Phase High-performance Liquid Chromatography Method Research Articles

A Two-Step Optimization Approach: Validated RP-HPLC Method for Determination of Gatifloxacin and Dexamethasone in Ophthalmic Formulation.

The growing technology of stationary phase chemistry has a great impact on the chromatographic system performance and analysis economics. In this context, a simple rapid reversed phase high-performance liquid chromatography method development is presented for the analysis of gatifloxacin (GFN) and dexamethasone sodium phosphate (DSP) in their ophthalmic formulation. A two-step optimization approach has been conducted using optimum chromatographic conditions as well as proper selection of stationary phase. The chromatographic separation was carried out using sodium phosphate buffer pH3.0±0.1 and acetonitrile 72:28v/v, respectively, with flow rate 1mLmin-1 and simultaneous detection at 243nm. Three different column technologies were investigated at the optimum set of the chromatographic conditions: Xbridge® bridged ethylene hybrid silica, Kinetex™ Core-Shell and the Onyx™ Monolithic stationary phase. The monolithic column has shown better chromatographic separation, based on system suitability testing as well as shorter analysis time and sensitivity. The proposed method was validated according to International Conference on Harmonization guidelines. The linearity was achieved for GFN and DSP in the range 0.58-120μgmL-1 and 0.50-120μgmL-1, respectively, with acceptable accuracy, precision and selectivity.

Chemical Standardization and Anti-Proliferative Activity of Ardisia elliptica Fruit against the HCT116 Human Colon Cancer Cell Line.

The present study is intended to carry out the chemical standardization and evaluation of the anti-proliferative activity of A. elliptica fruit extract. A. elliptica fruit powder was extracted with ethanol. The obtained extract was assessed for total phenolic content using the Folin–Ciocalteu method. Moreover, a simple, accurate, and precise reversed phase high-performance liquid chromatographic method was developed and validated to determine the embelin content of A. elliptica fruit extract. Then, the extract and embelin were investigated for their anti-proliferative effect against HCT-116 cells. Finally, the mechanisms of inhibition of the extract and embelin on the mRNA expression of pro-apoptotic genes Bad, Bax, and Caspase-8 and anti-apoptotic genes c-IAP1, Mcl-1, and XIAP were determined by real-time qRT-PCR. The phenolic content and embelin content of the extract were 5.20 ± 0.01 g of gallic acid equivalent per 100 g of dried fruit (g% GAE) and 5.57 ± 0.56 mg/g of extract, respectively. The extract and embelin showed strong anti-proliferative effects on HCT-116 cells with 50% inhibition concentration (IC50) values of 19.16 ± 1.09 µg/mL and 25.93 ± 1.75 µg/mL, respectively. The A. elliptica extract exhibited a significant increase in the mRNA level of Bad, Bax, and Caspase-8 and a significant decrease in c-IAP1, Mcl-1, and XIAP. Embelin showed a significant decrease in Mcl-1 and XIAP.

Selenium and Zinc Biofortification of Pleurotus eryngii Mycelium and Fruiting Bodies as a Tool for Controlling Their Biological Activity.

Pleurotus eryngii (DC:Fr.) Quel. is a cultivated mushroom of high culinary value and medicinal properties. Mycelium of P. eryngii is characterized by the ability of effective bio-elements absorption from growth media so it could be biofortified with trace elements with a functional activity in the human body. In this study, the ability of P. eryngii mycelia from in vitro cultures as well as fruiting bodies were investigated in terms of their effectiveness in zinc and selenium accumulation. The effect of Se and Zn biofortification on productivity, chemical compounds, and bio-elements content of P. eryngii was determined as well. To enhance Se and Zn content in P. eryngii fruiting bodies and mycelia, substrates were supplemented with sodium selenite, at a concentration of 50 mg L−1, zinc sulfate, and zinc hydro-aspartate at a concentration of 87.2 and 100.0 mg L−1, respectively. Mentioned Zn concentrations contained the same amount of zinc(II) ions, namely 20 mg L−1. The content of organic compounds include phenolic compounds and lovastatin, which were determined by a high-performance liquid chromatography with diode-array detector (HPLC-DAD) and reverse phase high-performance liquid chromatography (RP-HPLC) method with UV detection. The ability of P. eryngii to accumulate zinc and selenium from the culture medium was demonstrated. The degree of accumulation of zinc turned out to be different depending on the type of salt used. The present study also showed that conducting mycelium of P. eryngii in in vitro culture, with a higher content of zinc ions, can result in obtaining the materials with better antioxidant ability. The results of this study can be used to develop the composition of growing media, which ensures the production of biomass with the desired composition of elements.

Chemometric Approach to Develop and Validate RP-HPLC Method for Estimation of Erlotinib Hydrochloride in Nano Structured Lipid Carriers

Background: Erlotinib hydrochloride is a novel drug for the treatment of lung cancer. Objective: The objective of the present study was to design an uncomplicated and precise reverse phase high-performance liquid chromatography (RP-HPLC) method and optimize the chromatographic parameters using response surface methodology derived from Box Behnken design. The optimized method was validated for estimating Erlotinib from bulk and nanostructured lipid carriers (NLCs) formulation. Methods: Independent variables such as flow rate, injection volume and strength of the buffer were optimized in order to decrease retention time and curtail asymmetry factor of Erlotinib. Forced degradation studies were done to determine the stability of the drug. The developed method was validated as per ICH guidelines. Results: The optimized strength of ortho-phosphoric acid buffer by blending with Acetonitrile (80:20 v/v), flow rate and injection volume were found to be 25mM, 1ml/min, 20µL respectively. Linearity was observed in the concentration range of 1-6 µg/mL. The retention time of Erlotinib was found to be 3.717 minutes. The limit of detection and limit of quantification for Erlotinib were found to be 0.01ng/ml and 1ng/ml, respectively. Conclusion: The proposed method was found to be a simple and the best method for analysing Erlotinib in nanostructured lipid carriers. Chemometric approach was employed as an effective tool for optimising the chromatographic conditions of the proposed method.

Validated RP-HPLC method for the simultaneous determination of glucosamine sulphate and curcumin in cream formulation: A novel stability-indicating study

Purpose: To develop and validate a stability-indicating reverse phase-high performance liquid chromatography (RP-HPLC) method for the simultaneous determination of glucosamine sulphate (GS) and curcumin (Cur) in drug solution and formulation.Methods: The optimized chromatographic conditions were achieved by passing various compositions of mobile phases over different reverse phase chromatographic columns. Various validation parameters, including linearity, range, limit of detection (LOD), limit of quantification (LOQ), accuracy, precision, specificity and system suitability were performed and evaluated. Stability studies under stressed conditions were done to evaluate the effects of acid, alkali, oxidation, heat and degradation by UV light.Results: The validated method was linear over the concentration range of 0.094 to 1.5 mg/mL for GS and 0.125 to 1.5 mg/mL for Cur, with a correlation coefficient > 0.999. The Intra and inter-day precision were 1.9 % for GS and 0.5 % for Cur, while accuracy was 96 and 102 % for GS and Cur, respectively. Stability studies showed that GS was highly sensitive to acid, alkali and oxidation and less sensitive to heat and UV. Cur was stable against acid, heat and oxidation but sensitive to alkali and UV.Conclusion: The developed and validated method was precise and accurate for both GS and Cur and can potentially be utilized for their identification and quantification at industrial, research and quality control laboratories.

Development and validation of a RP-HPLC method for the simultaneous analysis of paracetamol, ibuprofen, olanzapine, and simvastatin during microalgae bioremediation

A rapid reverse phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous quantification of paracetamol, ibuprofen, olanzapine, simvastatin and simvastatin acid in the context of microalgae bioremediation. The method was validated according to the guidelines of the US Food and Drug Administration (FDA), the International Conference on Harmonization (ICH), and Eurachem with respect to system suitability, linearity, accuracy, precision, recovery, limits of detection and quantification, ruggedness, selectivity and specificity. The estimated limits of detection and quantification were, respectively, 0.03 and 0.10 µg mL−1 for paracetamol, 0.03 and 0.09 µg mL−1 for ibuprofen, 0.04 and 0.13 µg mL−1 for olanzapine, 0.27 and 0.83 µg mL−1 for simvastantin, and 0.05 and 0.14 µg mL−1 for simvastantin acid. The inter-day and intra-day precision results were within the acceptance limit of relative standard deviation (%RSD) of less than 2, and the percentage recovery was found to be within the required limits of 80–110%. The developed method is rapid, linear, precise, robust and accurate, and has been successfully applied to the determination of the above common pharmaceutical products during microalgae bioremediation.

Quality by Design (Qbd) Approach to Develop HPLC Method for Estimation of Gliclazide and its Impurity (Gliclazide Impurity A) in Bulk Drug

Background:Gliclazide Impurity A (GI-A) is one of the gliclazide impurities, as described in the European Pharmacopoeia.Objective:The objective of this study was to develop and validate simple, robust and accurate Reverse- Phase High-Performance Liquid Chromatography (RP-HPLC) method for estimation of gliclazide along with GI-A in bulk by optimising chromatographic parameters using Box Behnken design in response surface methodology.Methods & Results:Box Behnken design was employed for optimizing flow rate, injection volume and strength of the buffer in order to minimize retention time of both gliclazide and GI-A. The optimized strength of orthophosphoric acid buffer in a mixture of Acetonitrile (50:50 v/v), flow rate and injection volume were found to be 25mM, 1mL/min, 20 µL respectively. Linearity was observed in concentration range of 25-150 µg/mL (r2=0.999). The retention time of gliclazide and GI-A was found to be 5.799 minutes and 3.819 minutes, respectively. The limit of detection for Gliclazide and GI-A was found to be 0.0066, and 0.0075 µg/mL and the limit of quantification limit was found to be 0.0202, 0.0228 µg/mL, respectively. The developed method was validated as per the ICH guidelines.Conclusion:The proposed method is useful for best analysis of Gliclazide and GI-A in pharmaceutical dosage forms. QbD approach was found to be an effective tool for optimising chromatographic conditions of the proposed method.

Enhancement of total antioxidants and flavonoid (quercetin) by methyl jasmonate elicitation in tissue cultures of onion (Allium cepa L.)

The onion (<em>Allium cepa</em>) is a vegetable used extensively all over the world both for culinary purposes as well as in medicine. Its medicinal values are due to the high levels of biologically-active compounds present within the bulb. There are various phytochemicals of therapeutic importance found in <em>A. cepa</em>. Quercetin, a flavonoid, is one of these phytochemicals and it is a potent antioxidant. <em>Allium cepa</em> is a dietary supplement and is beneficial for diverse ailments, thus justifying its status as a valuable medicinal plant. Due to its medicinal significance, elicitation of total antioxidants and quercetin levels have been attempted to enhance their production in tissue callus cultures. This study reports in vitro enhancement of total antioxidants and quercetin in <em>A. cepa</em> using methyl jasmonate as an elicitor. A reverse phase-high performance liquid chromatography (RP-HPLC) method was used with an isocratic system and a flow rate of 1.0 mL min<sup>−1</sup> and a mobile phase of acetonitrile: 1% v/v acetic acid (60%:40% v/v). The detection wavelength was 362 nm and the retention time 8.79 minutes. Total antioxidant and quercetin contents were maximal with 100 µM of methyl jasmonate in leaf tissue callus cultures at 84.61 ±6.03% and 0.81 ±0.03 mg g<sup>−1</sup> dry cell weight, respectively. They decreased with further increases of methyl jasmonate at 200 µM. The increase in total antioxidant and quercetin contents were 2.3- and 13.9-fold, respectively. The optimization of methyl jasmonate as an elicitor, as well as the determination of a suitable concentration in <em>A. cepa</em> in callus cultures, will be helpful for enhanced production of various other secondary metabolites of therapeutic significance. This could be beneficial for the pharmaceutical and neutraceutical industries for herbal drug formulations.

Formulation and evaluation of thymoquinone niosomes: application of developed and validated RP-HPLC method in delivery system

A rapid, accurate, and sensitive reverse phase high-performance liquid chromatographic (RP-HPLC) method was developed and validated for the estimation of Thymoquinone (TMQ) in API as well as in noisome. The chromatograms were developed with the mobile phase – water: 2-propanol: methanol (50:45:5 v/v/v) as a solvent system at 254 nm. The method was validated as per ICH guidelines for different parameters and the recovery of TMQ was calculated in developed niosomes. Further, TMQ loaded niosomes (TMQNIOS) were prepared and evaluated for different parameters. The optimized TMQNIOS (F3) was further evaluated for surface morphology, in vitro drug release, permeation study, and confocal laser scanning microscopic (CLSM) study. The method showed linearity range between 6.25 and 100 µg/ml with low detection limit and quantitation limit with a value of 2.08 and 6.25 µg/ml. The developed formulations showed the vesicle size and encapsulation efficiency in the range of 157.32 ± 3.15 to 211.44 ± 5.23 nm and 59.32 ± 4.87 to 83.21 ± 3.55%, respectively. The drug release result showed the significant higher release from TMQNIOS in compared to TMQDIS, and the release kinetics data showed Higuchi's equation with highest regression coefficient values. The permeation study and the confocal laser microscopy study further confirmed the enhancement in permeation of TMQ in the intestinal mucosa.

Development and Validation of a New HPLC Method for Quantification of a Novel Antifungal Drug Based on 1,3,4-Thiadiazole and its Impurities

Chromatographic behavior of a new antifungal drug, (2-[(Z)-1-(3,5-diphenyl-1,3,4-thiadiazol-2(3H)-ylidene)-methyl]-3,5-diphenyl-1,3,4-thiadiazol-3-ium chloride (TDZ), and its impurities was studied using several types of columns, mobile phases and elution modes. A new reversed phase high-performance liquid chromatography (HPLC) method for the determination of TDZ and its impurities in a pharmaceutical substance and dosage form (tablets, 60 mg) was developed and validated. The optimal chromatographic separation of TDZ and its impurities was performed on a 5-μm C8 column (4.6 mm × 250 mm, I.D.). The mobile phase consisted of 0.03% TFA in water (pH 2.5) and acetonitrile under gradient elution condition (from 40:80 v/v to 80:20 v/v for 20 min) at a flow rate of 1.0 mL min−1. UV detection was set at a wavelength of 250 nm. The limit of detection (LOD) and quantification (LOQ) were 0.05 and 0.1 μg mL−1, respectively. The method showed good linearity (r2 > 0.999) over the assayed concentration range (0.1–1.4 μg mL−1 for quantification of impurities and 0.005–0.013 mg mL−1 for quantification TDZ) and demonstrated good intra- and inter-day precision and accuracy. The method was validated and successfully applied for quantitative determination of TDZ and its impurities in both the pharmaceutical substance and tablets.

Analytical Method Development and Validation for the Simultaneous Estimation of Aspirin, Clopidogrel and Rosuvastatin in Pharmaceutical Dosage Form

A new, simple, novel, accurate, precise, reliable, rapid and linear reverse phase high-performance liquid chromatography (RP-HPLC) method was developed and fully validated for simultaneous qualitative and quantitative estimation of Rosuvastatin (ROS), Clopidogrel (CLOP) and Aspirin (ASP) in bulk and pharmaceutical dosage form as per International Conference on Harmonization (ICH) guidelines. In the present work, good chromatographic separation was achieved by isocratic method using a Hypersil BDS C18 column (250 mm ×4.6, 5 μm) and a mobile phase consisting of KH2Po4 buffer pH-6.0: acetonitrile in the ratio 60:40, at a flow rate of 1 ml/min. The effluents obtained were monitored at 242nm with the UV-visible detector. The calibration curves obtained were linear (r2=0.999) over the concentration range of 7.5-22.5μg/ml and 1-3μg/ml for CLOP, ASP and ROS respectively. A run time of 7.0 minutes for each sample made it possible to analyze more than 200 samples per day. The retention time of ASP, CLOP and ROS was found to be 3.103 min, 4,277 min and 5.707 min respectively. The high recovery values (99%-101%) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveal that the method is precise therefore the method can be used for routine monitoring of CLOP, ASP and ROS in industry in the assay of bulk drug and dosage form.
 Keywords: RP-HPLC, Rosuvastatin, Clopidogrel, Aspirin, Method validation, ICH guidelines.

Characterization of covalent crosslinking strategies for synthesizing DNA-based bioconjugates

An adapted strategy from the conventional 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) crosslinking method was developed to form a covalently coupled phosphoramidated single stranded DNA (ssDNA). Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) results demonstrated that the phosphoramidated ssDNA conjugate is stable for several days, and that phosphoramidation occurred exclusively at the 5′ phosphate of ssDNA. A reversed phase high-performance liquid chromatography (RP-HPLC) method with UV detection was developed to determine the yield of conjugates. The methods coefficients of variation (%CV) were less than 6%, and biases ranged from − 5.1 – 1.2%. The conjugate yield via the conventional EDC method was 68.3 ± 2.2%, while that of the adapted EDC/Imidazole method was 79.0 ± 2.4% (n = 10). This study demonstrates a convenient one pot strategy for crosslinking biological molecules.

A SIMPLE LIQUID CHROMATOGRAPHIC METHOD FOR SIMULTANEOUS ESTIMATION OF AZITHROMYCIN, FLUCONAZOLE AND ORNIDAZOLE IN BULK AND PHARMACEUTICAL DOSAGE FORMS

Objective: The objective of the study was to develop and validate a new rapid and more sensitive Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method for the simultaneous estimation of azithromycin, fluconazole and ornidazole in bulk and pharmaceutical dosage forms.
 Methods: Separation was achieved with a cap cell pack C18 column (4.6 x 250 mm, 5μ) with an isocratic mobile phase containing a mixture of acetonitrile and phosphate buffer pH 4.8 [adjusted with ortho-phosphoric acid] (50:50 % v/v) at the flow rate of 1 ml/min and detection was monitored at 210 nm.
 Results: The retention time (Rt) of azithromycin, fluconazole and ornidazole were found to be 4.82±0.01, 5.25±0.01 and 6.33±0.01 min respectively. The precision was found with<1.5% of %RSD. The calibration curve was linear over the concentration ranging from 500-1000 µg/ml for azithromycin, 75-150 µg/ml for fluconazole and 375-750 µg/ml for ornidazole with the correlation coefficient (r2) of 0.999. The percentage recovery was found to be within the specified range i.e., 98-102 % for three drugs. Limit of detection (LOD) was found to be 5.810, 1.790 and 4.924 µg/ml, whereas Limit of quantification limits (LOQ) was found to be 9.834, 2.667 and 7.980 µg/ml, respectively.
 Conclusion: A simple isocratic liquid chromatographic method was developed and validated for simultaneous estimation of azithromycin, fluconazole and ornidazole in their formulations. Due to its simplicity, rapidness and specificity, it can be applied for routine quality control analysis of these drugs.

A A RAPID AND SENSITIVE RP-HPLC METHOD FOR THE QUANTITATIVE ANALYSIS OF EMPAGLIFLOZIN IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: The present study aims at developing an accurate precise, rapid and sensitive Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method for assessing Empagliflozin in bulk drug and in the pharmaceutical dosage form.
 Methods: The proposed method employs a Reverse Phase Shim Pack C18 column (250 mm × 4.6 mm id; 5 µm) using a mobile phase comprising of acetonitrile and water in the ratio of 60:40 v/v flushed at a flow rate of 1 ml/min. The eluents were monitored at 223 nm.
 Results: Empagliflozin was eluted at a retention time of 5.417 min and established a co-relation co-efficient (R2>0.999) over a concentration ranging from 0.0495-100µg/ml. Percentage recovery was obtained between 98-102% which indicated that the method is accurate. The Limit of Detection (LOD) and Limit of Quantitation (LOQ) were found at 0.0125µg/ml and 0.0495µg/ml, respectively.
 Conclusion: An RP-HPLC method which was relatively simple, accurate, rapid and precise was developed and its validation was performed for the quantitative analysis of empagliflozin in bulk and tablet dosage form (10 and 25 mg) in accordance to International Conference of Harmonization (ICH) Q2 (R1) guidelines. The proposed method may aid in routinely analyzing empagliflozin in pharmaceuticals.

Simultaneous Estimation of Saxagliptin and Dapagliflozin in Human Plasma by Validated High Performance Liquid Chromatography - Ultraviolet Method.

The fixed dose combination of saxagliptin and dapagliflozin is a recently approved antidiabetic medication. It is marketed under the brand name Qtern. The aim of this study was to develop a simple, rapid, sensitive, and validated isocratic reversed phase-high performance liquid chromatography (RP-HPLC) method for the simultaneous estimation of saxagliptin and dapagliflozin in human plasma using linagliptin as internal standard as per US-Food and Drug Administration guidelines. The method was performed on a Waters 2695 HPLC equipped with a quaternary pump. The analyte separation was achieved using an Eclipse XDB C18 (150 × 4.6 mm × 5 µm) column with a mobile phase consisting of 0.1% ortho phosphoric acid and acetonitrile (50:50) with pH adjusted to 5.0 at 1 mL/min flow rate. The analyte was detected at 254 nm. The retention time of the internal standard, saxagliptin, and dapagliflozin was 2.746, 5.173, and 7.218 min, respectively. The peaks were found to be free of interference. The method was validated over a dynamic linear range of 0.01 to 0.5 μg/mL and 0.05 to 2 μg/mL for saxagliptin and dapagliflozin, respectively, with a correlation coefficient of 0.998. The precision and accuracy of samples of six replicate measurements at lower limits of quantification level were within the limits. The analytes were found to be stable in human plasma at -28°C for 37 days. The stability, sensitivity, specificity, and reproducibility of this method make it appropriate for the determination of saxagliptin and dapagliflozin in human plasma.

RELATED SUBSTANCE METHOD DEVELOPMENT AND VALIDATION OF LOXAPINE SUCCINATE IN CAPSULE DOSAGE FORM BY REVERSE PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Objective: The present study gives a simple, rapid, and accurate stability indicating reverse phase high-performance liquid chromatography method for the determination of loxapine succinate and its related substance (related compound A) in capsule dosage form.
 Methods: Loxapine succinate and its related substance were attained on a C18 Purospher star (250 mm × 4.6 mm, 5 μm particle size) column at 254 nm detection wavelength, 1.0 ml/min as a flow rate, and 10 μl injection volume. Water:methanol: Triethylamine: Tetrahydrofuran (50:40:1:10) was used a mobile phase, and column oven temperature was 30°C.
 Results: The resolution between loxapine succinate and known unknown impurities was >2.0. The correlation coefficient (0.999) value indicates the linear relationship between the concentration and peak areas. The accuracy study was performed by spiking method. Loxapine succinate was exposed to the stress condition of hydrolysis (acid and base), oxidative, thermal, and photolytic degradation. Loxapine succinate was found to degrade unquestionably in acid and base stress condition and almost stable in oxidative, thermal, and photolytic conditions.
 Conclusion: The degradant products were well resolved from leading peak and its related compound A peak and any other unknown peak justifying the stability indicating capability of the method. The developed method was validated as per the ICH guidelines. This method is used for periodic analysis in laboratory.

AN INNOVATIVE WAY FOR EXTRACTION OF ESSENTIAL OIL COMPONENTS FROM PIMPINELLA ANISUM L. FRUITS

Objective: The aim of this article was to test the hypothesis about the possibility for extraction of essential oil components from the plant raw material (PRM) by the example of Pimpinella anisum L. fruits with two different perfluoro organic solvents, Novec 1230 and Novec 7100.
 Methods: For the studies, we used Pimpinella anisum L. fruits ground to obtain particle size between 0.1 and 0.5 mm. The study of qualitative and quantitative content of the extracts was carried out by the following method: simple maceration, PRM: extractant ratio 1:10 w/v; and circulation method for extraction using Soxhlet extractor, PRM: extractant ratio 1:5 w/v. Qualitative analysis of extracts was carried out by gas chromatography-mass spectrometry (GC-MS) method. Quantitative analysis of trans-anethole in the extracts was carried out by reverse phase high-performance liquid chromatography (RP HPLC) method.
 Results: The largest yield of anethole was observed for n-hexane, 84±4 %, and for Novec 7100, 81±4 %. In case of Novec 1230 solvent, the yield of anethole was noted to be three times less, 32±2 %. The yield of anethole under the conditions of solvents (Novec 1230 and Novec 7100) circulation method was up to 94±5 % within two hours.
 Conclusion: Adequacy of the working hypothesis about the possibility for extraction of essential oil components from Pimpinella anisum L. fruits by two different perfluoro organic solvents, Novec 1230 and Novec 7100, was verified experimentally. In the context of the solvents tested, it was found that the level of fluorination of the solvent’s molecule had a great influence on its extraction properties in regard to anethole and vegetable oil.

Bioanalytical method development and validation of garenoxacin mesylate in human plasma by RP-HPLC

A simple, precise, accurate Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method was developed for the estimation of Garenoxacin mesylate in human plasma using Ciprofloxacin Hydrochloride as an internal standard. Chromatographic conditions used are stationary phase Zorbax Eclipse XDB C18 (250x4.6 mm, 5m), Mobile phase 0.1% orthophosphoric acid and Acetonitrile in the ratio of 50:50 (% v/v) and flow rate was maintained at 1.0 ml/min, detection wavelength was 240 nm, injection volume of 50 µL and the column temperature was set to 30°C. The retention time of Garenoxacin mesylate was found to be 4.0 min. % Coefficient of Variation of the Garenoxacin mesylate was found to be 4.30. % Recovery was obtained as 98.97%. The linearity of the proposed method was established in the concentration range of 0.04 to 4 µg/ml (Correlation Coefficient = 0.999). The lower limit of quantification was 0.04 μg/ml (S/N Ratio 21) which reach the level drug possibly found in human plasma. Further, the reported method was validated as per the ICH guidelines and found to be well within the acceptable range.

VALIDATION AND QUANTITATIVE ANALYSIS OF CARMINE AND RHODAMINE B IN LIPSTICK FORMULATION

Objective: The objective of this study was to validate reversed phase-high performance liquid chromatography (RP-HPLC) method using photo-diode array detector for the simultaneous determination of carmine (CAR) and rhodamine B (RHO B) in lipstick products.
 Methods: CAR and RHO B were analysed using Cosmosil C18 column (250 mm x 4.6 mm i.d., 5 µm) using Shimadzu LC 20AD chromatograph equipped with photo-diode array (PDA) detector at 245-600 nm. The mobile phase used was acetonitrile-phosphate buffer pH 3.4 (5.5:4.5 v/v) delivered isocratically at flow rate of 1.0 ml/min, using column temperature at 35 °C. The optimized HPLC condition was validated by determining several characteristics including selectivity, linearity, sensitivity expressed by the limit of detection and limit of quantification, precision and accuracy.
 Results: The optimized HPLC condition could separate CAR and RHO B successfully with resolution value>2.0. The method was linear over studied concentration with a coefficient of determination (R2) 0.9999. The relative standard deviation (RSD) values were lower than those required RSD Horwitz and the recovery values were also acceptable, therefore, the developed method was precise and accurate.
 Conclusion: HPLC with PDA detector has been accurately and successfully used for quantitative analysis of CAR and RHO B. The developed method is simple and can be used for routine analysis of CAR and RHO B for quality assurance purposes.

SIMPLE AND RAPID HPLC METHOD FOR SIMULTANEOUS QUANTIFICATION OF LEVODOPA AND CARBIDOPA IN A FAST DISINTEGRATING TABLET FORMULATION

Objective: Fast disintegrating tablets (FDTs) are found helpful in dysphagia (difficulty in swallowing) especially in Parkinson patients. Levodopa is still the first choice in Parkinson disease treatment and is co-administered by carbidopa for better efficacy.
 Methods: In the present study, a rapid and simple isocratic Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for simultaneous quantification of levodopa and carbidopa in optimized Fast Disintegrating Tablets (FDTs). The linearity, precision, accuracy, limit of detection (LOD) and limit of quantification (LOQ) of the method were determined. FDTs were prepared using direct compression, dry and wet granulation and were optimized for faster disintegration time. Tablets thickness, weight, hardness, friability, drug content and dissolution profile were also evaluated.
 Results: A RP-HPLC system with C18 column and mobile phase 90:10 (v/v) phosphate buffer: methanol was used. The method linearity was found to be within the concentration range of 3.125-50 μg/ml for levodopa, and 3.125-25 μg/ml for carbidopa. The intra and inter-day precision and accuracy were acceptable. LOD and LOQ of levodopa-carbidopa were 0.2-0.8 μg/ml and 0.5-2.4 μg/ml, respectively. The total chromatographic run time was 5 min. The optimized FDTs hardness was 3.81±0.4 and tablets were disintegrated within 30 sec. Levodopa and carbidopa were dissolved in dissolution media within 5 min.
 Conclusion: Results indicated that this method was suitable for simultaneous quantification of levodopa and carbidopa in the presence of different ingredients of a pharmaceutical solid dosage form. Therefore, this method could be applied in pharmaceutical quality control for rapid quantification of structurally similar substances with different physicochemical properties.