Isocratic Reversed-phase High-performance Liquid Research Articles

DEVELOPMENT AND VALIDATION OF NOVEL RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF SALICYLIC ACID AND BECLOMETHASONE DIPROPIONATE IN A TOPICAL OINTMENT DOSAGE FORM

Objective: The objective of the study is to develop and validate an analytical method to simultaneously estimate salicylic acid (SA) and beclomethasone dipropionate (BD) in a topical ointment formulation. Methods: The simultaneous estimation of SA and BD in a topical ointment formulation was carried out by developing and validating a novel, accurate, and economical isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method. Separation was achieved by chromatographic technique using a prontosil HPLC C18 (250×4.6 mm) column with a particle size of 5 μm. The mobile phase utilized for this study includes methanol, acetonitrile, and 0.1% orthophosphoric acid in the ratio 50:35:15 v/v/v, respectively. The flow rate of 1 mL/min and column temperature of 28±2°C were set. The detection of the two drugs was carried out at 235 nm using an ultraviolet detector. It was observed that SA and BD were retained at 3.59 min and 6.00 min, respectively. Results: The RP-HPLC method was found to be linear with excellent correlation between peak areas and concentrations of 30–108 µg/mL for SA and 1–3.6 µg/mL for BD. The observed recovery data was obtained within the acceptance range of 98–102%, which confirmed the accuracy of the developed method. The two drugs, SA and BD, showed good resolution with a short analysis time of 7 min. Conclusion: The method was successfully created and validated in compliance with the recommendations of the International Conference on Harmonization for specificity, precision, linearity, accuracy, and robustness.

Development and Validation of RP-HPLC Method for the Estimation of Linezolid from Bulk and Formulations

A new, simple, rapid, selective, precise and accurate isocratic reverse-phase high-performance liquid Chromatography assay method has been developed to estimate Linezolid in tablet formulations. The separation was achieved using column Inert sustain C18 (250×4.6mm, 5µ) in the mobile phase consisting of methanol: 1% Acetic acid (50:50 v/v). The flow rate was 0.80mL/min-1, and the separated Linezolid was detected using a UV detector at 249nm. Column temperature 45°C and sample temperature ambient and injection volume 20µL. The retention time of Linezolid was noted to be 7.3min. The method was validated as per ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent.

Development and Validation of an RP-HPLC Method for the Quantitative Analysis of Triclosan in Human Urine

Triclosan (TCS), a synthesized chlorinated phenolic compound, is commonly utilized in consumable products as an antimicrobial agent. TCS has sparked widespread awareness because of its toxicity and possible negative effect on public health in recent years. In this study, a highly sensitive, fast, and cost-effective isocratic reversed-phase high-performance liquid chromatography (RP-HPLC) method coupled with solid-phase extraction for analysis of triclosan in human urine samples was developed. The method utilized methanol and water in a ratio of 90 : 10 as the mobile phase on a Phenomenex Luna 3 µm C18(2) 100 Å, 150 × 4.60 mm stationary phase, with a runtime of 5 minutes. The method showed good resolution of triclosan in the presence of the sample matrix. Validation of the method was performed according to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH). Linearity was tested over a range of 0.00625 µg/mL to 6.4 µg/mL, as accuracy recorded a recovery of 89.25%, 91.0%, and 92.75%. Limits of detection (LOD) and quantification (LOQ) were obtained to be 0.0173 µg/mL and 0.0525 µg/mL, respectively. The method proved to be robust over a temperature range of 26°C, 30°C, and 35°C and a flow rate of 0.5 ml, 1.0 ml, and 1.5 ml. The developed method was employed to detect and quantify triclosan in 153 urine samples, comprising 60 samples from Ibadan, Nigeria, and 93 samples from Kumasi, Ghana. Triclosan was detected in a total of 52 samples with an average content of 0.054588 µg/ml. This method can therefore be used for the routine analysis of triclosan in urine samples.

Validation of Liquid Chromatographic Analytical Method for Determination of Aspirin, Caffeine and Paracetamol in some Pharmaceutical Tablets Form in Iraqi market

In this research, an accurate, efficient and reproducible isocratic reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and validated for the determination of aspirin (ASP), caffeine (CAF), and Paracetamol (PCM). A mixture of drugs as standard material and in formulation tablets were separated using an phenomenex C18 column, (L, 15cm, I.D, 0.46cm, and Size of particle, 5μm), with the shimadzu RP – HPLC, model LC–20 - A, Japan. The eluent phase was optimized through the design, experiment. Elution was done by an eluent phase composed of water (H2O), methanol (MOH) and acetonitrile (ACN) mixture have a ratio of (60: 20: 20, V/V), with adjusted pH of 4.0 with acetic acid, have a pumped flow rate of 0.5mL/min. The drugs, separation were performed at 254nm using a UV-VIS - detector for 8 min. The time of elution for the drugs was recorded at (3.980, 4.366, and 6.894 min) for PCM, CAF and ASP respectively. The validation of the method was conducted in a range of concentration (1 - 100μg/mL) for PCM, CAF and ASP. The method was found to be robust, specified and resisting against the small experimental changes in the value of the flow rate, pH, and wavelength. The LOD were found to be 0.01μg/mL, 0.03μg/mL and 0.05μg/mL for PCM, CAF and ASP respectively. The values of LOQ were 0.033μg/mL, 0.099μg/mL and 0.165μg/mL for PCM, CAF and ASP respectively.

Drawbacks in the efficient monitoring of gold nanoparticle-based cisplatin delivery systems formation by HPLC-ICP-MS.

Since chemotherapy suffers many limitations related to side effects of anticancer drugs (e.g. cisplatin - CDDP), nanoparticles are probed as carriers in targeted drug delivery. Gold nanoparticles (AuNPs) are broadly investigated due to their biocompatibility, nontoxicity, and tunable surface. Despite many AuNPs-cisplatin systems (AuNP-CS) reports found in the literature, only a few include studies of their synthesis and formation efficiency using analytical tools providing simultaneously qualitative and quantitative analytical information. Therefore, this research continues our previous study of AuNP-CS formation investigated by capillary electrophoresis with inductively coupled plasma mass spectrometry (ICP-MS). Namely, it presents the analogical approach but employs the coupling of another separation technique: isocratic reversed-phase high-performance liquid chromatography. The study concerns the difficulties of analytical method optimization path and contains a discussion of the observed problematic issues related to the analysis and preparation of AuNP-CS. Moreover, the presented work confronts the performance and applicability of both tools for the scrutiny of AuNP-CS, especially considering the comparison of their resolution power.

"Development and Validation of an Isocratic Reverse-Phase High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Desloratadine and Montelukast sodium in Bulk Drug, Pharmaceutical Dosage Forms, and Human Plasma.”

"Development and Validation of an Isocratic Reverse-Phase High-Performance Liquid Chromatographic Method for the Simultaneous Determination of Desloratadine and Montelukast sodium in Bulk Drug, Pharmaceutical Dosage Forms, and Human Plasma.”

Stability Indicating Isocratic HPLC Method for Bilastine and Characterization of Forced Degradation Products by LC-MS/MS

Abstract: The present study aims to develop and validate a simple, precise, accurate, stability indicating and isocratic reversephase high-performance liquid chromatography (RP-HPLC) method for estimating Bilastine in bulk and synthetic mixture. Bilastine from its degradation products were well separated and estimated on Discovery C8 column (250 mm x 4.6 mm, 5μm) using methanol: 0.1% ortho-phosphoric acid (55:45 %v/v) as a mobile phase and detection was performed at 276 nm by PDA detector. The degradation of Bilastine was studied under different ICH recommended stress conditions. The developed stability-indicating method was validated for system suitability, linearity, accuracy, precision, robustness, detection limit and quantitation as per ICH guidelines. The method was linear over 25-150 μg/ml. Mathematically computed Limit of Detection and Limit of Quantitation were found to be 0.19μg/ml and 0.57μg/ml, respectively. It was discovered that Bilastine degrades under acid and oxidation conditions. By using LC-MS/MS analysis, the structure of Bilastine breakdown products created amid acidic and oxidative settings was elucidated. A complete fragmentation pathway of Bilastine was established to elucidate the degradation products' structures using LC–MS/MS. The elemental concentrations of oxidation products and their fragmentation products were studied using the acquired mass values. The overall data was utilized to describe the degradation of products and their fragmentation process. The developed method can be used for routine analysis of Bilastine in dosage form as it can estimate of Bilastine in presence of excipients and degradation products.

REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-ULTRAVIOLET-BASED APPROACH FOR METHOD DEVELOPMENT AND VALIDATION OF LACOSAMIDE ESTIMATION IN HUMAN SERUM

Objective: The main aim of the study was to develop and validate a simple, accurate, and rapid isocratic reverse phase high-performance liquid chromatographic method with UV detection for the determination of lacosamide, an antiepileptic agent, in human serum. Methods: Chromatographic separation was performed using a reverse phase chromatographic column (Zorbax SB-C18, 5 μm 4.6×250 mm) with a mobile phase being a mixture of potassium dihydrogen phosphate buffer (pH adjusted to 3.0 with orthophosphoric acid) and acetonitrile (ratio of 83:17 v/v) at a flow rate of 1.2 mL/min. UV detection was carried out at 210 nm and the sample temperature was maintained at 4°C. Results: Linear calibration curves in the range of 1.012–40.894 μg/ml gave a correlation coefficient of 0.9988. The intra-day (n=6) and inter-day (n=18) precision (expressed as relative standard deviation) were in the range of 0.79–2.485% and from 0.99 to 3.21%, respectively. The retention time (in minutes) of lacosamide and internal standard was approximately 8.785±0.19 and 3.985±0.77, respectively, with no matrix interference. The method was validated for system suitability, specificity, precision, accuracy, robustness, linearity, limit of detection, and limit of quantification following the International Conference on Harmonization guidelines. The method was further validated using sera of epileptic patients consuming lacosamide, and it was observed that the results matched with the patients’ clinical response. Conclusion: Our method developed to estimate serum lacosamide level is simple, cost-effective, and reliable for therapeutic drug monitoring.

Development & Validation of RP-HPLC Method for the Estimation of Doravirine in Bulk and Pharmaceutical Dosage Form

A simple, accurate, rapid and precise isocratic reversed phase high-performance liquid chromatographic method has been developed and validated for determination of Doravirine in tablets. The chromatographic separation was carried out on Dionex C18 (250 x 4.6mm, 5µ) with a mixture of methanol: 0.05M potassium dihydrogen phosphate (40:60%v/v) as a mobile phase at a flow rate of 1.5 mL/min. UV detection was performed at 306 nm. The retention time was 5.24 min for Doravirine. Calibration plot was linear (r2=0.999) over the concentration range of 200-600 µg/mL. The method was validated for accuracy, precision, specificity, linearity, robustness, LOD and LOQ. The proposed method was successfully used for quantitative analysis of tablets. No interference from any component of pharmaceutical dosage form was observed. Validation studies revealed that method is specific, rapid, reliable, and reproducible. The high recovery and low relative standard deviation confirm the suitability of the method for routine determination of Doravirine in bulk and tablet dosage form.

DoE Screening and Optimization of Liquid Chromatographic Determination of Nicotinic Acid and Six Statins: Application to Pharmaceutical Preparations and Counterfeit Detection.

An isocratic reversed-phase high performance liquid chromatographic method has been developed and validated to simultaneously determine nicotinic acid, pravastatin sodium, rosuvastatin calcium, atorvastatin calcium, pitavastatin calcium, lovastatin sodium and simvastatin sodium in focus on counterfeit drug detection. Thin-layer chromatography, nuclear magnetic resonance and mass spectrometry have been additionally performed to verify the identification of adulterants of counterfeit herbal medicines. Chromatographic separation was carried out on Inertsil® ODS-3 C18 (4.6 × 150mm, 5μm) with isocratic mobile phase elution containing a mixture of acetonitrile: methanol: 25mM potassium dihydrogen phosphate buffer, pH2.86 adjusted with 0.1M o-phosphoric acid (48: 30: 22, v/v/v), at a flow rate of 1mL/min and with UV detection at 238nm. The design of experiment methodology, Plackett-Burman and Box-Behnken designs, was used to screen and optimize the mobile phase composition. The validation of the method was also carried out under the International Conference on Harmonization guidelines. The developed method was sensitive, accurate, simple, economical and highly robust, in addition to the comprehensiveness and novelty of this method for separating the seven drugs. The results were statistically compared with the reference methods used Student's t-test and variance ratio F-test at P < 0.05.

Development and Validation of RP-HPLC Chromatographic Dissolution Method for the Simultaneous Estimation of Ramipril and Hydrochlorothiazide from Solid Dosage Formulation

The present study describes the dissolution method development and validation of Ramipril and Hydrochlorothiazide in tablet dosage form by HPLC Method.&#x0D; A simple, rapid, selective, reproducible and isocratic reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated as per ICH guidelines.&#x0D; Analysis was performed on a Thermo, Sunniest C8 (150 mm x 4.6 mm, 5 µm) with the mobile phase consisting of mixing 500 mL of buffer solution and 500 mL of acetonitrile at a flow rate of 1.0mL/min. UV detection was performed at 210nm and the Run time for Ramipril and Hydrochlorothiazide were 10 minutes. The calibration curve was linear (correlation coefficient = 1.000) in the selected range for both analytes.&#x0D; The optimized dissolution conditions include the USP Type 1 (Basket) rotation rate of 100 rpm and 750 mL of 0.1 N Hydrochloric acid as dissolution medium, at 37.0 ± 0.5°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 six standard replicates were well within the limits. The stability result shows that the drug is stable in the prescribed dissolution medium.

Evaluation of Perampanel in the Presence of its Degradation Products and Process-Related Impurities by Validated Stability-Indicating Reverse Phase High Performance Liquid Chromatography Method.

A novel stability-indicating isocratic reverse phase high-performance liquid chromatographic method was developed for the quantitative determination of perampanel in the presence of degradation products and its process-related impurities. Good resolution was achieved between the peaks corresponding to process-related impurities and degradation products from the analyte using Shim-pack GIST C18 column with mobile phase, potassium dihydrogen phosphate (pH 2.5; 30 mM)-acetonitrile (45: 55, v/v) at a wavelength of 294nm. Perampanel and its impurities were well-separated within a run time of 7min. To show the stability-indicating power of the method, forced degradation studies were performed on bulk samples of perampanel as per ICH prescribed stress condition using acid, base, hydrolytic, oxidative and photolytic degradation conditions. The degradation products were resolved from main peak and its impurities. The developed method was validated as per ICH guidelines with respect to specificity, linearity, limit of detection, limit of quantification, accuracy, precision and robustness. This method was also applied for assay and related substances determination of perampanel in bulk and pharmaceutical dosage form.

Novel Rapid Isocratic RP-HPLC Method for Simultaneous Estimation of Phenylephrine Hydrochloride, Paracetamol, Caffeine, Diphenhydramine Hydrochloride

Background: A tablet dosage form widely used in the treatment of cough and cold, containing phenylephrine hydrochloride, paracetamol, caffeine, and diphenhydramine hydrochloride as active pharmaceutical ingredient was selected for the development of a novel, rapid, simultaneous isocratic reversed phase-high performance liquid chromatography (RP-HPLC) method. Objective: The objective of this paper was to develop and validate a novel, rapid, simple, precise, accurate and reproducible RP-HPLC method for simultaneous estimation of phenylephrine hydrochloride, paracetamol, caffeine, diphenhydramine hydrochloride in bulk and pharmaceutical dosage form. Method: Optimized chromatographic conditions were an isocratic elution with prontosil C18-column (250×4.6 mm, 5 μ), methanol and 20mM phosphate buffer (55:45 v/v, pH 3 ) as mobile phase, flow rate 1.0 mL/min and UV detector set at λ max 220 nm. The method was validated for specificity, precision, linearity, accuracy, sensitivity, and robustness as per the International Council for Harmonization guidelines. Result: The retention times of phenylephrine hydrochloride, paracetamol, caffeine, diphenhydramine hydrochloride were found to be 2.8 min, 3.3 min, and 4.0 min and 7.3 min, respectively. This novel method was found to be rapid, simple, linear (R2&gt; 0.99), preciserelative standard deviation &lt; 2.0 %), accurate (recovery 98-102%), sensitive and robust. Conclusion: The proposed novel isocratic RP-HPLC method is rapid (short run time below 10min), highly selective, precise, accurate, sensitive and robust. The method was successfully applied for the simultaneous analysis of phenylephrine hydrochloride, paracetamol, caffeine, diphenhydramine hydrochloride in a pharmaceutical dosage form.

Detection and Quantitative Estimation of Toxic Acrylamide Levels in Selected Potatoes Chips and French Fries from the Libyan Market Using HPLC-UV Method

Acrylamide is a potential health hazardous compound occurring in baked and fried food as a result of excessive dry heating during the preparation and/or processing of foods. Exposure to a high level of acrylamide may cause cancer, neurotoxicity, and mutagenicity. In this study, an isocratic reversed-phase high-performance liquid chromatographic (HPLC) method using a C18 column was used for the determination of acrylamide in selected food. The mobile phase consisted of 0.1% formic acid in water: acetonitrile (98:02), and the flow rate was 1.0 mL min-1, elution was monitored at 200 nm. Validation in selected conditions showed that the chosen method is sensitive, selective, precise, and reproducible with a linear detector response for the determination of acrylamide. The limit of detection (LOD), and the limit of quantification (LOQ), were achieved at 0.41μg mL-1 and 1.25 μg mL-1respectively. The proposed method was also applied after validation to the most popular six brands of chips and French fries available in the Libyan market. Acrylamide was extracted by a simplified extraction method avoiding cleanup by solid-phase extraction (SPE), then analyzed by HPLC-UV. The highest level of acrylamide was found in one brand of chips with a concentration of 16.33 μg mL-1, whereas only one of the French fries products analyzed exhibited an acrylamide concentration of 10.26 μg mL-1.

DEVELOPMENT AND VALIDATION OF REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY ASSAY METHOD FOR ESTIMATION OF FENOFIBRATE IN TABLET DOSAGE FORM PREPARED USING CRYSTALLO-CO-AGGLOMERATES OF THE DRUG

The aim of the present study was to develop a simple isocratic reverse phase-high performance liquid chromatography (RP-HPLC) method and validate for the determination of fenofibrate in tablet dosage forms. RP-HPLC method was developed using Hi Q Sil C18 (250 cm × 4.6 mm, 5 μm) and mobile phase comprising 1 mM ammonium acetate buffer: Acetonitrile (10:90 v/v) at a flow rate of 1.0 mL/min. The detection was carried out at 290 nm. The retention time was found to be 6.15 ± 0.03 min. Validation of the method was performed for precision, accuracy, linearity, robustness, specificity and sensitivity to conform to the International Conference on Harmonization (ICH) guidelines. The data of linear regression analysis indicated a good linear response in the concentration range of 5 μg/mL–30 μg/mL with correlation co-efficient (R2) of 0.997. The developed method was found to be simple, sensitive, accurate and repeatable for assay of tablets of fenofibrate prepared using crystallo-co-agglomerates of the drug.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ESTIMATION METHOD FOR TRANSDERMAL DIFFUSION STUDY OF GRANISETRON

Objective: The study is aimed to develop a simple, isocratic and sensitive reversed-phase high-performance liquid chromatography method for the analysis of granisetron during transdermal permeation studies through porcine ear epithelium.&#x0D; Methods: A reversed-phase (C18) column was used with ultraviolet detection at 210 nm. Selected mobile phase contained 25% (v/v) of acetonitrile and 75% (v/v) of 0.25 mM potassium dihydrogen orthophosphate solution pH adjusted to 3.0 using 1% orthophosphoric acid solution.&#x0D; Results: Calibration curve showed good linearity over 0.1–30 μg/mL concentration range of porcine ear permeates in 80% ethanol and 20% water (blank permeates). The applicability of the method was demonstrated by the analysis of diffusion study samples of granisetron through porcine ear epithelium and the steady-state permeability flux (J) from ethanolic solution was found to be 4.707 µg/square cm/h.&#x0D; Conclusion: The developed method can be used in the analysis of diffusion study samples of granisetron transdermal formulations through the porcine ear epithelium.

BIO ANALYTICAL METHOD DEVELOPMENT AND VALIDATION FOR SIMULANEOUS ESTIMATION OF NITAZOXANIDE AND OFLOXACIN IN HUMAN PLASMA BY RP-HPLC

An isocratic Reversed-Phase High Performance Liquid Chromatography method has been developed for rapid and simultaneous separation and estimation of two antibiotics, namely, nitazoxanide and ofloxacin, in human plasma. Separation was carried out on Altima C8 (150 x 4.6 mm, 5µ) column using a mobile phase of 0.1% ortho phosphoric acid: acetonitrile (50:50, V/V) at 260 nm. The retention time of nitazoxanide and ofloxacin was noted to be 4.850 and 7.949 min, respectively. The average % recovery for nitazoxanide and ofloxacin were 98.012 % and 94.176 %, respectively and reproducibility was found to be satisfactory. The linearity was investigated in the concentration range of 0.02-2 µg/ml (r2=0.9996) for nitazoxanide and 0.008-0.8 µg/ml (r2=0.9998) for ofloxacin. The lower limits of quantification were 0.0196 µg/ml and 0.0079 µg/ml for nitazoxanide and ofloxacin, respectively, which reach the level of both drugs possibly found in human plasma. The proposed method can be applied for etermination of nitazoxanide and ofloxacin from dosage forms during pharmacokinetic study.

Development and validation by statistical treatment of stability indicating RP-HPLC method for quantification of Orlistat in Orlistat-loaded solid dispersion

BackgroundMost of the analytical methods reported for the estimation of Orlistat were complex, expensive, and deficient in reproducibility with no or very less informative regarding various statistical methods and equations used for the validation purpose. This study provides a fast, accurate, descriptive, and precise isocratic reversed phase high-performance liquid chromatographic (HPLC) method using Waters Spherisorb 5 μm Octadecyl-silica-2 (250 × 4.6 mm) column, for the estimation of Orlistat in bulk drug and pharmaceutical formulations with minimized drug extraction steps. The drug was detected in an analytical column with mobile phase comprising a mixture of methanol, acetonitrile, and 2% phosphoric acid in the ratio of 85:14:1 v/v/v at flow rate of 1 ml/min with elution monitoring at 215.0 nm.ResultsThe retention time for Orlistat was found to be 5.9 min with sharp and proper peak. The linearity was covered over the concentration range of 1.00–10.00 μg/ml (r2 = 0.9997) with a limit of detection and limit of quantitation 0.06 and 0.2 μg/ml, respectively. The developed analytical technique was found to be validated for all the parameters within the acceptance criteria of ICH guidelines. The mean ± standard deviation (SD) recoveries of Orlistat were 99.87 ± 0.45.ConclusionThe optimized method was well precise, accurate, sensitive, stability indicating, and tested with all statistical parameters. Thus, the method can be conveniently used in quality control and routine analysis of Orlistat containing solid dispersions and other formulations. The main advantage of the developed method was its high specificity for the estimation of Orlistat in presence of various degradation products resulting from stress conditions and formulation excipients.

Analysis of Aspirin, Prasugrel and Clopidogrel in Counterfeit Pharmaceutical and Herbal Products: Plackett-Burman Screening and Box-Behnken Optimization.

An isocratic reversed-phase high-performance liquid chromatographic method has been developed and validated for the simultaneous determination of aspirin, prasugrel HCl and clopidogrel bisulfate in the presence of clopidogrel-related compound (impurity-A) in focus on counterfeit. This method was used to determine counterfeited antiplatelet drugs in two substandard Indian pharmaceutical products sold on the market in Yemen and two traditional herbal medicines sold on the market in China. Thin layer chromatography and mass spectrometry of counterfeit herbal medicines have additionally been carried out to verify the identification of adulterants. Chromatographic separation was performed on Inertsil ® ODS-3 C18 (4.6 × 250mm, 5μm) with isocratic mobile phase elution containing a mixture of acetonitrile: (25mM) potassium dihydrogen phosphate buffer, pH2.7 adjusted with 0.1M o-phosphoric acid (79: 21, v/v), at a flow rate of 1mL/min and UV detection at 220nm. Designs of experiment methodology, Plackett-Burman and Box-Behnken designs were used for the screening and optimization of the mobile phase composition. The method validation was also performed in accordance with the International Council on Harmonization (ICH) guidelines. The method developed for routine analysis was found to be sensitive, simple, accurate and highly robust. The results were statistically compared to reference methods using Student's t-test and variance ratio F-test at P<0.05.

Development of a reversed phase-high performance liquid chromatographic method for the analysis of glucosamine sulphate in dietary supplement tablets

Glucosamine sulphate can be taken orally from the dietary supplement tablets for the treatment of osteoarthritis. In these tablets, the determination of glucosamine sulphate concentration is very important for the identification of fraudulent products and quality-control studies. In this study, a simple, rapid, accurate and sensitive method was developed for the determination of glucosamine sulphate in the dietary supplement tablets by using isocratic reversed-phase high-performance liquid chromatography with diode array detector (RP-HPLC-DAD). The separation process was performed by Prodigy™ C18 reversed-phase column using an acetonitrile:H2O mixture (50:50 (v/v), pH 4.0) as mobile phase. The method developed was optimized and subsequently validated. Method validation was performed to illustrate the reliability of new analytical method through various validation parameters including linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), selectivity and stability. Relative standard deviation (%) values for the intra-day and inter-day precision studies were low. The calibration curve was linear in the glucosamine sulphate concentration range of 0.05-2.5 mg.L-1. In this method, recovery values were obtained in the range between 85.15% and 118.86% and LOD and LOQ values were determined to be 0.014 mg.L-1 and 0.046 mg.L-1, respectively. Furthermore, all results confirmed that the new method is suitable for the determination of glucosamine sulphate in dietary supplement tablets.