Chromatography Research International The latest articles from Hindawi Publishing Corporation © 2014 , Hindawi Publishing Corporation . All rights reserved. High-Performance Liquid Chromatography Determination of Meloxicam and Piroxicam with Ultraviolet Detection Mon, 27 Oct 2014 11:53:21 +0000 A simple accurate and sensitive high-performance liquid chromatographic method for the determination of meloxicam and piroxicam concentrations in small volume plasma samples has been developed. Following a liquid extraction using chloroform, samples were separated by reversed-phase high-performance liquid chromatography on an XBridge C18 column (4.6 × 250 mm) and quantified using ultraviolet detection at 360 nm. The mobile phase was a mixture of water with glacial acetic acid (pH 3.0) and acetonitrile (50 : 50), with a flow rate of 1.0 mL/min. The standard curve ranged from 5 to 10,000 ng/mL for meloxicam in bearded dragon (Pogona vitticeps) plasma and piroxicam in crane (Grus rubicunda) plasma. Intra- and interassay variability for meloxicam and piroxicam were less than 10% and the average recovery was greater than 90% for both drugs. This method was developed in bearded dragon and crane plasma and should be applicable to any species, making it useful for those investigators dealing with small sample volumes, particularly when conducting pharmacokinetics studies which require multiple sampling from the same animal. Sherry Cox, Joan Hayes, Jason Yarbrough, Tamara Veiga-Parga, and Cheryl Greenacre Copyright © 2014 Sherry Cox et al. All rights reserved. Development and Validation of Stability-Indicating RP-UPLC Method for Simultaneous Determination of Related Substances of S(−)Amlodipine and S(−)Metoprolol Succinate in Fixed Dose Combination Tablet Dosage Form Mon, 20 Oct 2014 07:07:47 +0000 A novel, rapid, accurate, sensitive, precise, and stability-indicating reverse-phase ultra-performance liquid chromatographic (RP-UPLC) method was developed and validated for determination of related substances of S(−)Amlodipine and S(−)Metoprolol Succinate in fixed dose combination tablet dosage form. The chromatographic separation was achieved with the use of Acquity UPLC HSS T3, 1.8 μm, 2.1 × 100 mm analytical column at 45°C employing a gradient elution. Mobile phase consisting of mobile phase-A (solution containing 5.0 gm of sodium dihydrogen phosphate monohydrate per liter of water and Acetonitrile in the ratio of 95 : 5) and mobile phase-B (Acetonitrile) was used at a flow rate of 0.5 mL min−1 with injection volume of 10 μL and the detection was done at 232 nm using UV detector. The retention times of S(−)Metoprolol Succinate and S(−)Amlodipine were found to be 2.8 minutes and 8.1 minutes, respectively. During method validation all the parameters were evaluated as per ICH guidelines, which remained well within acceptable limits. This method can be used for the estimation of related substances of S(−)Amlodipine and S(−)Metoprolol Succinate in fixed dose combination tablet dosage form. Suresh Shitole, Mukund Gurjar, Mahesh Shah, Srikant Pimple, Gobardhan Bal, and Rahul Patel Copyright © 2014 Suresh Shitole et al. All rights reserved. Analysis of Selected Amino Acids in Different Varieties of Wheat Available in Punjab, Pakistan Thu, 16 Oct 2014 10:47:13 +0000 A validated method is proposed to check amino acids variability among eighty-nine wheat samples collected from Punjab province of Pakistan during 2012-2013. Orthophthalaldehyde along with 2-mercaptoethanol was used as a derivatizing reagent that showed florescence at detection wavelength of Ex of 340 nm and Em of 450 nm under suitable pH range of 9-10. RP-HPLC-FLD system employed was Agilent 1100 series equipped with Eclipse XDB C-18 column (2.1 × 150 mm, 5 µ) and column temperature was maintained at 40°C. The maximum concentration of aspartic acid, glutamic acid, leucine, arginine, and histidine was found in Vehari (0.496 g/100 g), Rajanpur (1.292 g/100 g), Rahim Yar khan (0.60 g/100 g), Bahawalpur (0.662 g/100 g), and Narowal (0.377 g/100 g), respectively, while the minimum in Narowal (0.13 g/100 g), Vehari (0.706 g/100 g), Narowal (0.339 g/100 g), Muzaffargarh (0.14 g/100 g), and Rahim Yar Khan (0.088 g/100 g) among the samples obtained from districts. Wheat variety Pb-11 contained relatively high aspartic acid (0.297 g/100 g), glutamic acid (0.897 g/100 g), and leucine (0.484 g/100 g) whereas variety Ass-11 had arginine (0.895 g/100 g) and histidine (0.266 g/100 g). The amino acids were found to vary as follows: aspartic acid 0.130–0.496, glutamic acid 0.706–1.292, leucine 0.321–0.6, arginine 0.118–0.895, and histidine 0.088–0.377 g/100 g flour. The accuracy was in the range of 95.88–100.67%, whereas the RSD for precision was not more than 1.40 for all amino acids. Sumera Zafar, Narjis Naz, Saliha Nazir, Mateen Abbas, and Abdul Muqeet Khan Copyright © 2014 Sumera Zafar et al. All rights reserved. Extraction of Artemisinin, an Active Antimalarial Phytopharmaceutical from Dried Leaves of Artemisia annua L., Using Microwaves and a Validated HPTLC-Visible Method for Its Quantitative Determination Wed, 01 Oct 2014 12:50:05 +0000 A simple, rapid, precise, and accurate high-performance thin-layer chromatographic method coupled with visible densitometric detection of artemisinin is developed and validated. Samples of the dried Artemisia annua leaves were extracted via microwaves using different solvents. This method shows the advantage of shorter extraction time of artemisinin from leaves under the influence of electromagnetic radiations. Results obtained from microwave-assisted extraction (MAE) were compared with hot soxhlet extraction. Chromatographic separation of artemisinin from plant extract was performed over silica gel 60 F254 HPTLC plate using n-hexane : ethyl acetate as mobile phase in the ratio of 75 : 25, v/v. The plate was developed at room temperature 25 ± 2.0°C. Artemisinin separation over thin-layer plate was visualized after postchromatographic derivatization with anisaldehyde-sulphuric acid reagent. HPTLC plate was scanned in a CAMAG’s TLC scanner 3 at 540 nm. Artemisinin responses were found to be linear over a range of 400–2800 ng spot−1 with a correlation coefficient 0.99754. Limits of detection and quantification were 40 and 80 ng spot−1, respectively. The HPTLC method was validated in terms of system suitability, precision, accuracy, sensitivity (LOD and LOQ), and robustness. Additionally, calculation of plate efficiency and flow constant were included as components of validation. Extracts prepared from different parts of the plant (leaves, branches, main stem, and roots) were analyzed for artemisinin content, in which, artemisinin content was found higher in the leaf extract with respect to branches and main stem extracts; however, no artemisinin was detected in root extract. The developed HPTLC-visible method of artemisinin determination will be very useful for pharmaceutical industries, which are involved in monitoring of artemisinin content during different growth stages (in vitro and in vivo) of A. annua for qualitative and quantitative assessment of final produce prior to commercial-scale processing for assessment of cost-benefit ratio. Himanshu Misra, Darshana Mehta, Bhupendra Kumar Mehta, and Dharam Chand Jain Copyright © 2014 Himanshu Misra et al. All rights reserved. Determination of Strong Acidic Drugs in Biological Matrices: A Review of Separation Methods Mon, 29 Sep 2014 12:11:40 +0000 Strong acidic drugs are a class of chemical compounds that normally have high hydrophilicity and large negative charges, such as organophosphatic compounds and organosulphonic compounds. This review focuses on sample preparation and separation methods for this group of compounds in biological matrices in recent years. A wide range of separation techniques, especially chromatographic method, are presented and critically discussed, which include liquid chromatography (e.g., ion-pair and ion-exchange chromatography), capillary electrophoresis (CE), and other types. Due to the extremely low concentration level of target analytes as well as the complexity of biological matrices, sample pretreatment methods, such as dilute and shoot methods, protein precipitation (PP), liquid-liquid extraction (LLE), solid-phase extraction (SPE), degradation, and derivatization strategy, also play important roles for the development of successful analytical methods and thus are also discussed. Lingli Mu, Feifan Xie, Sanwang Li, and Peng Yu Copyright © 2014 Lingli Mu et al. All rights reserved. HPLC-UV Method Development and Validation of Potato Sprout Inhibitor 1,4-Dimethylnaphthalene Using Different Systems Tue, 16 Sep 2014 09:59:51 +0000 1,4-Dimethylnaphthalene (1,4-DMN) is effective sprout suppressant used in potato stores in many countries in the world. High residue levels of this compound on the potatoes and in other environmental samples are considered for human health and environmental risks. Determination of the residue requires specific analytical methods to be developed and validated. In this study, HPLC-UV was selected for validating a separation method based on reversed phase for the analysis of 1,4-DMN using 2-methylnaphthalene (2-MeN) as internal standard testing three HPLC systems. Under the same chromatographic conditions, all three systems achieved good separation on a Jones column (Hypersil ODS 5 μm, 250 mm × 4.6 mm) at ambient temperature isocratically using 70% acetonitrile as mobile phase at a flow rate of 1.5 mL min−1, 20 μL injection volume, a run time of 10 min, and a detection wavelength of 228 nm. All three systems showed high precision, good linearity, and low limit of detection (LOD) and quantification (LOQ); particularly, the SpectraSERIES UV100-autosampler system offered lower values of LOD (0.001–0.004 μg mL−1) and LOQ (0.002–0.013 μg mL−1) for both compounds. This system can be used for the quantitative determination of 1,4-DMN residue in potato and environmental samples. Nidhal S. Mohammed, T. H. Flowers, and H. J. Duncan Copyright © 2014 Nidhal S. Mohammed et al. All rights reserved. Photocatalytic Degradation of Trifluralin, Clodinafop-Propargyl, and 1,2-Dichloro-4-Nitrobenzene As Determined by Gas Chromatography Coupled with Mass Spectrometry Sun, 31 Aug 2014 10:54:31 +0000 Phototransformation is considered one of the most key factors affecting the fate of pesticides. Therefore, our study focused on photocatalytic degradation of three selected pesticide derivatives: trifluralin (1), clodinafop-propargyl (2), and 1,2-dichloro-4-nitrobenzene (3). The degradation was carried out in acetonitrile/water medium in the presence of titanium dioxide (TiO2) under continuous purging of atmospheric air. The course of degradation was followed by thin-layer chromatography and gas chromatography-mass spectrometry techniques. Electron ionization mass spectrometry was used to identify the degradation species. GC-MS analysis indicates the formation of several intermediate products which have been characterized on the basis of molecular ion, mass fragmentation pattern, and comparison with NIST library. The photocatalytic degradation of pesticides of different chemical structures manifested distinctly different degradation mechanism. The major routes for the degradation of pesticides were found to be (a) dealkylation, dehalogenation, and decarboxylation, (b) hydroxylation, (c) oxidation of side chain, if present, (d) isomerization and cyclization, (e) cleavage of alkoxy bond, and (f) reduction of triple bond to double bond and nitro group to amino. Niyaz A. Mir, A. Khan, M. Muneer, and S. Vijayalakhsmi Copyright © 2014 Niyaz A. Mir et al. All rights reserved. Validated HPLC Method for Quantification of Pregabalin in Human Plasma Using 1-Fluoro-2,4-dinitrobenzene as Derivatization Agent Sun, 17 Aug 2014 12:21:00 +0000 In this study, a sensitive, simple, and reliable HPLC method for quantification of pregabalin in human plasma was developed and validated. 1-Fluoro-2,4-dinitrobenzene was used as precolumn derivatization agent. For chromatography, an analytical reversed phase (C18) column and a mixture of Na2HPO4 10 mM (pH 8.0)—methanol (35 : 65 v/v) were used as stationary and mobile phase, respectively. Detection was performed using a UV detector tuned at 360 nm. The linearity of the method was tested over the concentration range 1–4500 ng/mL in 500 μL of human plasma and satisfactory results were obtained (r2 > 0.999). The method showed good precision and accuracy in terms of within—between days relative standard deviations and percent deviation from nominated values (in the range of 4.3–12.7% and 2.6–8.0%, resp.). The limit of quantification of the method was found to be 1 ng/mL which is better than previously reported method and indicates its potential application for sensitive bioanalysis. Reza Ahmadkhaniha, Siavash Mottaghi, Mohammad Zargarpoor, and Effat Souri Copyright © 2014 Reza Ahmadkhaniha et al. 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.