laboratories. Sachin E. Potawale, Satish Y. Gabhe, and Kakasaheb R. Mahadik Copyright © 2014 Sachin E. Potawale et al. All rights reserved. Simultaneous Quantification of Glibenclamide, Simvastatin, and Quercetin by Using LC-UV Method and Its Application to Pharmacokinetic Study in Rats Mon, 16 Dec 2013 08:53:46 +0000 A sensitive, precise, and simple LC method for the simultaneous quantification of glibenclamide, simvastatin, and quercetin in rat plasma has been developed and validated. The chromatographic separation was achieved on a cyano column (250 mm × 4.6 mm, 5 µm) maintained at room temperature, using isocratic elution with methanol : acetonitrile : 10 mM potassium dihydrogen orthophosphate, pH adjusted to 4.5 with o-phosphoric acid (8 : 32 : 60, v/v) and detected using UV-VIS detector. Plasma samples were deproteinated with 0.1% perchloric acid and acetonitrile for extraction of the glibenclamide, simvastatin, and quercetin which resulted in their high recoveries. LC calibration curves based on the extracts from the rat plasma were linear in the range of 50–1000 ng mL−1 for all the three drugs. The limit of quantification was 50 ng mL−1. The described method was successfully applied to study the pharmacokinetics of glibenclamide, simvastatin, and quercetin following oral administration, in combination to Sprague-Dawley rats. Vijay Duppala, Ranjeet Prasad Dash, Mehul N. Jivrajani, Sandeep Kumar Thakur, Nirav M. Ravat, and Manish Nivsarkar Copyright © 2013 Vijay Duppala et al. All rights reserved. RP-HPLC Method for Determination of Several NSAIDs and Their Combination Drugs Wed, 11 Dec 2013 13:16:39 +0000 An RP-HPLC method for simultaneous determination of 9 NSAIDs (paracetamol, salicylic acid, ibuprofen, naproxen, aceclofenac, diclofenac, ketorolac, etoricoxib, and aspirin) and their commonly prescribed combination drugs (thiocolchicoside, moxifloxacin, clopidogrel, chlorpheniramine maleate, dextromethorphan, and domperidone) was established. The separation was performed on Kromasil C18 (250 × 4.6 mm, 5 μm) at 35°C using 15 mM phosphate buffer pH 3.25 and acetonitrile with gradient elution at a flow rate of 1.1 mL/min. The detection was performed by a diode array detector (DAD) at 230 nm with total run time of 30 min. Calibration curves were linear with correlation coefficients of determination . Limit of detection (LOD) and Limit of quantification (LOQ) ranged from 0.04 to 0.97 μg/mL and from 0.64 to 3.24 μg/mL, respectively. As an application tool of quality by design, full factorial experimental design was used for the testing of robustness of the method. The prediction profiler correlating various parameters and responses was established from the results of design of experiments (DOE). Prinesh N. Patel, Gananadhamu Samanthula, Vishalkumar Shrigod, Sudipkumar C. Modh, and Jainishkumar R. Chaudhari Copyright © 2013 Prinesh N. Patel et al. All rights reserved. Stress Degradation Studies on Flupirtine Maleate Using Stability-Indicating RP-HPLC Method Wed, 04 Dec 2013 18:00:51 +0000 With the objective of developing an advanced method for rapid separation with shorter runtime, a simple, precise, and accurate stability-indicating isocratic RP-LC method coupled with PDA detector was developed for the quantitative determination of flupirtine maleate in bulk and in capsule dosage form. Good resolution between the peaks for degradation products and the analyte was achieved on a Waters Agilent XDB C18 ( mm, 5 μm) column using mobile phase containing a mixture of phosphate buffer pH 3.36 and acetonitrile in the ratio of 65 : 35. The eluted compounds were monitored at 344 nm and the flow rate employed for the present investigation was 1 mL/min. The newly developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision, and robustness. The method may be employed for the assay determination of flupirtine maleate in pharmaceutical dosage forms. Singaram Kathirvel, Rachakonda Sujatha, Mutukuri Risheela Pandit, and Achanti Suneetha Copyright © 2013 Singaram Kathirvel et al. All rights reserved. A Comparative Validation Study of Fluconazole by HPLC and UPLC with Forced Degradation Study Wed, 04 Dec 2013 13:30:47 +0000 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. Hetal Jebaliya, Madhavi Patel, Yashwant Jadeja, Batuk Dabhi, and Anamik Shah Copyright © 2013 Hetal Jebaliya et al. All rights reserved. Recycle HPLC: A Powerful Tool for the Purification of Natural Products Wed, 06 Nov 2013 14:48:28 +0000 Natural compounds occur as various isomeric or closely related structures in biological matrices. These compounds are difficult to separate from the complex mixtures, and hence, the need for effective and innovative separation techniques arises. Recycle HPLC allows the recycling of sample, in part or full, and increases the separation efficiency of the process while keeping the peak dispersion to a minimum. Recycling in an HPLC system has been used in the isolation and purification of different types of natural products including enantiomers, diastereomers, epimers, positional isomers, and structurally related or unrelated compounds having similar retention characteristics. The present paper overviews the development of instrumentation and setup of recycle HPLC and its applications in the separation of natural products. Jasmeen Sidana and Lokesh Kumar Joshi Copyright © 2013 Jasmeen Sidana and Lokesh Kumar Joshi. All rights reserved. Trends and Advances in Separation and Detection of SSRIs and SNRIs in Biological Matrices Thu, 31 Oct 2013 18:35:58 +0000 Nowadays antidepressant drugs like selective serotonin reuptake inhibitors (SSRIs) and selective norepinephrine reuptake inhibitors (SNRIs) represent the first choice in the treatment of moderate to severe depressive illness, various phobias, and personality disorders. In spite of the therapeutic aspects, they often produce very severe and toxic effects in deliberate and accidental cases of poisoning. These are also considered as date-rape drugs used for drugged victims for raping or robbing. Therefore, in recent years, their analyses in different biological matrices for clinical and toxicological analysis purposes has been a target worthy of interest. Thus, the review focuses on recent advancements of various separation techniques like chromatography and electrophoresis that are concernd with the determination of selective serotonin reuptake inhibitor and selective norepinephrine reuptake inhibitor drugs and their metabolites in various biological matrices. In addition to this, a critical discussion on analytical approaches has also been incorporated, suggesting their applicability and limitations for further implementations. Thus, this paper will definitely help in the selection and development of proper analytical methodologies to achieve satisfactory results, better scientific understanding, and test interpretation. Ruchita Das and Y. K. Agrawal Copyright © 2013 Ruchita Das and Y. K. Agrawal. All rights reserved. Development and Validation of a Stability-Indicating RP-HPLC Method for the Simultaneous Estimation of Guaifenesin and Dextromethorphan Impurities in Pharmaceutical Formulations Mon, 23 Sep 2013 09:36:30 +0000 A sensitive, stability-indicating gradient RP-HPLC method has been developed for the simultaneous estimation of impurities of Guaifenesin and Dextromethorphan in pharmaceutical formulations. Efficient chromatographic separation was achieved on a Sunfire C18, 250 × 4.6 mm, 5 µm column with mobile phase containing a gradient mixture of solvents A and B. The flow rate of the mobile phase was 0.8 mL min−1 with column temperature of 50°C and detection wavelength at 224 nm. Regression analysis showed an r value (correlation coefficient) greater than 0.999 for Guaifenesin, Dextromethorphan, and their impurities. Guaifenesin and Dextromethorphan formulation sample was subjected to the stress conditions of oxidative, acid, base, hydrolytic, thermal, and photolytic degradation. Guaifenesin was found stable and Dextromethorphan was found to degrade significantly in peroxide stress condition. The degradation products were well resolved from Guaifenesin, Dextromethorphan, and their impurities. The peak purity test results confirmed that the Guaifenesin and Dextromethorphan peak was homogenous and pure in all stress samples and the mass balance was found to be more than 98%, thus proving the stability-indicating power of the method. The developed method was validated according to ICH guidelines with respect to specificity, linearity, limits of detection and quantification, accuracy, precision, and robustness. Thummala V. Raghava Raju, Noru Anil Kumar, Seshadri Raja Kumar, Annarapu Malleswara Reddy, Nittala Someswara Rao, and Ivaturi Mrutyunjaya Rao Copyright © 2013 Thummala V. Raghava Raju et al. All rights reserved. Quantification of Amiridine in Human Plasma by High-Performance Liquid Chromatography Coupled with Electrospray Tandem Mass Spectrometry Sun, 22 Sep 2013 14:48:57 +0000 The aim of this study was to develop and validate a high-performance liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for analysis of the amiridine in human plasma. The analyte and internal standard (IS), zolpidem, were extracted from human plasma by solid phase extraction (SPE with SOLA cartridges) and separated on a Zorbax SB-C18 column using methanol and 0.2% formic acid in water as mobile phase. Detection was performed using an electrospray ionization source and mass spectrometric positive multireaction monitoring mode (+MRM) at a voltage capillary of +2000 V. The assay was linear over the concentration range 0.5–200 ng/mL with the lowest limit of quantification (LLOQ) of 0.5 ng/mL. The method also afforded satisfactory results in terms of the sensitivity, specificity, precision (intra- and interday %), accuracy, recovery, and the stability of the analyte under various conditions. The method can be successfully applied to pharmacokinetic studies. Igor I. Miroshnichenko and Angelina I. Platova Copyright © 2013 Igor I. Miroshnichenko and Angelina I. Platova. All rights reserved. Development and Validation of an RP-HPLC Method for Estimation of Chlorpheniramine Maleate, Ibuprofen, and Phenylephrine Hydrochloride in Combined Pharmaceutical Dosage Form Wed, 24 Jul 2013 09:32:18 +0000 The objective of this paper is to develope a simple, precise, accurate, and reproducible reversed phase high performance liquid chromatographic method for the quantitative determination of chlorpheniramine maleate, ibuprofen, and phenylephrine hydrochloride in combined pharmaceutical dosage form. Analysis was carried out using acetonitrile : mathanol : phoshphate buffer (50 : 20 : 30, v/v/v, pH 5.6) mobile phase at 1.0 mL/min flow rate and Sunfire C 18 column (5 μm × 250 mm × 4.6 mm) as stationary phase with detection wavelength of 220 nm. The retention times of chlorpheniramine maleate (CPM), ibuprofen (IBU), and phenylephrine hydrochloride (PHE) were 4.2 min, 13.6 min, and 2.7 min, respectively. The proposed method was validated with respect to linearity, accuracy, precision, specificity, and robustness. The linearity for chlorpheniramine maleate, ibuprofen, and phenylephrine hydrochloride was in the range of 0.5–2.5 μg/mL, 25–125 μg/mL, and 1.25–6.25 μg/mL, respectively. The % recoveries of all the three drugs were found to be 99.44–101.61%, 99.39–101.79%, and 98.66–101.83%. LOD were found to be 32, 120, and 68 ng/mL for CPM, IBU, and PHE, respectively. The method was successfully applied to the estimation of chlorpheniramine maleate, ibuprofen, and phenylephrine hydrochloride in combined pharmaceutical dosage form. Pinak M. Sanchaniya, Falgun A. Mehta, and Nirav B. Uchadadiya Copyright © 2013 Pinak M. Sanchaniya et al. All rights reserved. A Stability Indicating UPLC Method for the Determination of Levofloxacin Hemihydrate in Pharmaceutical Dosage Form: Application to Pharmaceutical Analysis Wed, 19 Jun 2013 11:40:27 +0000 A reliable and sensitive isocratic stability indicating RP-UPLC method has been developed and validated for quantitative analysis and content uniformity study of levofloxacin hemihydrate in tablets. An isocratic method for analysis of levofloxacin hemihydrate was archived on ACQUITY UPLC BEH C18 (100*2.1) mm particle size 1.7  columns within shorter runtime of 4 min with a flow rate of 0.400 mL/min and using a photodiode array detector to monitor the eluate at 294 nm. The mobile phase consisted of acetonitrile-buffer (23 : 77 v/v), (buffer: 20 mM K2HPO4 + 1 mL triethylamine in 1 L water, by orthophosphoric acid). Response was a liner function of drug concentration in the range of 0.5–80 g/mL () with a limit of detection and quantification of 0.1 and 0.5 g/mL, respectively. Accuracy (recovery) was between 99.77% and 101.55%. The drug was subjected to oxidation, hydrolysis, photolysis, and thermal degradation. Degradation products resulting from the stress studies did not interfere with the detection of levofloxacin hemihydrate, and the assay is stability indicating. Batuk Dabhi, Bhavesh Parmar, Nitish Patel, Yashwantsinh Jadeja, Madhavi Patel, Hetal Jebaliya, Denish Karia, and A. K. Shah Copyright © 2013 Batuk Dabhi et al. All rights reserved. Isolation of Low Abundance Proteins and Cells Using Buoyant Glass Microbubble Chromatography Wed, 12 Jun 2013 15:13:14 +0000 Conventional protein affinity chromatography relies on highly porous resins that have large surface areas. These properties are ideal for fast flow separation of proteins from biological samples with maximum yields, but these properties can also lead to increased nonspecific protein binding. In certain applications where the purity of an isolated protein is more important than the yield, using a glass solid phase could be advantageous as glass is nonporous and hydrophilic and has a low surface area and low nonspecific protein binding. As a proof of principle, we used protein A-conjugated hollow glass microbubbles to isolate fluorescently labeled neurofilament heavy chain spiked into serum and compared them to protein A Sepharose and protein A magnetic beads (Dynabeads) using an anti-neurofilament protein antibody. As expected, a greater volume of glass bubbles was required to match the binding capacity of the magnetic beads and Sepharose resins. On the other hand, nonspecific protein binding to glass bubbles was greatly reduced compared to the other resins. Additionally, since the glass bubbles are buoyant and transparent, they are well suited for isolating cells from biological samples and staining them in situ. Steingrimur Stefansson, Daniel L. Adams, and Cha-Mei Tang Copyright © 2013 Steingrimur Stefansson et al. All rights reserved. HPTLC-Densitometric Analysis of Eperisone Hydrochloride and Paracetamol in Their Combined Tablet Dosage Form Sun, 19 May 2013 15:37:37 +0000 A simple, precise, accurate, and reliable HPTLC method has been developed and validated for the analysis of EPE-Eperisone hydrochloride and PCM-Paracetamol in their combined dosage form. Identification and analysis were performed on 100 mm × 100 mm layer thickness 0.2 mm, precoated silica gel G60-F254 aluminum sheet, prewashed with methanol, and dried in an oven at 50°C for 5 min. Toluene : methanol : ethyl acetate : glacial acetic acid (4 : 3.5 : 2.5 : 0.05) (v/v/v/v) was used as mobile phase. Calibration plots were established showing the dependence of response (peak area) on the amount chromatographed. The validated calibration ranges were 200–700 ng/spot and 1300–4550 ng/spot for EPE and PCM with correlation coefficient (R2) 0.994 and 0.996, respectively. Average % recovery was between 98.61–100.94% and 99.18–100.57% for EPE and PCM, respectively. The spots were scanned at 248 nm in a reflectance mode. The proposed method was validated as per ICH guidelines and successfully applied to the estimation of EPE and PCM in their combined tablet dosage form. Nirav Uchadadiya, Falgun Mehta, and Pinak Sanchaniya Copyright © 2013 Nirav Uchadadiya et al. All rights reserved. Stability Study of Darunavir Ethanolate Tablets Applying a New Stability-Indicating HPLC Method Wed, 15 May 2013 09:16:32 +0000 Chemical and physical degradation of drugs may result in altered therapeutic efficacy and even toxic effects. Therefore, the aim of this work was to study the stability of darunavir and to develop and validate a liquid chromatography (LC) method to determine darunavir in raw material and tablets in the presence of degradation products. The novel method showed to be linear from 6.0 to 21.0 μg/mL, with high precision (CV < 2%) and accuracy (recuperation of 99.64%). It is simple and reliable, free of placebo interferences. The robustness of the method was evaluated by a factorial design using seven different parameters. Forced degradation study was done under alkaline, acidic, and oxidative stress at ambient temperature and by heating. The LC method was able to quantify and separate darunavir and its degradation products. Darunavir showed to be unstable under alkaline, acid, and oxidative conditions. The novelty of this study is understanding the factors that affect darunavir ethanolate stability in tablets, which is the first step to unravel the path to know the degradation products. The novel stability-indicating method can be used to monitor the drug and the main degradation products in low concentrations in which there is linearity. Josilene Chaves Ruela Corrêa, Cristina Helena dos Reis Serra, and Hérida Regina Nunes Salgado Copyright © 2013 Josilene Chaves Ruela Corrêa et al. All rights reserved. A New Validated Stability Indicating RP-HPLC Method for Simultaneous Estimation of Pyridoxine Hydrochloride and Meclizine Hydrochloride in Pharmaceutical Solid Dosage Forms Tue, 07 May 2013 11:33:50 +0000 A simple, specific, accurate, precise stability indicating reversed-phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the simultaneous determination of pyridoxine hydrochloride (PYH) and meclizine hydrochloride (MEH). An isocratic separation of PYH and MEH were achieved on C 18, 250 × 4.6 mm ID, 5 μm particle size columns at column oven temperature 37°C with a flow rate of 0.5 mL min−1 and using a diode array detector to monitor the detection at 254 nm. The mobile phase consisted of buffer : acetonitrile : trifluoroacetic acid at a ratio of 30 : 70 : 0.1 (v/v). The retention times of PYH and MEH was found to be 5.25 and 10.14 min, respectively. Suitability, specificity, linearity, accuracy, precision, stability, and sensitivity of this method for the quantitative determination of the drugs were proved by validation in accordance with the requirements laid down by International Conference on Harmonization (ICH) Q2 (R1) guidelines. The proposed method is reliable and robust and can be used as quality control tool for the estimation of these drugs in combined pharmaceutical solid dosage forms. Md. Saddam Nawaz Copyright © 2013 Md. Saddam Nawaz. All rights reserved. Implementation of QbD Approach to the Analytical Method Development and Validation for the Estimation of Propafenone Hydrochloride in Tablet Dosage Form Thu, 18 Apr 2013 08:30:45 +0000 Chromatographic and spectrophotometric methods were developed according to Quality by Design (QbD) approach as per ICH Q8(R2) guidelines for estimation of propafenone hydrochloride in tablet dosage form. QbD approach was carried out by varying various parameters and these variable parameters were designed into Ishikawa diagram. The critical parameters were determined by using principal component analysis as well as by observation. Estimated critical parameters in HPTLC method include solvent methanol, mode of detection absorbance, precoated aluminium backed TLC plate (10 cm 10 cm), wavelength: 250 nm, saturation time: 20 min, band length: 8 mm, solvent front: 70 mm, volume of mobile phase: 5 mL, type of chamber: 10 cm 10 cm, scanning time: 10 min, and mobile phase methanol : ethyl acetate : triethylamine (1.5 : 3.5 : 0.4 v/v/v). Estimated critical parameters in zero order spectrophotometric method were solvent methanol, sample preparation tablet, wavelength: 247.4 nm, slit width: 1.0, scan speed medium, and sampling interval: 0.2, and for first order derivative spectrophotometric method it was scaling factor: 5 and delta lambda 4. The above methods were validated according to ICH Q2(R1) guidelines. Proposed methods can be used for routine analysis of propafenone hydrochloride in tablet dosage form as they were found to be robust and specific. Monika L. Jadhav and Santosh R. Tambe Copyright © 2013 Monika L. Jadhav and Santosh R. Tambe. All rights reserved. A Validated New Gradient Stability-Indicating LC Method for the Analysis of Doripenem in Bulk and Injection Formulation Tue, 09 Apr 2013 13:22:29 +0000 A sensitive, precise, specific, linear, and stability-indicating gradient HPLC method was developed for the estimation of doripenem in active pharmaceutical ingredient (API) and in injectable preparations. Chromatographic separation was achieved on C18 stationary phase with a mobile phase gradient consisting of acetonitrile, methanol, and pH 5.2 phosphate buffer. The mobile phase flow rate was 0.8 mL/min, and the eluted compounds were monitored at 210 nm. The method is linear over the range of 0.335 to 76.129 µg/mL. The correlation coefficient was found to be 0.999. The numbers of theoretical plates and tailing factor for doripenem were 53021 and 0.9, respectively. Doripenem was subjected to the International Conference on Harmonization (ICH) prescribed hydrolytic (acid, base, and neutral), oxidative, photolytic, and thermal stress conditions. Among all the above-mentioned conditions, the drug was found to be stable under photolytic degradation. Peak homogeneity data for doripenem in the chromatograms from the stressed samples obtained by use of the photodiode array detector demonstrated the specificity of the method for analysis of doripenem in presence of the degradation products. The performance of the method was validated according to the present ICH guidelines for specificity, limit of detection, limit of quantification, linearity, accuracy, precision, and robustness. Singaram Kathirvel and Garikapati Devalarao Copyright © 2013 Singaram Kathirvel and Garikapati Devalarao. All rights reserved. Validated Stability Indicating RP-HPLC Method for Simultaneous Estimation of Codeine Phosphate and Chlorpheniramine Maleate from Their Combined Liquid Dosage Form Sun, 31 Mar 2013 08:12:17 +0000 The present paper describes the development of quick stability indicating RP-HPLC method for the simultaneous estimation of codeine phosphate and chlorpheniramine maleate in the presence of its degradation products, generated from forced degradation studies. The developed method separates codeine phosphate and chlorpheniramine maleate in impurities/degradation products. Codeine phosphate and chlorpheniramine maleate and their combination drug product were exposed to acid, base, oxidation, dry heat, and photolytic stress conditions, and the stressed samples were analysed by proposed method. The proposed HPLC method utilizes the Shimadzu HPLC system on a Phenomenex C18 column (, 5 μ) using a mixture of 1% o-phosphoric acid in water : acetonitrile : methanol (78 : 10 : 12) mobile phase with pH adjusted to 3.0 in an isocratic elution mode at a flow rate of 1 mL/min, at 23°C with a load of 20 μL. The detection was carried out at 254 nm. The retention time of codeine phosphate and chlorpheniramine maleate was found to be around 3.47 min and 9.45 min, respectively. The method has been validated with respect to linearity, robustness, precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). The developed validated stability indicating HPLC method was found to be simple, accurate, and reproducible for the determination of instability of these drugs in bulk and commercial products. Ramakrishna Kommana and Praveen Basappa Copyright © 2013 Ramakrishna Kommana and Praveen Basappa. All rights reserved. Separation of Cyclic Dipeptides (Diketopiperazines) from Their Corresponding Linear Dipeptides by RP-HPLC and Method Validation Thu, 14 Feb 2013 10:43:10 +0000 Simple, rapid, sensitive, precise, and accurate methods for detection and separation of seven diketopiperazines (DKPs), cyclo(Gly-Gly), cyclo(DL-Ala-DL-Ala), cyclo(L-Asp-L-Phe), cyclo(L-Asp-L-Asp), cyclo(Gly-L-Phe), cyclo(L-Pro-L-Tyr), and cyclo(L-Arg-L-Arg), from their corresponding linear dipeptides and related amino acids L-Phe and L-Tyr by reversed-phase high-performance liquid chromatography (RP-HPLC) were established. Moreover, for the racemic DKP cyclo(DL-Ala-DL-Ala) and dipeptide DL-Ala-DL-Ala, separation of the diastereomers was achieved. All methods can be performed within 15 min. For all DKPs, dipeptides, and amino acids, linear ranges with correlation coefficients greater than 0.998 were determined. Lowest limits of detection were found to be between 0.05 and 10 nmol per 10 μL injection, depending on the substance. For all tested substances intrarun and interrun precision ranged from 0.5 to 4.7% and 0.7 to 9.9% relative standard deviation, and accuracy was between −4.2 and 8.1% relative error. Short-term and freeze-thaw stabilities were 93% or greater for all substances. Recovery rate after heat treatment was determined to be at least 97%. These methods will be useful for quantitative determination of DKPs and their potential biodegradation products: dipeptides and amino acids Mareike Perzborn, Christoph Syldatk, and Jens Rudat Copyright © 2013 Mareike Perzborn et al. All rights reserved. New Developments in Liquid Chromatography Mass Spectrometry for the Determination of Micropollutants Mon, 17 Dec 2012 14:59:12 +0000 The combination of liquid chromatography (LC) with mass spectrometry (MS) in the environmental field has appeared as a valuable tool for the determination of micropollutants. Several groups of compounds have been considered as particularly relevant (e.g., pharmaceuticals, hormones and other endocrine-disrupting, personal care products and their metabolites, flame retardants, surfactants, and plasticizers, among others) since the same ones are continuously being released in the environment mainly as a result of the manufacturing processes, the disposal of unused or expired products, and the excreta. Because these micropollutants are not completely removed in the environment, very specific and sensitive analytical procedures are needed for their identification and quantification. High performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) (or LC-MS2) and especially time-of-flight mass spectrometry (TOF/MS), has allowed that many environmental contaminants that are highly polar or nonvolatile or have a high molecular weight to be analyzed or identified. In this work we present an overview focused on the developments of liquid chromatography mass spectrometry applied to the analysis of the main classes of micropollutants in aqueous and solid environmental samples. Various aspects of methodologies based on these techniques, including sample preparation (extraction/preconcentration) and matrix effects, are discussed. Zoraida Sosa-Ferrera, Cristina Mahugo-Santana, and José Juan Santana-Rodríguez Copyright © 2012 Zoraida Sosa-Ferrera et al. All rights reserved. Analytical Method Development and Validation of Pharmaceutical Analysis Using Chromatographic Techniques Sun, 04 Nov 2012 15:35:12 +0000 Bengi Uslu, Henk Lingeman, Sibel A. Ozkan, Meehir Palit, and Burcu Dogan-Topal Copyright © 2012 Bengi Uslu et al. All rights reserved. Ion Suppression Study for Tetracyclines in Feed Thu, 27 Sep 2012 19:11:43 +0000 Ion suppression in analysis of tetracyclines in feed was studied. The conventional analysis consists of a liquid extraction followed by a clean-up step using solid phase extraction (SPE) technique and analysis of the tetracyclines by liquid chromatography and mass spectrometric detection. Various strategies for extraction and cleanup were tested in the present work, and the effectiveness to decrease the ion suppression on the MS/MS signals was evaluated. Four sample treatment methods were tested with five different feed samples. Extraction solvents tested were McIlvaine buffer and a mixture of McIlvaine buffer dichloromethane (3 : 1). SPE cartridges for cleanup were Oasis HLB, Oasis MCX, and Oasis MAX. The effectiveness of the methods was evaluated in terms of decreasing the ion suppression effect but also of decreasing the variability of ion suppression between samples. The method that provided the most satisfactory results involved a clean-up step based on SPE using mixed-mode cation exchange cartridges (Oasis MCX). Joaquim Chico, Frederique van Holthoon, and Tina Zuidema Copyright © 2012 Joaquim Chico et al. All rights reserved. Analysis of Some Biogenic Amines by Micellar Liquid Chromatography Sun, 09 Sep 2012 16:03:35 +0000 Micellar liquid chromatography (MLC) with the use of high performance liquid chromatography (HPLC) was used to determine some physicochemical parameters of six biogenic amines: adrenaline, dopamine, octopamine, histamine, 2-phenylethylamine, and tyramine. In this paper, an influence of surfactant’s concentration and pH of the micellar mobile phase on the retention of the tested substances was examined. To determine the influence of surfactant’s concentration on the retention of the tested amines, buffered solutions (at pH 7.4) of ionic surfactant—sodium dodecyl sulfate SDS (at different concentrations) with acetonitrile as an organic modifier (0.8/0.2 v/v) were used as the micellar mobile phases. To determine the influence of pH of the micellar mobile phase on the retention, mobile phases contained buffered solutions (at different pH values) of sodium dodecyl sulfate SDS (at 0.1 M) with acetonitrile (0.8/0.2 v/v). The inverse of value of retention factor (1/𝑘) versus concentration of micelles (𝐶𝑀) relationships were examined. Other physicochemical parameters of solutes such as an association constant analyte—micelle (𝐾ma)—and partition coefficient of analyte between stationary phase and water (hydrophobicity descriptor) (𝑃swΦ) were determined by the use of Foley’s equation. Irena Malinowska and Katarzyna E. Stępnik Copyright © 2012 Irena Malinowska and Katarzyna E. Stępnik. All rights reserved. Development and Validation of a Stability Indicating LC Method for the Assay and Related Substances Determination of a Proteasome Inhibitor Bortezomib Wed, 29 Aug 2012 16:09:32 +0000 A novel, simple, sensitive, stability indicating HPLC method was developed and validated for quantification of impurities (process related and degradants) and assay determination of bortezomib. Stability indicating power of the method was established by forced degradation experiments and mass balance study. The chromatographic separation was achieved with Waters SymmetryShield RP18 column using gradient elution using the mobile phase-A consists of a mixture of water-acetonitrile-formic acid (715 : 285 : 1, v/v/v) and the mobile phase-B consists a mixture of methanol-water-formic acid (800 : 200 : 1, v/v/v), respectively. The developed method is validated for parameters like precision, accuracy, linearity, LOD, LOQ, and ruggedness. Central composite experimental design (CCD) was applied to check the robustness of the method. The stability tests were also performed on drug substances as per ICH norms. Kasa Srinivasulu, Mopidevi Narasimha Naidu, Kadaboina Rajasekhar, Murki Veerender, and Mulukutla Venkata Suryanarayana Copyright © 2012 Kasa Srinivasulu et al. All rights reserved. Separation of Polyphenols from Jordanian Olive Oil Mill Wastewater Wed, 13 Jun 2012 10:06:02 +0000 This research aims at separation of polyphenols from Jordanian olive mill wastewater which have possible applications in pharmaceutical industry. The phenolic compounds were isolated using silica column chromatography based on using different solvents after extracting the acidified solution with n-hexane and ethyl acetate. The structural elucidation of the separated compounds was achieved using 1H-NMR, 13C-NMR and mass spectrometry. The concentrations of these compounds were determined by GC-MS after derivatization with N, O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). The concentrations of the main isolated phenolic compounds in the Jordanian olive mill wastewater were ferulic acid (93.6 mg/L), trans-cinnamic acid (105.3 mg/L), p-coumaric acid (117.0 mg/L), vanillic acid (128.7 mg/L), caffeic acid (140.4 mg/L), tyrosol (210.6 mg/L), and hydroxytyrosol (315.9 mg/L). Ahmad A. Deeb, Manar K. Fayyad, and Mahmoud A. Alawi Copyright © 2012 Ahmad A. Deeb et al. All rights reserved.